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piggyBac (PB) is a DNA transposon that mediates transposition in various animal cells. As an efficient tool, PB transposons are applied to various transgenic research and optimized for different species, which is an essential means to enhance its versatility. To construct a universal PB transposase (PBase) vector for ovine transgene manipulation, the gene expression box of PBase with optimized bias for sheep codons was inserted into the pBNW-TP1 vector. The vector pBNW-TP2 with a single plasmid was successfully constructed. Then, pBNW-TP2 was transfected into ovine fibroblasts and mammary epithelial cells. The stable transfected cell lines were selected by G418 and the PB transposition sites were determined by Tail-PCR in the stable transfected cell lines. The transposition efficiency was verified by student's t-tests for cell clones with methylene blue staining. The results showed that pBNW-TP2 successfully guided the production of transgenic ovine fibroblasts and mammary epithelial cell lines. The PB transposition test indicated that the pBNW-TP2 vector can be specifically integrated into the TTAA sites of the sheep genome. The statistical analysis of methylene blue staining results suggested that the transgenic efficiency mediated by the pBNW-TP2 vector increased significantly. In summary, we verified and analyzed characteristics of the universal PB transposon vector pBNW-TP2 for sheep in this study, which will provide a scientific basis for future transgenic research mediated by the PB transposon in ovine somatic cells.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of adipogenesis. Our previous study showed that unlike human and mouse PPARγ transcripts, several chicken PPARγ transcript variants contain upstream open reading frames (uORFs) in their 5'untranslated region (5'TR). To decipher the role of uORFs in post-transcriptional regulation of chicken PPARγ gene, we constructed wild-type (psiCHECK2-cPPARγ3-5'UTR-WT) and a uORF mutant (the upstream ATG (uATG) was mutated to stop codon TGA) 5'UTR reporters (psiCHECK2-cPPARγ3- 5'UTR-Mut) of chicken PPARγ transcript variant 3 (cPPARγ3). These two reporters were individually transfected into immortalized chicken pre-adipocytes (ICPA) and DF1 cells, and the renilla luciferase (hRluc) activity and mRNA expression level were detected by reporter assay and qRT-PCR. The results showed that the hRluc activity of the mutated 5'UTR was significantly higher than that of the wild-type 5'UTR in ICPA cells (P<0.01), and the hRluc activity of the mutated 5'UTR tended to be higher than that of the wild-type 5'UTR in DF1 cells, but this difference did not reach statistical significance (P>0.05). The qRT-PCR analysis showed, in ICPA cells, the hRluc mRNA expression was significantly lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct (P<0.01). In DF1 cells, the hRluc mRNA expression tended to be lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct, but this difference did not reach statistical significance (P>0.05). To further gain insight into the post-transcriptional regulation of cPPARγ3 by the uORF, we constructed the expression plasmids bearing the full-length coding region of chicken PPARγ gene plus either wild-type or mutant uORF 5'UTR (pcDNA3.1-cPPARγ3-WT and pcDNA3.1-cPPARγ3-Mut). These two constructed PPARγ expression plasmids were individually transiently transfected into both ICPA and DF1 cells, and PPARγ mRNA and protein levels were assayed by qRT-PCR and western blotting. The result showed that in both cell lines, PPARγ mRNA expression was significantly lower in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.05). In contrast, western blot analysis showed that PPARγ protein level was significantly higher in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.001). Taken together, our results demonstrate that the uORF in 5'UTR of the cPPARγ3 inhibits its translation.

Objective: To construct an eukaryotic expression plasmid for AY358935 gene and explore its function., Methods: cDNA of the AY358935 gene was amplified by reverse transcription-PCR and cloned into pGEM-Teasy. The pGEM-T-AY was validated by sequencing and served as a template for the construction of eukaryotic expression plasmid. The pcDNA3.1-AY recombinant was validated by double enzyme digestion and used for transient transfection of M14 cells. Expression of the AY358935 protein and proliferation of the M14 cells were determined respectively by Western blotting and 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) colorimetry., Results: The amplicons of RT-PCR were confirmed to have similar size with the cDNA fragment of the AY358935 gene as well as cloned region of pcDNA3.1-AY. The cloned region of pGEM-T-AY was sequenced to be identical with cDNA sequence of the AY358935 gene. M14 cells were transfected by the AY358935 gene, pcDNA3.1 and liposomes, respectively. After 48 h, expression of the AY358935 protein in M14 cells transfected with the AY358935 gene was significantly higher than other two groups. They also had a significantly higher absorbance value (A=0.74) than other two groups (A=0.39 and 0.46, respectively; P<0.05)., Conclusion: An eukaryotic expression plasmid of the AY358935 gene was successfully constructed. Product of the AY358935 gene may promote the proliferation of M14 cells.

Objective: To investigate the role of allograft inflammatory factor-1 (AIF-1) in colorectal cancer (CRC) progression and explore the possible mechanism., Methods: The expression levels of AIF-1 in 70 CRC tissues and paired adjacent tissues were detected using immunohistochemistry and Western blotting, and the correlation of AIF-1 expression with the clinicopathological features of the patients was analyzed. In the CRC cell line SW480, the functional role of AIF-1 in regulating tumor progression was investigated by transfecting the cells with an AIF-1-overexpressing plasmid (AIF-1) and a negative control plasmid (NC). EdU proliferation assay and flow cytometry were used to assess the cell proliferation and cell cycle changes; Transwell migration assay and Annexin V-APC/7-AAD apoptosis assay kit were used to analyze the cell migration and apoptosis. The changes in the biological behaviors of the cells were observed after application of SB203580 to block the p38 MAPK pathway. The expression levels of CDK4, cyclin D1, P21, P27, MMP2, MMP9, Bax, Bcl2, Bcl-xl, p38 and p-p38 were detected using Western blotting., Results: AIF-1 was down-regulated in CRC tissues compared with the adjacent normal tissues, and its expression level was positively correlated with lymph node metastasis (P=0.008), TNM stage (P=0.003) and tumor size (P=0.023). Overexpression of AIF-1 in SW480 cells significantly reduced EdU-positive cells and caused obvious cell cycle arrest in G1 phase (P<0.05). AIF-1 overexpression resulted in significantly lowered protein expressions of CDK4 and cyclin D1, enhanced expressions of P21 and P27, attenuated cell migration ability (P<0.001), and decreased protein levels of MMP2 and MMP9. AIF-1 overexpression also induced obvious apoptosis of SW480 cells (P<0.01), significantly increased the protein levels of Bax and p-p38, and decreased the protein levels of Bcl-2 and Bcl-xl; SB203580 significantly attenuated the apoptosis-inducing effect of AIF-1 overexpression., Conclusion: AIF-1 plays the role of a tumor suppressor in CRC by inhibiting cell proliferation, suppressing cell migration and inducing cell apoptosis. AIF-1 overexpression promotes the apoptosis of CRC cells by activating the p38 MAPK pathway.

Objective: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties., Methods: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods., Results: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity., Conclusions: LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.

Objective: To explore the stability of resistant phenotypes and changes of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene system on four Shigella strains in the absence of antibiotics. Methods: Four clinical isolated Shigella strains that resistant to different antibiotics were consecutive passaged for 90 times without antibiotics. Agar dilution method was used to determine the minimum inhibitory concentration of Shigella strains. After sequence analysis with PCR, CRISPR Finder and Clustal X 2.1 were applied to identify the changes of CRISPR loci in the Shigella strains. Results: After the consecutive transfer of 90 generations, sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums increased. Mel-sf1998024/zz resistance to ampicillin, cephalexin, cefotaxime, chloramphenicol decreased, mel-s2014026/sx resistance to norfloxacin, trimethoprim decreased, mel-sf2004004/sx drug resistance to ampicillin, cefuroxime, cefotaxime, chloramphenicol, trimethoprim decreased and mel-sf2013004/bj resistance to chloramphenicol decreased. The spacer of which matched gene codes Cas and its upstream repeat in 3'end of CRISPR3 got lost in mel-sf1998024/zz and mel-sf2013004/bj. Conclusions: Shigella strains could reduce or lose their resistance to some antibiotics after consecutive transfers, without the interference of antibiotics. CRISPR3 locus had dynamic spacers in Shigella strains while CRISPR3 locus and cas genes might have been co-evolved.

Objective: We constructed Bordetella bronchiseptica QH0814 hcp mutant to characterize its pathogenicity.[Methods] Through the homologous recombination mediated by a suicide plasmid pRE112, we acquired the mutant QH0814Δhcp successfully. Then, we evaluated the growth condition, the ability of adhesion and invasion, the median lethal dose (LD50) and the infection capacity., Methods: Through the homologous recombination mediated by a suicide plasmid pRE112, we acquired the mutant QH0814Δhcp successfully. Then, we evaluated the growth condition, the ability of adhesion and invasion, the median lethal dose (LD50) and the infection capacity., Results: There was no significant variation of the growth rate between the mutant and the parental strain. Compared with the parental strain, the adherence ability of the mutant did not change remarkably. However, the invasion ability decreased significantly. Mice lethal test showed that the LD50 of the mutant was higher than that of the parental strain. Correspondingly, the bacterial colonization of the mutant in mice blood, lung and liver was much less than that of the parental strain., Conclusion: The knocking-out of the hcp gene had no influence on bacterial growth, but it could attenuate significantly the invasion and colonization of the bacterium. Therefore, the gene may play a role in the pathogenesis of Bordetella bronchiseptica.

Objective: We studied the functions and characteristics of hfq gene in Mesorhizobium huakuii 7653R in adverse environment and symbiotic with its host plant., Methods: The hfq mutant of 7653R was constructed via homologous recombination with small cloned fragments on suicide plasmids pK19mob to insert target gene. We applied 7653RΔhfq to characterize stress tolerance and symbiosis with host plant, in comparison with the complementary strains 7653R △hfq-C and the wild type., Results: Mutant 7653RΔhfq presented lower growth rate, and higher mortality after heat shock-pretreated than that of the wild type, as well as the decreasing adaptability under the stress of 4.5% ethanol and 50 mmol H2O2. The defection of hfq affected the expression of some sRNAs in 7653R. Moreover, the mutant displayed significant reduced nodulation ability and nitrogenase activity compared with the wild type., Conclusion: As a crucial post transcriptional regulatory factor, hfq plays an important role in Mesorhizobium Huakuii 7653R on both processes of stress resistance and symbiosis with the host plant Astragalus sinicus L.

Objective: We aimed at co-expressing heterologous D-hydantoinase and N-carbamoylase in Bacillus subtilis, and evaluating the feasibility of producing D-p-hydroxyphenylglycine by the recombinant B. subtilis whole-cell catalysis., Methods: The Paco expression cassette was combined with the coding sequence of hyd or sd1 gene as an artificial gene to express D-hydantoinase. The PAE expression cassette was combined with the coding sequence of adc gene as an artificial gene to express N-carbamoylase. The D-hydantoinase and N-carbamoylase co-expression plasmids pHCS(sd1+adc) and pHCY(hyd+adc) were constructed, using plasmid pHP13 as carrier; the co-expression plasmids pUCS(sd1+adc) was constructed, using plasmid pUB110 as carrier. The additional copy of acoR and sigL gene was integrated at chromosome. The skf and sdp gene were knocked out in B. subtilis. All recombinant strains bearing co-expression plasmid were characterized by analyzing whole-cell catalysis activity., Results: In the recombinant strains with plasmid pHCY and with pHCS, the whole-cell catalytic activity reached 0.21 U/mL and 0.31 U/mL, respectively. After the over-expression of acoR, sigL, and high-copy-number pUCS, the whole-cell catalytic activity reached 1.0 U/mL., Conclusion: Overexpression of acoR, sigL and the deletion of skf, sdp genes had significant effects on the catalysis activity of recombinant whole-cell.

Objective: In this study, we constructed recombinant Kluyveromyces lactis strains to produce phospholipase C (PLC) of Bacillus cereus. The recombinant enzymes were purified and characterized., Methods: We cloned the PLC encoding gene bcplc of Bacillus cereus. And the amplified fragments were inserted into pKLAC1 to obtain expression plasmids. K. lactis harboring the above plasmids was cultivated to express PLC that was purified by HisTrapTM affinity chromatography and characterized., Results: PLC of B. cereus was cloned and expressed in K. lactis. The recombinant enzyme had shown activity of 19251 U/mg when using p-nitrophenyl phosphorycholine as substrate. Purified PLC exhibited optimum temperature at 80 °C and optimal pH at 9.0. The recombinant enzyme was stable below 40 °C and pH between 7.0 and 8.0. Cu2+ and Co2+ inhibited its activity whereas Zn2+, Mn2+, Ca2+ and Mg2+ stimulated its activity., Conclusion: It is the first time to express and characterize the PLC gene in K. lactis. These research results provide reference for the study of recombinant PLC.

Objective: To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3)., Methods: We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy., Results: We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3., Conclusion: This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.

To study the effect of interferon(IFN)against hepatitis B virus(HBV)by silencing phospholipid scramblase (PLSCR)1in HepG2 cells. siRNA specific for PLSCR1 was designed and transfected in HepG2 cells. The inhibitory effect of siRNA was determined using semi-quantitative polymerase chain reaction(PCR)and western blotting 48hpost-transfection.HepG2 cells treated with IFN were co-transfected with plasmids expressing HBV1.3and siRNA targeting PLSCR1.Total RNA of HepG2 cells was isolated and the mRNA level of PLSCR1 measured by reverse-transcription semi-quantitative PCR. The expression of HBsAg in culture supernatants was determined by enzyme-linked immunosorbent assay. Expression of PLSCR1 was inhibited by siRNA911 in HepG2cells.Compared with the control, the level of HBsAg decreased in the cell supernatants of cells transfected with HBV1.3plasmid or NC-siRNA + HBV1.3plasmid.Compared with cells not treated with IFN, the level of HBsAg did not change significantly in the supernatants of cells transfected with siRNA + HBV1.3plasmid and treated with IFN. Inhibition of PLSCR1 could decrease the antiviral activity of IFN against HBV. These data suggest that PLSCR1 has an important role in the inhibition of HBV replication due to IFN.

Objective To obtain the PP7 bacteriophage-like particles carrying the peptide of prostatic acid phosphatase PAP 114-128 , and prove that they retain the original biological activity. Methods First, the plasmid pETDuet-2PP7 was constructed as follows: the gene of PP7 coat protein dimer was amplified by gene mutation combined with overlapping PCR technology, and inserted into the vector pETDuet-1. Following that, the plasmid pETDuet-2PP7-PAP 114-128 was constructed as follows: the PP7 coat protein gene carrying the coding gene of PAP 114-128 peptide was amplified using PCR, and then inserted into the vector pETDuet-2PP7. Both pETDuet-2PP7 and pETDuet-2PP7-PAP 114-128 were transformed into E.coli and expressed. The expression product was verified by SDS-PAGE, double immunodiffusion assay and ELISA. Results The gene fragment of PP7 coat protein dimer was obtained by overlapping PCR using Ex Taq DNA polymerase, and the antigenicity of its expression product was the same as that of the coat protein of wild-type PP7 bacteriophage. Moreover, the PAP 114-128 peptide epitope that was displayed on the surface of PP7 bacteriophage was identical with the corresponding epitope of natural human PAP, and it was able to induce high levels of antibodies. Conclusion The gene of PP7 coat protein dimer with repeated sequences can be prepared by gene mutation combined with overlapping PCR. Based on this, PP7 bacteriophage-like particles carrying PAP peptide can be prepared, which not only solves the problem of the instability of the peptides, but also lays a foundation for the study on their delivery and function.

The purpose of this study was to construct recombinant full-length hepatitis E virus(HEV)fused with enhanced green fluorescent protein(EGFP),and assess its infectivity in A549 cells. Two fragments from the full-length HEV genome and the EGFP gene were amplified by PCR. The EGFP gene was inserted downstream of the HEV ORF2 and then cloned into the pGEM® -7Zf(+)vector containing the T7 and SP6RNA polymerase promoters, producing pGEM-HEV-EGFP. The construction of the pGEM-HEV-EGFP recombinant plasmid was confirmed by restriction enzyme digest and sequencing. The pGEM-HEV-EGFP recombinant plasmid was transfected into A549 cells to assess infectivity using Lipofectamine. EGFP expression was observed at 24hpost-transfection,and expression of the HEV ORF2 was detected by immunofluorescence, confirming the presence of the HEV ORF2 and EGFP fusion protein. Cytopathic effects were observed at day seven post-transfection. The infectivity of pGEM-HEV-EGFP was confirmed by the presence of fluorescence after three continuous passages. The recombinant pGEM-HEV-EGFP vector was successfully constructed and effectively infected A549 cells, which will facilitate future studies on the mechanisms of HEV infection and pathogenesis.

Objective: In order to obtain piericidin intermediates of low toxicity by metabolic engineering, we studied the function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1., Methods: The methyltransferase pieB2 gene disrupted Streptomyces piomogeues var. Hangzhouwanensis was constructedby double crossover recombination. The methyltransferase gene pieB2 was also PCR amplified and cloned into the plasmid pET28a for overexpressing N-(His)6-tag PieB2 in E. coli BL21(DE3)/pLysE. The recombinant PieB2 was purified by affinity chromatography via AKTA FPLC system. The PieB2 catalyzed reactions were performed using SAM and demethyl-piericidinas substrates., Results: The disruption mutant LQ-9 produced demethyl-piericidininstead of piereicidin A1, which was restored by in trans complementation of the pieB2 gene. The N-(His)6-tag PieB2 was expressed in E. coli in soluble form and was successfully purified via Ni2+ mediated affinity chromatography. In vitro biochemical experiments showed that PieB2 could convert demethyl-piericidininto piereicidin A1 in the presence of SAM. The demethyl-piericidin intermediat showed an attractive biological activities as well as piericidin A1., Conclusion: We confirmed that PieB2 is function as a SAM dependent methyltransferase in the biosynthetic gene cluster of piericidin A1.

This study was designed to prepare a monoclonal antibody against Orf virus 118 protein and explore the biological properties of ORFV118 using this antibody. We constructed a recombinant plasmid pET33b-ORFV118 that contained a full-length ORFV118 gene. The plasmid was transformed into E.coli BL21,and the expression of ORFV118 was induced by isopropyl-β-d-thiogalactoside (IPTG).Prokaryotic ORFV118 was purified via Ni-NTA affinity chromatography and was subsequently used as an antigen to immunize mice. An anti-ORFV118 antibody was prepared using hybridoma technology. The titer and specificity of this antibody were tested by an indirect ELISA and Western blot/immunohistochemistry, respectively. We successfully obtained three antibody-secreting hybridomas,1A2,3B5,and 5D10.The titers of the three hybridomas were 1:10000,1:6400,and 1:8000.The monoclonal antibody (mAb),1A2 and the highest titer was selected for further research. The mAb 1A2,an IgG1 type antibody was bonded to its immunizing antigen, both the eukaryotic and natural ORFV118 with high specificity. The immunohistochemical analysis showed that the focal specific staining was restricted to the epidermal layer and subcutaneous tissue, which conformed to the characteristics of an ORFV infection. The mAb 1A2 recognized ORFV118with high specificity. Further study of mAb 1A2 will facilitate our understanding of ORFV118 and provide potentially novel methods for the diagnosis, prevention, and treatment of Orf.

Objective: To prokaryotic express and identify the recombinant plasmids containing the genes or gene segments which coding secreted or peripheral membrane protein of Schistosoma japonicum ., Methods: A number of 28 pET32 (+) previous constructed recombinant plasmids containing these genes or segments were transferred into E. coli BL21 (DE3) strain for culture via the CaCl 2 transformation method, and then induced by IPTG for prokaryotic expression. Then, the bacteria were treated by broking the wall with ultrasonic, and the bacterial suspension, supernatant and precipitation were detected with 12% SDS-PAGE. The size and distribution of these target proteins were determined. Finally, the expression of the recombinant fusion protein was further identified by the protein microarray combined with anti-His tag fluorescent antibody., Results: Under the inducing conditions of 0.1 mmol/L IPTG, 37 ℃, 200 r/min and 4 h, these fusion proteins were highly expressed in E. coli BL21 (DE3). The SDS-PAGE results showed all the 28 recombinant fusion proteins were expressed in different degrees, among which 2 were distributed in the supernatant, and the other 26 were distributed in precipitation, and the sizes of the fusion proteins were the same as that of prediction. The fluorescent signals of His antibody were successfully detected by confocal laser scanner., Conclusions: A series of recombinant plasmids containing genes or gene segments that coding secreted protein and peripheral membrane protein of S. japonicum have been successfully expressed by using an efficient prokaryotic expression system of E. coli. It has established a foundation for the high throughput immunoscreening of the vaccine candidates or diagnostic antigens of S.japonicum.

Quinolone antibacterial drugs, developing from the treatment of urinary tract infection in early time and now from the treatment of intestinal infection and respiratory infection, have been widely used in clinical, animal husbandry and aquaculture. Bacteria gradually become resistant to them and resistance mechanism is more and more complicated. Quinolone resistance mechanism is mainly divided into chromosome mediated resistance and plasmid mediated resistance, the latter plays an important role in spreading of antibiotic resistance. In 1998, plasmid mediated quinolone resistance mechanism was reported for the first time, namely the qnr gene mediated fluoroquinolone resistance mechanism. qnr genes can spread rapidly in different bacteria, which causes the infection difficult to control, makes the nosocomial infection popular in a wide range. In addition, qnr genes are usually associated with β-lactamase resistance gene. They exist in complex integron and integrate with the other varieties of resistance genes, which narrows the space of clinical medicine choose or drug combinations use to treat related bacterial infection and brings us a serious challenge. In this review, we provide a detailed overview for the historical discovery, classification, the resistance mechanisms of qnr genes, and the prevalence of those genes in China.

Objective: To clone the biosynthetic gene clusters for secondary metabolites, we developed the genetic modification system and constructed a genomic library of Streptomyces luteosporeus NRRL 2401., Methods: The genetic modification system was developed by using conjugal transfer vectors pSET152, pPM927 and pJTU1278 which were transferred from Escherichia coli ET12567/pUZ8002 to S. luteosporeus. The genomic library of S. luteosporeus NRRL 2401 was constructed by the fosmid vector pCClFOS, with E. coli EP1300 -T1 as the host strain. A PCR-based method was then developed for screening the biosynthetic gene clusters of secondary metabolites in the constructed genomic library., Results: Vectors pSET152, pPM927 and pJTU1278 were successfully transferred into S. luteosporeus for genetic modification, with pSET152 presenting the highest transformation efficiency. The constructed genomic library of S. luteosporeus NRRL 2401 contained 2880 clones with an average -35 kb inserted DNA fragment in each clone, indicating the 99.99% coverage of the genome in the library. In this genomic library, we detected 9 clones containing possible indolmycin biosynthesis genes by the PCR-based screening method., Conclusion: A stable, efficient genetic modification system and high-quality genomic library could be used for discovery of the biosynthetic gene clusters for secondary metabolites in S. luteosporeus NRRL 2401.

Objective: To explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system., Methods: An eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively., Results: The eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis., Conclusion: The ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.

The full-length coding sequence (cds) of jasmonate-zim-domain protein (AsJAZ1) gene was cloned from Aquilaria sinensis, the prokaryotic vector was constructed and the recombinant proteins expression was induced to provide the basic material for interactive proteins screen and gene function research. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length cds of AsJAZ1 gene was amplified using RT-PCR method and subcloned into pET-28a vector. The recombinant plasmid identified by restriction enzyme digestion and nucleotide sequencing was transformed into E. coli BL21(DE3). Inducing with 0.5 mmol•L⁻¹ IPTG at 37 ℃ for 4 hours, a fusion protein about 39 kDa was maximumly obtained. AsJAZ1 fusion protein had been expressed successfully mainly in the form of inclusion bodies and only a very small amount was secreted into the cytoplasm in the supernatant., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Human adenovirus type 41 (HAdV-41) is considered to be a "fastidious adenovirus". E1-deleted HAdV-41 cannot be rescued or amplified in 293 cells. To propagate recombinant HAdV-41 in 293 cells, the backbone plasmid pAdbone41 was reconstructed. That is, the E3 coding sequence of HAdV-41 was deleted and replaced with the HAdV-5 E4orf6 gene; and the E1A enhancer of HAdV-5 was inserted upstream of the E4 promoter of HAdV-41. Novel adenoviral plasmid pAd41E4EE-GFP was generated by homologous recombination of the shuttle plasmid pSh41-GFP with the modified backbone plasmid in the Escherichia coli BJ5183 strain. Adenovirus HAdV-41-E4EE-GFP was rescued by transfecting 293 cells with linearized pAd41E4EE-GFP. After seven rounds of propagation, viruses were purified by the CsCl ultracentrifugation method. HAdV-41-E4EE-GFP in 1.0 ml with a particle titer of 8 x 10(10) vp/mL was obtained which had a particle-to-infectious ratio of 50 : 1. The genome of HAdV-41-E4EE-GFP was confirmed by restriction analyses and polymerase chain reaction. These results showed that a novel HAdV-41 vector system was established in which recombinant HAdV-41 could be constructed and packaged in 293 cells.

Background and Objective: WIF-1 is an important tumor-suppressing gene in lung cancer, and its encoding protein WIF-1 can reduce proliferation and promote apoptosis by inhibiting Wnt/β-catenin signaling in lung cancer. This study constructs a eukaryotic expression plasmid carrying WIF-1 using FDA-approved clinical plasmid pVAX and explores the anti-tumor effect of pVAX-WIF-1 on A549 lung cancer cells in vitro and vivo., Methods: The DNA fragment of human WIF-1 coding sequence was amplified by PCR and was cloned into the multiple cloning sites of eukaryotic expression vector pVAX to construct pVAX-WIF-1. A recombinant plasmid was transfected into lung cancer A549 cells, and the expression of WIF-1 genes was verified by Western blot after transfection. Subsequently, the effect of pVAX-WIF-1 on cell apoptosis and proliferation was identified by MTT assay, staining A549 cells with Hoechst 3235, and flow cytometry. Finally, the A549 subcutaneous xenograft was established to detect the effect of pVAX-WIF-1 on lung tumor growth in vivo., Results: The results of restriction enzyme digestion, PCR, and sequencing indicated that eukaryotic expression plasmid pVAX-WIF-1 was successfully constructed. The protein expression level of WIF-1 was increased in the transfected A549 cells. Further results showed that transfection with pVAX-WIF-1 significantly inhibited proliferation and promoted apoptosis in A549 cells. Moreover, pVAX-WIF-1 significantly inhibited the tumor growth of the A549 subcutaneous xenograft in vivo., Conclusions: The recombinant eukaryotic expression vector pVAX-WIF-1 was successfully constructed. Transfection with pVAX-WIF-1 could significantly inhibit proliferation and promote apoptosis of lung cancer A549 cells and also effectively inhibit the tumor growth of the A549 subcutaneous xenograft in vivo. Our research can contribute to clinical applications of WIF-1 in lung cancer gene therapy.

To explore a new method for stable expression of virus-like particles (VLPs) of the severe fever with thrombocytopenia syndrome (SFTS) virus, an expression plasmid for the membrane glycoprotein (GP) and nucleocapsid protein (NP) of the SFTS virus was constructed by fusion of the two proteins via a serine residue, and a yellow fluorescence protein (YFP) gene was introduced into the plasmid as a reporter. CHO-K1 cells were transfected with this plasmid, and stable cell lines constructed using the limited dilution method. Cellular colonies were hand-picked based on YFP with the help of fluorescence microscopy and expanded without selection pressure. Stability of cell lines was evaluated by monitoring of fluctuation of the intensity of YFP for 40 passages. VLP production was characterized using an indirect fluorescence assay, immunoblotting, and electronic microscopy. We showed that GP and NP fusion proteins could be assembled into VLPs in vivo, and that VLPs had similar morphologies to virus particles. Selected cell lines were stable for YFP expression: no significant fluctuation was detected in 40 passages. These data demonstrated the effectiveness of this new method for expression of structural proteins of the SFTS virus and screening for stable cell lines. Our results could provide new concepts for the production of biopharmaceuticals.

According to the sequence of P450 cDNA of Eleutherococcus senticosus, specific primers were designed. Frokaryotic ex pression vector pET30a-P450 was constructed and the prokaryotic expression conditions were optimized. Results showed that the BL21 after being transformed with the recombinant expression vector accumulated the high amount of recombinant protein. SDS-PAGE analysis showed that the recombinant protein was about 53 kDa. The recombinant accumulated the highest amount of recombinant protein af ter IPTG (1 mmol x L(-1)) at 27-37 degrees C for 24 h. Consequently P450 gene of E. senticosus could be expressed successfully by prokaryotic expression vector pET30a-P450. Induction temperature, IPTG concentration, medium type and amount of induction time could all influence the expression of target protein, but the impact strength was different.

Objective: The mechanisms of mesenchymal stromal cells (MSCs)-mediated treatment of liver damage have been unclear. Two major mechanisms, which involve paracrine effects and/or direct trans-differentiation, have been proposed. To clarify which mechanism is more important, we planned to construct a recombinant plasmid expressing green fluorescent protein (GFP) driven by the cytomegalovirus (CMV) promoter and Pseudomonas aeruginosa exotoxin 40 (PE40) driven by the albumin (alb) promoter, which can induce cell death as soon as MSCs differentiate into hepatocytes., Methods: To construct the recombinant eukaryotic expression vector pFlag-CMV-GFP-TM-albp-PE40, GFP, transmembrane domain of DLL1, alb promoter and PE40 were obtained by PCR and were inserted into pFlag-CMV-1. The expression of GFP was observed under a fluorescence microscope and the killing effect on hepatocytes was analyzed by flow cytometry., Results: The recombinant plasmid inducing cell death in hepatocytes was successfully constructed, suggesting that the plasmid could be employed to study the mechanism of MSCs-mediated treatment on liver damage., Conclusion: This study might provide a promising tool for revealing the mechanism of MSCs-mediated treatment on liver diseases.

Objective: To express the SpiC protein of Salmonella pullorum and establish an indirect ELISA method with SpiC protein as antigen., Methods: The 384 bp spiC gene of Salmonella pullorum was amplified by PCR from the genomic DNA and cloned into pET30a vector. The recombinant plasmid pET30a-spiC was transformed into competent E.coli BL21(DE3) cells and induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Indirect ELISA based on purified SpiC protein was applied to detect 144 clinical serum samples., Results: SDS-PAGE and Western blotting confirmed that a soluble recombinant His-SpiC protein of 19.2 ku was expressed in BL21(DE3) cells. SPF chicken antibodies against GST-SpiC could recognize His-SpiC, indicating that His-SpiC had a good immunogenicity. The indirect ELISA that we established using His-SpiC protein as coating antigen for detecting antibodies against SpiC could differentiate infected from vaccinated animals (DIVA)., Conclusion: The recombinant His-SpiC was successfully expressed and the indirect ELISA with it as coating antigen could be used as DIVA method for the related vaccine of pullorum disease.

Objective: To evaluate the combination of lipopolysaccharide-amine nanopolymersomes (LNPs), as a gene vector, with target gene and the transfection in bone marrow mesenchymal stem cells (BMSCs) so as to provide a preliminary experiment basis for combination treatment of bone defect with gene therapy mediated by LNPs and stem cells., Methods: Plasmid of bone morphogenetic protein 2 (pBMP-2)-loaded LNPs (pLNPs) were prepared. The binding ability of pLNPs to pBMP-2 was evaluated by a gel retardation experiment with different ratios of nitrogen to phosphorus elements (N/P). The morphology ofpLNPs (N/P = 60) was observed under transmission electron microscope (TEM) and atomic force microscope (AFM). The size and Zeta potential were measured by dynamic light scattering (DLS). The resistance of pLNPs against DNase I degradation over time was explored. The viability of BMSCs, transfection efficiency, and expression of target protein were investigated after transfection by pLNPs in vitro., Results: At N/P ≥ 1.5, pLNPs could completely retard pBMP-2; at N/P of 60, pLNPs was uniform vesicular shape under AFM; TEM observation demonstrated that pLNPs were spherical nano-vesicles with the diameter of (72.07 ± 11.03) nm, DLS observation showed that the size of pLNPs was (123 ± 6) nm and Zeta potential was 20 mV; pLNPs could completely resist DNase I degradation within 4 hours, and such protection capacity to pBMP-2 decreased slightly at 6 hours. The cell survival rate first increased and then decreased with the increase of N/P, and reached the maximum value at N/P of 45; the cytotoxicity was in grade I at N/P ≤ 90, which meant no toxicity for in vivo experiment. While the transfection efficiency of pLNPs increased with the increase of N/P, and reached the maximum value at N/P of 60. So it is comprehensively determined that the best N/P was 60. At 4 days, transfected BMSCs expressed BMP-2 continuously at a relatively high level at N/P of 60., Conclusion: LNPs can compress pBMP-2 effectively to form the nanovesicles complex, which protects the target gene against enzymolysis. LNPs has higher transfection efficiency and produces more amount of protein than polyethylenimine 25k and Lipofectamine 2000.

Object: To study the resistance mechanisms to quinolones in Aeromonas hydrohila isolated from aquatic animals., Methods: The drug-resistant spectrum of 23 strains was determined. Quinolone-resistance determining regions of gyrA and parC genes in both screened and in-vitro induction drug-resistant strains were analyzed. Then the detection of quinolone drugs relative efflux pump genes qepA, oqxA and mdfA was performed. The qnrA, qnrB, qnrC, qnrD and qnrS genes were also analyzed at the same time., Results: All organisms were resistant to more than 5 drugs; 39.1% (9/23) of the isolates were quinolone resistant, of which 55.6% (5/9) were enrofloxacin resistant. All the enrofloxacin-resistant isolates harbored qnrS gene, but none of the enrofloxacin-resistant strains harbored qnrA, qnrB, qnrC , qnrD genes and the efflux pump genes of qepA, oqxA and mdfA. AH19 possessed the gyrA and parC genes double mutation, plasmid-mediated quinolone resistance gene qnrS and efflux pump, 3 drug resistance mechanisms simultaneously, while the two drug-resistant mechanisms of AH4, AH7 and AH20 were gyrA and parC genes double mutation and qnrS gene. GyrA gene mutation and qnrS gene occurred in AH6. Compared to the strain ATCC7966, the in-vitro induction drug-resistant strain ATCC7966-QR had both the gyrA and parC genes mutation., Conclusion: The mechanisms of resistance to quinolones in the A. hydrophila isolates of this study mainly depended on the existence of plasmid-mediated gene qnrS and the variation of the target site of quinolone drugs, whereas, the drug resistance mechanism relying on the efflux pump system only existed in individual strains.

Objective: To construct an efficient gene knock-out system for Paenibacillus polymyxa SC2., Methods: Temperature sensitive plasmid pRN5101 was transformed into P. polymyxa SC2 by electrotransformation. A mutant SC2-E was obtained, in which pmxE was disrupted by homologous recombination. To confirm whether pmxE was knocked out, we used antibacterial activity assay and high performance liquid chromatography to analyze the ability of mutants synthesizing polymyxin., Results: We developed an efficient gene knock-out system for P. polymyxa SC2. Plasmid of pRN5101 could replicate at 28 degrees C and suicide at 39 degrees C in SC2. Mutants lost the ability of synthesizing polymyxin, indicating that pmxE gene was successfully knocked out., Conclusion: The constructed gene knock-out system for P. polymyxa provides a high-efficiency tool to detect genes function for P. polymyxa.

Objective: To design and package shRNA expressing lentiviral particles targeting B7-H1, and evaluate their inhibitory effect on B7-H1 expression in U251 cells., Methods: Three shRNAs targeting B7-H1 was designed and the sense and antisense primers were produced by chemical synthesis. After annealing, they were linked into restriction enzyme digested pLKO.1 vector. Confirmed by DNA sequencing, the lentiviral particles were packaged and applied to infect U251 cells. qRT-PCR and Western blotting were used to detect the B7-H1 mRNA and protein levels respectively., Results: qRT-PCR and Western blotting showed that two of the three shRNAs effectively knocked-down B7-H1 expression in U251 cells., Conclusion: The packaged lentiviral particles can specifically inhibit B7-H1 expression, which will be helpful for further functional study on B7-H1.

Objective: To construct and identify a lentiviral vector for mBTLA (mouse B and T lymphocyte attenuator) expression., Methods: The entire coding sequence of mBTLA gene was amplified from pET-28a-mBTLA plasmid, and then mBTLA gene was transferred into pMD18-T plasmid before cloning into a lentiviral transfer vector. Liposome was used to package lentiviral particles in 293T cells. After lentiviral particles packaging, morphological changes of 293T cells were observed by fluorescence microscope. The recombinant plasmid was identified using RT-PCR and sequencing. Lentiviral titer was detected by 50% tissue culture infectious dose (TCID50;) assay. RT-PCR and Western blotting were used to detect mBTLA mRNA and protein expression., Results: pMD18-T-mBTLA and pSL6-mBTLA plasmids were successfully constructed and digested for electrophoresis appearing a near 1 kb strip which matched the size of mBTLA coding sequence. Gene sequencing and alignment analysis further confirmed mBTLA coding sequence to be successfully integrated into the plasmid vector. Morphological observation and supernatant PCR amplification of 293T cells confirmed that Lenti-mBTLA lentiviral packaging was successful, and the Lenti-mBTLA lentiviral titer was 1.3×10(8); pfu/mL. RT-PCR and Western blotting demonstrated that the Lenti-mBTLA vector could effectively express mBTLA mRNA and protein., Conclusion: The lentiviral vector which can effectively express mBTLA mRNA and protein has been successfully constructed.

Objective: To investigate the fitness costs of the New Delhi Metallo-beta-lactamase-1 (bIaNDM-1) bearing plasmid pNDM-BJ01 in Acinetobacter lwoffii strain 10621, and to evaluate the potentials of the sustainahility and expansions among the Acinetobacter lwoffii population., Methods: We obtained the spontaneous mutant of A. lwoffii missing pNDM-BJ01 10621NDM-1(-) via serial passages of the wild strain 10621NDM1(-) on the antibiotics-free media. We then compared the in vitro fitness of these two strains by measuring the growth curves, the ability of biofilm formation and the survival rates in nutrient-free PBS buffer., Results: Plasmid pNDM-BJ01 was unstable in A. lwoffii strain 10621, and 11 passages will be enough to get it deleted from the ancestral strain. We found no significant difference in the growth curves either at 26 or 37 degrees C for 10621NDM-1(+) and 10621NDM-1(-). The biofilm formation ability of the plasmid free derivate 10621NDM-1(-) was significant higher than its resistant ancestor 10621NDM-1(+) at 26 degrees C, whereas the latter showed higher ability of biofilm formation at 37 degrees C. Strain 10621NDM-1(+) was vulnerable in the nutrient-free PBS buffer, with less than 5% survival rate on the first day and dying out on the sixth day, whereas 10621NDM-1(-) survived till the seventh day., Conclusion: blaNDM-1 bearing plasmid pNDM-BJ01 possesses significant fitness costs in A. lwoffii strain 10621, and it will get depleted easily if the antibiotic press released. pNDM-BJ01-free 10621NDM-1(-) strain has higher fitness than its ancestor, 10621NDM-1(+), which implies the limited expansion potential of plasmid pNDM-BJ01 in A. lwoffii.

Objective: Accumulation of tanshinton in Salvia miltiorrhiza are enhanced by exogenous application of jasmonates. The core JA signaling module COI1/JAZ/MYC2 play a central role on control of downstream gene expression in the JA pathway. To obtained the antibody of SmJAZ, SmJAZ recombinant protein was expressed in Escherichia coli and optimal expression was performed., Method: The full-length SmJAZ1 ORF was sub-cloned in a prokaryotic expression vector pET32a. The recombinant fusion protein had high expression level in BL21 (DE3) strain of E. coli, and SDS-PAGE analysis showed its molecular weight was about 24 kDa., Result: The induction of E. coli [pET32-JAZ1] in different temperature, induction time, IPTG concentrations and IPTG adding time of E. coli were performed. The induction time and the induction temperature are positively related trends with SmJAZ1 protein expression, and IPTG concentration had no significant impact in protein expression, whereas IPTG adding time had significant impact on protein expression., Conclusion: Shaking the culture at 30 degrees C until the A600 is approximately 0.9 (2 h in LB), and add IPTG to a final concentration of 0.1 mmol x L(-1), and then the optimal expression of SmJAZ1 recombinant protein were accumulated after the induction time of 20 h.

Aim: To clone human secreted IL-16 cDNA, construct its prokaryotic expression vector and express it in E.coli DH5α., Methods: The secreted IL-16 gene fragment from the cDNA library of human peripheral blood mononuclear cells (PBMC) was amplified by PCR. After purified, the product was cloned into pUC18 T-vector. The recombinant plasmid was confirmed by PCR, endonuclease digestion and sequencing analysis and then subcloned into prokaryotic expression vector pMAL-C2. Then the recombinant expression plasmid pMAL-IL-16 was transformed into E.coli DH5α. The expression of secreted hIL-16 was induced with IPTG and identified by SDS-PAGE and Western blotting., Results: Using PCR, we obtained the human secreted IL-16 cDNA fragment which was 393 bp and encoded 130 amino acids. The prokaryotic expression vector pMAL-IL-16 we constructed was successfully transformed into E.coli DH5α, and under the induction of IPTG, we found the expression of the recombinant fusion protein with relative molecular weight (M(r);) being 56 000 as expected and confirmed by SDS-PAGE and Western blotting., Conclusion: We successfully cloned the human secreted IL-16 cDNA and constructed its prokaryotic expression vector and expressed it in E.coli, which is helpful for further purification of human secreted IL-16 protein.

Objective: We developed a homologous recombination system to make marker-free mutants in Mtb in an easy screening way., Methods: The plasmid pSL002 harboring recE and recT-like protein gp60 and gp61 was transformed into wt Mtb in order to increase the recombing efficiency. The two homologous arms of target gene were amplified and cloned into pSL001. The purified homologous arms-loxP-gfp-hyg-loxP fragments were further transformed into Mtb (with pSL002 inside) competent cells to obtain the double cross over (DCO) mutants. The plasmid pSL003 was transformed into DCO competent cells to excise the two loxP sites flanked region. The plasmid pSL002 and pSL003 were removed via the counter selection marker sacB., Results: An efficient homologous recombination system was developed. Three different marker free mutants: Rv1364c phosphatase domain, PstP extracellular domain and PstP transmembrane domain were created by the system developed in this study. The efficiency to obtain DCO was varying from 25% to 62.5%, while the efficiency from DCO to KO was close to 100%. The gfp reporter was used to screen SCO, DCO and KO., Conclusion: The homologous system shortens the complicated screening process to 3 month in Mtb. This is the fastest allelic exchange system reported so far, providing novel deletion strategy to the repertoire of mycobacterial genetic tools for constructing unmarked mutations.

Objective: To increase the leavening ability in the lean dough, the maltose utilization ability of baker's yeast was enhanced., Methods: A 1.7kb PGK1 promoter and terminator were ligated and inserted into vector Yep-C to give the expression plasmid named Yep-CP. Then a 1.7kb DNA fragment containing the open reading frame and terminator of mal62 gene was amplified from Saccharomyces cerevisiae BY-14 by PCR, and inserted into Yep-CP to generate recombinant plasmid Yep-CPM. To express mal62 gene properly in S. cerevisiae, the recombinant expression plasmids Yep-CPM with copper resistance gene as the selection marker for yeast transformation were introduced into S. cerevisiae BY-14. The resulting yeast transformant BYCPM was screened on YEPD with 4 mmol/L CuSO,and identified by colony PCR. Target protein was detected by qRT-PCR, and the enzyme activities and the leavening ability of the recombinant strain BYCPM were determined to confirm whether functional expression was achieved., Results: The maximum maltase activity of recombinant strain BYCPM was 15%-52% higher than that of control strain BY-C. Leavening ability and specific leavening ability of recombinant strain BYCPM were 40% and 5.6% higher than that of the control strain BY-C, respectively., Conclusion: The mal62-overexpression was an effective way of increasing the maltase activity and the ability of glucose depression in the baker's yeast. Moreover, recombinant strain BYCPM could further enhance the leavening ability and produce more gas while consume lesser carbon source than the control strain.

Objective: To construct prokaryotic expression vectors suitable for tandem affinity purification to study protein-protein interactions in bacteria., Methods: Two tandem affinity tag sequences, including the coding sequences of Protein G and streptavidin binding protein (SBP), as the N- and C- terminus of fusion proteins were designed and de novo synthesized. Constitutive expression vectors pNTAP and pCTAP were constructed using pUC18 as the backbone deleted of the lacI gene., Results: Two expression vectors pNTAP and pCTAP were successfully constructed, pNTAP showed substantial expression of the built-in tag protein GFPuv not only in Escherichia coli BL21 (DE3) but also in enterohemorrhagic Escherichia coli O157:H7 and Shigella flexneri 5a., Conclusion: Of the two recombinant expression vectors successfully constructed, pNTAP can express the model protein for tandem affinity purification and could be used for studies of protein-protein interactions in some gram-negative pathogenic bacteria such as Escherichia coli and Shigella flexneri.

Objective: To investigate the possibility of increasing the yield of hyaluronic acid by constructing duplication hasABC of chromosome recombinant in Streptococcus equi subsp. Zooepidemicus with a thermosensitive delivery vector system pJR700., Methods: We amplified a 4147 bp DNA fragment of hyaluronic acid synthase operon hasABC genes from chromatosome of S. zooepidemicus using PCR. This DNA fragment was subcloned into the pJR700 at ClaI sites to result in recombinant plasmid pXL32. The recombinant plasmid was transformed into S. Zooepidemicus by electroperation. The homologous recombination was induced by growing the bacteria at 37 degrees C, and transformants were selected according to kanamycin resistance for 3 rounds. Then the culture was shifted to grow at 30 degrees C without antibiotics for 4 rounds to induce excision of the pJR700 indicated fragment. Colonies with kanamycin sensitivity were selected by plating on THY agar at 37 degrees C. The hasABC recombinant of S. Zooepidemicus was identified through RT-PCR with primers homologous to the flanking regions. HA titers were measured by the modified carbazole assay., Results: We constructed successfully the duplication hasABC of chromosome recombinant of S. Zooepidemicus and the HA titer production by recombinants harboring duplication hasABC was 34% higher than that of the wild type at 24 h in shake flask culture., Conclusion: The thermosensitive delivery vector of pJR700 could be used to construct the streptococcal hasABC recombinant strain for increasing the yield of HA in S. Zooepidemicus.

Aim: To construct recombinant clostridium sporogenes modified with the extracellular domain of human oncogene HER2/neu, to lay a foundation for further study of its antitumor effect., Methods: The extracellular domain (ECD) of HER2/neu gene was attached to the downstream of promoter and signal sequence of clostridia endo-1, 4-glucanase (eglAp) by SOE-PCR to construct fusion gene eglAp-HER2/neu, which was then inserted into E.coli-clostridia shuttle plasmid pIMP1 to construct recombinant plasmid pIMP1-eHER2/neu. The recombinant plasmid was firstly transformed into E.coli DH5α.Then the correct construct was identified and introduced into C. sporogenes by electroporation. Positive clones were selected by erythromycin resistance, bacteria PCR were used for verification., Results: Restriction map and sequencing result showed that the sequence and ORF of fusion gene eglAp-HER2/neu in recombinant plasmid pIMP1-eHER2/neu was correct. Bacteria PCR results indicated that the recombinant plasmid pIMP1-eHER2/neu was successfully transformed into C.sporogenes. After more than 20 passages under antibiotic pressure, C.sporogenes transformants could stably carry the recombinant plasmid pIMP1-eHER2/neu., Conclusion: Stable C.sporogenes transformants with the recombinant plasmid pIMP1-eHER2/neu are successfully acquired, which laid a foundation for further anti-tumor study.

Objective: To construct the recombinant plasmid containing S1 gene of new type of reovirus, and to study the expression of protein sigma1 in Vero cells., Methods: The recombinant plasmid, named pC-S, was constructed by cloning S1 gene into vector pCAGGS/MCS. Then Vero cells were transfected with pC-S and collected at 24, 48, 72 h post transfection followed by SDS-PAGE and Western-Blot assay., Results: Results both SDS-PAGE and Western-Blot assay indicated that sigma1 protein could be expressed well and the highest expression level was 72 h post transfection., Conclusions: Sigma1 protein could be expressed well in Vero cells by transfected with recombinant plasmid containing S1 gene, and could give some implications for subsequent research on virus-host interactions.

Objective: To establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method., Method: Human ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed., Result: The recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity., Conclusion: A phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.

Aim: To construct recombinant co-expression adenovirus vector of SEA and CD80 genes regulated by mouse TERT(telomerase reverse transcriptase, TERT) promoter and to observe the expression of SEA and CD80 in the Hepa1-6 cells mediated by it., Methods: Using AdEasy adenovirus system, the core promoter region of mTERT was subcloned to shuttle plasmid pShuttle2 and Myc-Max response element was inserted upstream of it to regulate the expression of SEA and CD80. The recombinant co-expression adenovirus vector of SEA and CD80 genes was constructed and named as Ad-MMRE-mTERT-BIS. Hepatoma cell line Hepa1-6 and fibroblast cell line NIH3T3 were infected by recombinant adenovirus at MOI(multiplicity of infection)of 100, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining., Results: SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on NIH3T3 cells., Conclusion: The recombinant co-expression adenovirus vector of SEA and CD80 gene regulated by mTERT promoter was sucessfully constructed and make targeting-expression of SEA and CD80 on the surface of hepatoma cells, which lays the foundation for further research on application of SEA and CD80 in targeted genetherapy for hepatoma.

Aim: Used RFP gene to construct a RNA interference vector for convenience to obtain the good effective hairpins sequence., Methods: NUDC and RFP genes were cloned into pDs vector separately, resulting in pDs-NUDC- RFP. as above, human U6 promoter and 9 hairpins sequence of NUDC were cloned into the pDs- NUDC-RFP vector separately.The RNA interfererence vectors target to 9 points of NUDC were constructed. Construct- ed recombinant vectors and then were identified by restrictive digestion and DNA sequencing.293T cells were transfected with the recombinant DNA samples by the liposome complex method, and then fluorescence photographs were taken., Results: Enzyme digestion and DNA sequencing showed that the target gene and shRNA fragments were correctly inserted in pDs vector. fluorescence photographs showed that shNUDC-A is the best effective fragment., Conclusion: The NUDC gene targeted shRNA and its vector are successfully constructed.

Objective: To develop a system to rescue virus by intracellular expression of T7 RNA Polymerase., Methods: The gene of T7 RNA Polymerase was amplified and cloned to VR1012 by molecular biological technology. The expression plasmid VR-1a was then identified. VR-1a and EV71 infectious plasmid were co-transfected in Vero cell. CPE was observed and viral gene viral antigen were detected., Results: The gene of T7 RNA Polymerase was successfully cloned into vector VR1012. Vero cell developed to CPE after being transfected VR-1a and EV71 infectious plasmid. EV71 gene was amplified by RT-PCR from the culture. EV71 antigen was also detected by ELISA., Conclusion: The method can be used to rescue virus. It could apply to immunologic research of EV71 DNA vaccine.

Objective: To investigate the expression characteristics of Gluc-Fluc dual luciferase plasmid after its transfection into MB49 bladder cells., Methods: pAAV2neoCAG-Gluc-2A-Fluc and pAAV2neo-Gluc plasmids were separately transfected into MB49 cells via LipofectamineTM2000. The Gluc activity in the cell culture supernatant and the Fluc activity in the cells were detected by luminometer and Lumina Imaging system., Results: The luminometer result showed that the activity of Gluc in the supernatant increased gradually in a cell number- and time-dependent manner, while Fluc activity in the cells increased with the cell number but not with time. The Lumina Imaging system showed that Gluc-Fluc was successfully expressed in MB49 bladder cells and cell lines with stable Gluc-Fluc expression were obtained after G418 selection., Conclusion: Gluc in the dual luciferase plasmid retains its expression characteristics. Due to the advantages of Fluc in localization in living imaging and the easy quantitative detection of Gluc, the dual luciferase plasmid, after transfection in MB49 bladder cells, allows reliable and dynamic detection of tumor growth in animal models.

Aim: To construct the ureI and ctB-ureI eukaryotic expression plasmid with pIRES-RD2 and study the expressing characters of those plasmids in HEK 293., Methods: We got the ureI and ctB-ureI gene from the procaryotic expression plasmid pET32a(+)-ureI and pET32a(+)-ctB-ureI by restriction enzyme BglII and XhoI, respectively.The pET32a(+)-ctB-ureI had been designed that ureI fused with Cholerae toxin B gene (ctB) at the 5'terminal of ureI to increase the immunogenicity of ureI, and then inserted those gens to eukaryotic expression plasmid pIRES-RD2 by ligase. After sequenced, the pIRES-RD2-ureI and pIRES-RD2-ctB-ureI were identical with the targets. These plasmids were transfected HEK293T cell strain by Lipofetmiane 2000. Then studied the transfection ratio of those cells by Fluorescence microscope, studied the expression features and the immunoreactivity of those cells by Immunofluorescence and Immunohistochemisty with the special antibodies., Results: Fluorescence microscope showed about 80% cells had been transfected the target plasmids, and Immunofluorescence and Immunohistochemisty demonstrated that the transfected cells expressed the recombinant proteins which located at the cytoplasm of HEK 293 strain after 48-72 hours. And the expressed products could react with the special antibodies, respectively., Conclusion: We successfully constructed the ureI and ctB-ureI eukaryotic expression plasmid with pIRES-RD2, which were pIRES2-DsRed2-ureI and pIRES2-DsRed2-ctB/ureI. Both these two plasmids could express ureI or ctB-ureI in HEK 293 cell, and the expressed products had immunoreactivity, respectively.

Hsc70-SNCG fusion protein cDNA fragment containing signal peptide sequence of Igkappa, MMP9, and P37 was inserted into the vector pVAX1 to construct recombinant plasmid pVAX-Igkappa-Hsc70-SNCG, pVAX-MMP9-Hsc70-SNCG, and pVAX-P37-Hsc70-SNCG. Three eukaryotic vectors were constructed and verified by restriction enzyme digestion and sequencing. After transfection with recombination plasmids in QM-7 cells, the transient expression and secretion of three fusion proteins were detected by ELISA and Western Blot. The results suggested that Hsc70-SNCG carrying three different signal peptides could be expressed and secreted by transfected cells, and three signal peptides effectively directed secretion of fusion protein by QM-7 cells. BALB/c mice were immunized by three plasmids using gene gun system. The serum levels of anti-SNCG antibodies in mice were measured by ELISA. The results showed that three secreted plasmids could stimulate humoral immune responses to SNCG in mice, which depended on the secreted expression levels induced by signal peptides.

To construct eukaryotic expression vector of general type in reverse genetics systems of non-segmented negative strand RNA viruses, the multiple cloning site of the plasmid pVAX1 was replaced with HamRz cDNA sequence, a 9 sites linker and HdvRz cDNA sequence through the sequential addition of three adapters, the insertion of which could generate the correct 3' and 5' terminal sequences of the primary viral genomic RNA transcript and facilitate the assembly of the complete viral cDNA sequence. The sequences of the three adaptors were correct after identifying by restriction endonuclease digestions and sequencing. The constructed eukaryotic expression vector could not only be used to assemble viral genome, but also provide the basis for establishing the reverse genetic system rapidly.