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Copyright of Journal of China Medical University is the property of Journal of China Medical University Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of China Medical University is the property of Journal of China Medical University Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of China Brewing is the property of China Brewing Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective To construct the eukaryotic expression plasmid of Homerl gene and its transfection and identification in HT22 cells. Methods We extracted the whole RNA from APP/PSI mouse hippocampus, and Homerl gene coding sequence was obtained by reverse transcription. After being isolated and purified though 1 % agarose gel electrophoresis, Homerl gene coding sequence was inserted into the eukaryotic expression vector p3 X FLAG-CMV-10 polyclonal site area, then the p3 X FLAG-CMV-10-Homerl eukaryotic expression vector was obtained. The Homerl recombinant plasm叫 was identified by restriction enzyme digestion and sequencing. HT22 cells were randomly divided into three groups, and the first two groups were transfected with p3 X FLAG-CMV-10-Homerl recombinant plasmid and p3 X FLAG-CMV-10 empty vector; the cells in the blank control group were not transfected. Western blotting was used to detect the expression of Homerl protein. Results We successfully constructed the p3 xFLAG-CMV-10-Homerl gene eukaryotic expression recombinant plasmid. The sequencing results showed that the recombinant sequence was completely identical to the target gene The expression of Homerl protein in the p3 X FLAG-CMV-10-Homerl recombinant plasmid group was significantly higher than that in p3 X FLAG-CMV-10 empty vector group and blank control group (P < 0. 05) . Conclusion We constructe the p3 xFLAG-CMV-10-Homerl eukaryotic recombinant plasmid and transfect it into HT22 cells successfully, which lays a foundation for the study of the effect of Homerl protein on Aβ clearance in brain and its role in the pathogenesis of Alzheimer disease. [ABSTRACT FROM AUTHOR]

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C ( WT / L127P ) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C ( WT / L127P ) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.

Copyright of Journal of Fuzhou University is the property of Journal of Fuzhou University, Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective To study the function of matrix metalloproteinase 2( MMP2) gene, and to construct the wild type and G1207 A mutant plasmids of MMP2 gene. Methods Wild type and mutation site G1207 A of MMP2 gene were amplified by polymerase chain reaction( PCR) and site-directed mutagenesis, and then we cloned into p UC57 plasmid and identified them by DNA restriction endonuclease profiling and plasmid sequencing. Results The length of PCR amplification products of MMP2 gene wild type and G1207 A mutation was 3 416 bp, which was consistent with the theoretical value.By the DNA restriction endonuclease analysis and gene sequencing, the sequences of recombinant plasmids p UC57-MMP2 and p UC57-p. Arg403 His were consistent with those of the Gen Bank( NCCT000016. 10). Among the G1207 A mutants, except that the G of 1 207 site was substituted by A, the other sequences were identical to the wild type sequences of the MMP2 gene. Conclusion The wild type plasmid p UC57-MMP2 and the mutant plasmid p UC57-p. Arg403 His are successfully constructed. [ABSTRACT FROM AUTHOR]

Objective To construct the eukaryotic expression vector, encoding major histocompatibility complex class I -related chain A gene (MICA), for the further research of transfecting Tca8113-Tb cell line (a metastatic cell line of brain metastasis from human tongue cancer Tca8113 cells in nude mouse), and to establish a stable MICA overexpression oral squamous cell line. Methods cDNA of MICA gene from pCMV-SPORT6-MICA was amplified by PCR, and subcloned into eukaryotic expression vector pEGFP-N 1 marked with green fluorescent protein (GFP). The recombinant plasmid was sequenced and transfected into Tca8113-Tb cell line by lipofectamine™ 2000. After screen culture by G418, stable tranfected Tca8113-Tb cell line was established using definite dilution method. The expressions of GFP protein was viewed directly with fluorescence microscopy and the overexpression of MICA was identified by RT-PCR, real time PCR and immunocytochemistry. Results The MICA gene was amplified by PCR and then cloned into the vector, whose sequence was identical to that in the GenBank. The transfected cells showed the expression of GFP. And the overexpression of MICA gene in transfected cells was detected by RT-PCR, real time PCR and immunocytochemistry. Conclusion The recombinant eukaryotic expression vector pEGFP-Nl-MICA has been constructed successfully and stably expressed in Tca8113-Tb cell line, providing a foundation for further studies on the function of MICA in vitro. [ABSTRACT FROM AUTHOR]

PURPOSE: To clone human 4-1BBL cDNA, which is a member of the tumor necrosis factor (TNF) ligand superfamily,construct an eukaryotic expression vector,pEFGPF-C1-4-1BBL, and detect the expression of 4-1BB ligand(4-1BBL) in Tca8113 cells. METHODS: Toral RNA was isolated from PBMC lymph-cell, 4-1BBL cDNA was amplified by RT-PCR, PCR products of 4-1BBL were inserted into pEGFP-C1 to form the expression vector of pEGFP-C1-4-1BBL Twenty-four hours after transfection of PEGFP-C1-4-1BBL into Tca8113 cell line by lipofectamine 2000, the expressions of GFP protein and 4-1BBL were detected by fluorescence microscopy, RT-PCR and Western blot, respectively. Transfected cells were selected in medium containing G418 (400 μg/ml) and termed as Tca8113/4-1BBL, using definite dilution method, the cell line of Tca8113/4-1BBL was constructed. RESULTS: the sequence of 4-1BBL, 768 bp, was confirmed by sequencing which was identical to be the human 4-1BBL mRNA in GenBank. The recombined expression vector PEGFP-C1-4-1BBL was determined by restriction enzyme digestion and PCR assay. Green fluorescence protein was expressed in Tca8113 cells that transferred by PEGFP-C1-4-1BBL A 271 bp fragment of RT-PCR product was detected using the 4-1BBL specific primers. Western blot showed a belt about 27.5kD protein. CONCLUSION: Construction of human 4-1BBL transfectant cell line has a great value for further study of 4-1BBL, which provides preparation of monoclone antibody of 4-1BBL. Supported by National Natural Science Foundation of China (Grant No. 30672335), Guangdong Natural Science Foundation (Grant No.06027977) and Foundation of Shenzhen Bureau of Science, Technology and Information (Grant No.200601008). [ABSTRACT FROM AUTHOR]

Objective: To construct pEGFP-N1-HBsAg-ROP2 recombinant expression plasmid and transfect HEK293T cells for expression, and pay a way for Toxoplasma gondii nucleic acid vaccine development., Methods: According to the HBsAg gene sequence and pcDNA3-p30-ROP2 recombinant plasmid restriction sites, the HBsAg gene was amplified by PCR. The HBsAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene. The HBsAg-ROP2 fragment was amplified by PCR and digested with Hind Ⅲ and Kpn Ⅰ to clone into the pEGFP-N1 eukaryotic expression vector and construct the recombinant pEGFP-N1-HBsAg-ROP2. The expression vector was transfected into HEK293T cells based on the identification of PCR amplification, restriction endonucleases and sequencing., Results: The PCR product of HBsAg was about 700 bp, which was consistent with the theoretical value. Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3HBsAg-ROP2 recombinant plasmid. The recombinant plasmid pEGFP-N1-HBsAg-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBsAg-ROP2 fragment. The results of sequencing showed that the sequence was 99.84% identical with the published sequence in GenBank. The target plasmid was successfully transfected into HEK293T cells, and the expression was correct, the protein concentration was 3.08 mg/ml., Conclusions: The recombinant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.

Molecular cloning is one of the most important and widely used technologies in molecular biology research. Generally, the target DNA fragment and the vector are separately digested by restriction enzyme, then purified and recovered, and then ligated with DNA-ligase. For some very short gene fragments (<300 bp), the recovery efficiency of the purified fragment is very low after digestion and cleavage, leading to the difficulty in its inserting into the expression vector. To address this issue, we developed a cloning method based on restoration of antibiotic resistance in constructing recombinant plasmid, which proved highly efficient in cloning very short gene fragments.