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Ribonuclease inhibitor (RI) is an acidic 50 kD protein with a high content of leucine and cysteine residues. RI inhibits RNases of the pancreatic type. A variant of RI was cloned from human fetal liver cDNA library by polymerase chain reaction (PCR). Compared with the reported RI, only two variations Arg(359)Ala and Leu(365)Pro were found in RIv amino acid sequence. Recombinant RIv has been expressed both in Escherichia coli and silkworm insect cells (Bombyx mori). The recombinant RIv exhibited inhibition activity on ribonucleolytic activity of RNase A in vitro system.

Baculovirus GP64 envelope glycoprotein is a specific major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. For promoter activity analysis in the baculovirus gp64 gene, two DNA fragments containing 437 and 439 bp upstream of 5' ends of the BmNPV and AcMNPV gp64 ORF were amplified by polymerase chain reaction and cloned, respectively. The sequence analysis indicated that two gp64 genes have both early (CAGT) and late (A/GTAAG) transcriptional start sites. By use of the plasmids with a reporter luciferase gene (Luc) driven by gp64 promoter to transfect insect cells, transient expression assay showed that pBmgp64Luc had high expression levels in permissive Bm-N cells and very low levels in non-permissive Sf-21 cells, while pAcgp64Luc had relatively high expression levels both in permissive Sf-21 cells and in non-permissive Bm-N cells. Furthermore, the transcription of both gp64 promoters appeared to be transactivated by 2.4-4 times in corresponding permissive cells by corresponding viral factors, separately. By inserting BmNPV homologous region-3 (hr3) into the downstream of luciferase reporter gene driven by gp64 promoter, it enhanced transcription from both gp64 promoters by 13 - 22 times in Bm-N cells and over 7000 - 14,000 times in Sf-21 cells, respectively. In the presence of BmNPV hr3, correspondingly, the viral factors transactivated the transcriptional activity from two promoters by about 73 - 78 times in corresponding permissive cells. It suggested that BmNPV hr3 plays an important role in co-activation with viral factors onto the gp64 promoter besides the functions of viral DNA origin and enhancer.

DNA helicases are essential for replication of baculoviruses. It was found that the helicase gene promoter of Bombyx mori nuclear polyhedrosis virus, including 510 bp upstream of ATG, had both early and late RNA initiation sites and could be recognized by cellular RNA polymerase. Transient expression assays in uninfected Sf-21 cells indicated that the helicase gene promoter could be classified as a delayed-early gene promoter. Deletion analysis by PCR showed that the regulation region of its basic transcription was mainly within -510 to -410 bp upstream of ATG. However, the basic activity was still detected with a deletion to -98 bp relative to ATG. In the presence of viral factors, deletion between -510 to -410 bp relative to ATG did not significantly reduce the promoter activity compared to the full-length promoter (510 bp). The remarkable reduction in the promoter activity was observed with continuous deletions. It suggests, therefore, that cis-acting elements responsive to viral factors are mainly located within the range of -410 to -309 bp upstream of ATG.

Factor C is an endotoxin-sensitive, intracellular serine protease zymogen which initiates the coagulation cascade system in the horseshoe crab hemolymph. The special lipopolysaccharide (LPS) binding activation of FC makes it a potential drug for anti-LPS treatment and has a high commercial value. Based on the sequence of reported FC from Japan horseshoe crab, two pairs of primers were designed. The total RNA was extracted from amebocytes of Chinese Tachypleus tridentatus and the cDNA was separated into two parts and were cloned using RT-PCR, respectively. FCs from different geographical areas showed high homology in sequence. The whole FC cDNA was cloned into pET-28a (+) containing T7 promoter and recombinant expression plasmid pET-FC was constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3). Recombinant FC was expressed as inclusion body when the expression strain was induced with 1 mmol/L IPTG. Refolded recombinant FC was confirmed to be active by bacteriostatic assay in vitro. The results of Western blot also suggested the recombinant FC may be able to cleave itself partly and produced an extra immunoblot band.

Recombinant bacteria exhibiting high enzymatic activities were obtained by cloning and coexpression of both caiB gene and caiE gene in the host of E. coli Bl21(DE3), which encode carnitine dehydratase and a protein related to the synthesis of cofactor for carnitine dehydratase, respectively. In order to coexpress these two genes, compatible and incompatible two plasmids system were used, the difference between them was also studied. After induction with IPTG, both caiB and caiE genes were coexpressed in both compatible two plasmids system and incompatible two plasmids system. In the former system, the expressed products accounted for 17% and 10% of the total proteins in the host; in the later system, the proportion was 39% and 20%, respectively. The activity of carnitine dehydratase in E. coli Bl21(DE3) with either coexpression system is about 2.3 times than that in E. coli Bl21(DE3) with only pET28-caiB. The plasmids stabilities in these two systems were the same, all needed the help of antibiotic selective pressure.

Oxygen-derived free radicals are thought to be involved in the pathogenesis of a wide range of neurological disorders. Targeted delivery of CuZn-SOD to neurons in central nervous system may have therapeutic value in such diseases. The gene encoding human CuZn-SOD was fused to tetanus toxin fragment C geneto construct a fusion gene, then it was cloned into prokaryotic expression vector pET-22b( ) and baculovirus vector pFastBacHTb, and was expressed in E.coli and Tn-5B1-4 cells, respectively. The recombinant fusion protein has a subunit molecular mass of 68 kD and is recognized by both anti-CuZn-SOD and anti-tetanustoxin antibody. By pyrogallol autooxidation assay, it is shown that the CuZn-SOD moiety retains substantial enzymatic activity, where the TTC moiety might deliver the fusion protein to neurons in central nervous system. So, CuZn-SOD/TTC may be a useful agent for the targeted delivery of CuZn-SOD to neurons.

Heat-shock protein A subunit(HspA), an effective immunogen may stimulate the immunoresponse in human body against challenge of H.pylori. The B subunit of cholera toxin (CtxB) has been proved to be a potent mucosal immunogen, actas an adjuvant for vaccine targeted for delivery to the mucosal-associated lymphoid tissue. A recombinant plasmid expressing bivalent antigen of HspA and CtxB subunit was constructed as follows. hspA and ctxB gene was amplified by PCR. The DNA products of hspA and ctxB were inserted in the prokaryotic expression vector pET-22b( ), respectively, and then the resulted recombinant plasmid expressinga fusion protein named HCT was transformed into the E.coli strain BL-21(DE3). hct gene was measured to be 708 base pairs long, and the fusion protein encoding a polypeptide of 236 amino acid residues, corresponded to a calculated molecular masses of 30 kD. Western blot analysis of the recombinant protein HCT confirmed that it could be specifically recognized by the serum of H.pylori-infected patients. HspA and HCT labelled (125)I were orally administered into the stomach of mice, respectively, and the radioactivity of (125)I in serum of each mouse was assayed at intervals 15 min, 30 min, 60 min, 90 min and 120 min. The result indicated that there were high radioactivity counts in the groups of HCT than that of HspA(P 0.001). This result suggests that the CtxB may enhance the volume of HspA absorbed from the intestine of mice, therefore the recombinant fusion protein HCT may be an effective oral vaccine for prevention and treatment against the infection of H.pylori.

The promoter of the helicase gene, including 510 bp upstream of ATG,was cloned and sequenced, and was found that it had both early and late RNA initiation sites. The initiation codon ATG was deleted by using point mutation. Luciferase gene, as a reporter gene, was fused with the promoter region to construct the plsmid pBm hel 510 luc. When pBm hel 510 luc was transfected into Bm-5 and Sf-21 cell lines, the helicase gene promoter was recognized by cellular RNA polymerase and transactivated by viral factors. Baculovirus homologous regions (hrs) act as viral DNA replication start sites, which also have been shown to alter the rate of transcription for cis-linked promoters. BmNPV hr3 was cloned into a downstream site of luc gene, to study the effect of this enhancer on hel 510 promoter activity. The transient expression in transfected insect cell lines and silkworm larvae indicated that hr 3 could enhance the transcriptional level of hel 510 promoter by about 7 000 and 1 000 fold, respectively.

Three new cDNAs named NL1, NL2 and NL3 were cloned from the total RNA of Naja naja atra by RT-PCR. The protein sequences encoded by them showed 77%, 72% and 98% structure identity to cobrotoxin which is a postsynaptic neurotoxin from Taiwan cobra (Naja naja atra), respectively. The five conservative residues, Tyr(25), Lys(27), Trp(29), Arg(33) and Lys(47), essential for the function of cobrotoxin were also found in the three cDNAs. The NL3, the most homologous to cobrotoxin, was expressed in E.coli BL21(DE3) by cloning into pET28b+. The expressed product was insoluble inclusion bodies and could be purified up to 90% purity in the range of 6 mg per liter culture cells by a single affinity step. The purified protein was refolded in vitro and the toxicity was assessed to be less than the native cobrotoxin in mice.

The CtxB gene encoding cholerae toxin subunit B was amplified from Vibrio cholerae genomic DNA by PCR. The result of sequencing indicated that CtxB gene encodes 124 amino acid residues. The sequence of CtxB gene was almost the same as that of reported except for the codon of Thr 62. The expression plasmid pGEX-CTXB was constructed by inserting CtxB gene into plasmid pGEX-4T-2, containing gst gene, immediately downstream of the T7 promoter. The expressed plasmid was introduced into E.coli BL21(DE3) cells and expression strain CTXB/BL21 was selected. SDS-PAGE analysis revealed that the GST-CTXB fusion protein was highly expression and accumulated up to 36% of bacterial soluble proteins after the induction by IPTG. A fusion protein of 40 kD was expressed as inclusion body. The fusion protein was refolded and purified. The purified fusion protein was cut by thrombin to obtain the purified CTXB protein.

In order to reduce the human anti-murine antibody (HAMA) in radioimmunotherapy, the gene encoding the heavy chain variable region (V(H)) of the murine monoclonal antibody with high specificity and affinity to carcinoembryonic antigen was fused to the human C(gamma3) gene to construct the murine-human chimeric heavy chain antibody gene, then was linked to core-streptavidin which can specifically bind to biotin, facilitating its purification and radioisotope labeling. The fusion gene was expressed in E.coli at high level, accounting for 24% of the total bacteria protein, and was characterized by SDS-PAGE and Western-blots. When using RIA the content of inclusion bodies was denatured and followed by renaturation, it was shown by using RIA to possess ability to bind to its specific antigen of CEA. Using horseradish peroxidase (HRP) labeled biotin as antibody in Western-blots, one band of 70 kD only was detected, demonstrating the fusion protein not only had the ability to bind CEA, but also could bind biotin specifically.

The fragment C of tetanus toxin was amplified from Clostridium tetani DNA by PCR. This fragment was cloned into expression vector pET-28a(+),under the control of the T7 promoter. Expression of this plasmid in E.coli resulted in the production of a protein consisting of 6xHis of the vector fused to the N-terminal 451 amino acids of tetanus toxin. After induction with 1 mmol/L IPTG, TTC was expressed in E.coli BL21(DE3). The protein product accounted for 8.2% of the bacteria total protein in soluble form, SDS-PAGE and Western blot analysis of TTC recombinant protein confirmed this result. The expression products were also purified by Ni(2+)-IDA-Sephrose 6B column. Immunization of mice with rTTC resulted in the production of antibodies that were able to protect mice against a challenge with tetanus toxin furthermore, rTTC in vivo appeared to be able to undergo retrograde axonal transport.

Human endostatin cDNA was cloned from total RNA of normal Chinese liver cell line L02 by RT-PCR. Endostatin DNA sequence encoded 184 amino acid residues. Five base pairs and 3 amino acid residues are different from that reported, it may be due to interspecies difference. The endostatin cDNA was inserted into the pET-28a(+) containing T7 promoter. The recombinant plasmid was transformed the E.coli BL21(DE3). Recombinant human endostatin was highly expressed as inclusion body when the expression strain BL-ENDO was induced with 1 mmol/L IPTG. Result of SDS-PAGE analysis revealed that recombinant human endostatin was accounted for up to 25% of soluble protein in E.coli. Purified and refolded recombinant human endostatin was active in inhibiting tumor growth and metastasis.

The human persephin (PSP) was expressed in Tn-5B1-4 insect cells using the Bac to Bac baculovirus expression system. The expressed product amounted for 20% of total cellular soluble proteins. The expressed product was purified by Ni(2+) affinity chromatography and the activity assays showed that it could significantly prolong the survival of spinal cord neurons.

A 1 131 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with primers designed according to published SmAct2 encoding Schistosoma mansoni actin. Sequence analysis indicated that this fragment, with 92% homology to SmAct2, was a complete open reading fragment (ORF) of actin gene of Schistosoma japonicum (Chinese strain). This gene was cloned into the expression vector pET28a( ) and subsequently expressed in Escerichia coli. SDS-PAGE revealed that the molecular weight of this expressed product was 47 kD. Western blotting showed that the recombinant protein had good reactivity with the rabbit serum immunized with Sj worm antigen, indicating that this gene encode actin of Schistosoma japonicum(Chinese strain).

To set up an in vitro amidating system, a recombinant human calcitonin with a glycine at C termial (mhCT-Gly) was used as the amidating substrate of recombinant rat peptidylglycine alpha-amidating monooxygenase (rPAM). First, the mhCT-Gly gene was synthesized and cloned into a fusion expression vector to get an expression plasmid pGEXCT. The GST-fused mhCT-Gly was highly expressed in E.coli BL21(DE3) harboring the pGEXCT, and was purified rapidly by affinity chromatography. Second, using the method of ultrafiltration, the rPAM was prepared from the supernatant of cultured transfectant CHO cells which express rPAM stably. Finally, the in vitro amidating experiments were carried out using GST-mhCT-Gly as substrate and the prepared rPAM. The results of dot blot with the specific antibody and of mass spectrum assay indicated that amidating product GST-hCT-NH(2) could be easily detected. This study provides a useful method for the amidation of recombinant products in vitro.

The glial cell line-derived neurotrophic factor(GDNF) plays a very important role in the regeneration of the nervous system. Based on the results of the X-ray structure analysis of rat GDNF, human GDNF gene was modified with deletion and insertion mutagenesis by using PCR methods. The various mutants were all highly expressed in E.coli. The recombinant proteins were purified and their survival-promoting activities were determined by using cultures of the spinal cord neurons of embryonic mouse. The results showed that the "cystine knot motif" was critical for the maintenance of GDNF structure the alpha-helix, finger 1 and finger 2 region were critical for GDNF neurotrophic activity and the N-terminus of human GDNF was not essential for its biological functions.

The fragment of Hyphantria cunea nuclear polyhedrosis virus (HcNPV), which contains cysteine protease gene(CP) and chitinase gene (ChiA),was inserted into plasmid PCRII to construct transfer vector pHcCVdel. Recombinant transfer vector pHcCvpolh was constructed by inserting the fragment of HcNPV polyhedrin gene (polh) into the EcoRI site of the pHcCVdel. By contransfection of the pHcCvpolh and HcNPV-PTTH(+) DNA, which contained Bombyx mori prothoracicotropic hormone gene (PTTH) into SPIM cells, the region from +76 downstream of ChiA gene initiation codon to +20 downstream of CP gene initiation codon was substituted by the polh gene, and the recombinant virus HcNPVpolh(+)CP(-)ChiA(-)PTTH(+) was produced. The recombinant virus, in which CP gene and ChiA gene were inactive, could express PTTH gene and form polyhedra. SPIM cells infected with the recombinant virus revealed that CP and ChiA gene were not essential for viral replication and had no significant effect on polyhedron formation, but the infected cells survived 2 days more than those infected with HcNPV and HcNPVPTTH(+). Therefore, the expression time of foreign gene may be prolonged when CP gene and ChiA gene were inactivated.

A fused construct formed by an anti-CEA single-chain antibody gene with a core-streptavidin gene(ScFv-CS)was inserted into the donor plasmids pFastBacHTa and expressed in Tn-5B1-4 cells. SDS-PAGE analysis revealed that the molecular weight expressed product of ScFv-CS were 41 kD. Using horseradish peroxidase(HRP)labeled biotin as antibody product Western blot, one band of 41 kD only was detected. It was also shown by RIA that the expression product possessed the ability to bind to its specific antigen of CEA. These results showed that the fusion protein not only had the ability to bind CEA, but also could bind biotin specifically.

Recombinant human serum albumin (rHSA) was produced in methylotrophic yeast Pichia pastoris. By optimization of expression, about 150 mg/L of rHSA was obtained from broth of Pichia pastoris GS115/HAS (his+Mut(S)) supernatant. The rHSA was isolated and purified by hollow-fiber ultrafiltration, Phenyl-Sepharose hydrophobic chromatography and antibody-immunoadsorbent chromatography. Finally, rHSA was purified to electrophoretic purity.

The potency of salmon calcitonin (sCT) is higher but the structural homology between sCT and human calcition (hCT) is only 50%. Based on the comparison of the structure between sCT and hCT, we have designed and synthesized a hCT analogue (mhCT-2) by solid phase method, using air oxidation in diluted solution to obtain a peptide with intramolecular disulfide bond. Through HPLC purification, we obtained a capillary electrophoresis-homogeneous mhCT, results of analysis of its mass spectrum and N-terminal sequencing were in accordance with the theoretical values. The results of calcitonin bioassay by estimating the blood calcium levels in rats showed that the potency of mhCT-2 was around 2 000 IU/mg, one order of magnitude higher than that of hCT. In RIA assay, we have found that the immunoactivity of mhCT-2 and hCT was much different because of their different binding abilities to anti-hCT antibody. This indicated that the conformation of mhCT-2 was changed as compared with hCT. In rat osteoporosis model, the results showed that pharmacologic effects of mhCT-2 was the same as that of sCT. The synthetic mhCT-2 seems promising to be a clinically useful peptide with high potential in osteoporosis therapy, because it is similar in biological properties to, but less immunogenic than sCT.

Total RNA was isolated from normal Chinese human liver cell line L02. A mutated osteoprotegerin/osteoclastogenesis Inhibitory Factor(OPG/OCIF) cDNA was amplified by RT-PCR using the total RNA as template and was inserted into pBS-sk plasmid. Sequence analysis showed that the OPG/OCIF cDNA from L02 cells was a mutated OPG/OCIF gene which had a nonsense mutation(Ochre) at the codon of Gln(394). The OPG/OCIF isoform was 8 amino acid residues less at the C terminal than the OPG/OCIF reported. The 3'fragment of OPG/OCIF gene from genomic DNA of cell line 293(ATCC CRL 1573) was also cloned. Sequence analysis indicated that the sequence of genomic DNA was the same as that of cDNA. The mutated OPG/OCIF gene was inserted into yeast expression plasmid pPIC3.5K, and the recombinant plasmid was used to transform Pichia pastoris GS115. SDS-PAGE analysis revealed that the human OPG/OCIF isoform was highly expressed and accumulated up to over 30% of soluble protein of yeast after the induction by methanol for 3 to 5 days. The longer the transformant was induced, the higher the ratio of glycosylated protein was.

A fragment of CYP6B2 gene was obtained from the midgut cDNA library of the fifth instar larvae of the ballworm (Helicoverpa armigera) with RT-PCR. The product of 627 bp was cloned and sequenced. The results of dot blotting analysis of mRNA proved that CYP6B2 is expressed at higher level (mRNA) in an insecticide-resistant strain than in a susceptible strain, showing that the regulation was at transcription level. The mRNA is induced by pyrethroid in susceptible, but not in the insecticide-resistant strains. We guess that mutation in the 5' upstream regulator of the insecticide-resistant strain may lead to the difference in the expression level between resistant and susceptible strains.

The human glial cell derived neurotrophic factor (GDNF) was expressed in Tn-5B1-4 insect cells using the Bac to Bac baculovirus expression system, accounting for 30% of total cellular soluble proteins. The expressed product was purified by affinity chromatography and the activity assays showed that it could significantly promote the survival of dopaminergic neurons. The study can be the basis for further structural-functional analysis of GDNF.

A cDNA encoding acidic phospholipase A(2)I(A.aAPLA(2)I)from Agkistrodon acutus was inserted into a bacterial expression vector and effectively expressed in E.coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column Superose(TM)12. The enzymatic acti-vity and platelet aggregation inhibiting effect of the expressed A.aAPLA(2)I is close to those of denatured-refolded native acidic PLA(2) from Agkistrodon halys Pallas, and has the same hemolytic activity as denatured-refolded basic phospholipase A(2) from Agkistrodon halys Pallas. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

The cDNA encoding nerve growth factor (NGF) precursor from Agkistrodon halys Pallas (a Chinese snake species) was cloned into pBacPAK8 a baculovirus shuttle vector. The recombinant shuttle vector pBacPAK-NGF was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The silkworm larvae were infected with the recombinant virus and collected 5 days later. The SDS-PAGE and the NGF activity by bioassay of PC12 cells have shown a high expression level of NGF of good biological activity in the larvae.

The cDNA encoding the mature annexin V was isolated by using RT-PCR method from total RNAs of fresh human placenta. The result of sequencing indicated that the sequence of isolated annexin V cDNA was the same as the reported nucleotide sequence of annexin V. The annexin V cDNA was cloned into expression plasmid pET24a(+) under T7 promoter and then transformed into E.coli BL21(DE3). SDS-PAGE analysis revealed that the human annexin V was highly expressed and accumulated up to 38% of total bacterial proteins in soluble form after the induction by 1 mmol/L IPTG. The purified human annexin V can significantly prolong activated partial thromboplastin time (APTT).

Fusion protein of Newcastle disease virus is a glycoprotein with antigenicity. The cDNA of fusion gene of NDV strain F48E8 was cloned by RT-PCR, and the sequence of F48E8 was compared with other two virulent strains, Australia-victoria and Italien. Results show that the sequences of N-terminus are very similar, and cysteine residues and the potential glycosylation sites are relatively conserved. However, there are a few differences in the region of signal peptides.

The cDNA encoding the human copper, zinc-superoxide dismutase(Cu, Zn-SOD) was amplified from the human liver by RT-PCR and sequenced. The cloned human Cu, Zn-SOD cDNA was ligated into expression vector pET-22b(+) under T7 promotor. After 3 h induction with 1 mmol/L IPTG, human Cu, Zn-SOD was highly expressed in E.coli BL21(DE3). The expression product was up to 30% of the total protein of the bacteria in soluble form, which had specific SOD activity.

Recombinant Bm-BacScFv virus which contains a cDNA encoding anti-CEA single-chain antibody was generated by cotransfection into Bm N cells with the transfer plasmid pBacPAK-(His)(6)-ScFv and the modified Bombyx mori nuclear polyhedrosis virus Bm-BacPAK genomic DNA. Bombyx mori cells and larvae were infected by the recombinant virus, respectively. The anti-CEA single-chain antibody were expressed both in Bombyx mori cells and larvae, the former accounted for 6% of the total cell protein, the later 0.3 mg per larvae. The histidine-tagged ScFvs were respectively purified with nickel-iminodiacetic acid affinity chromatography. The purity of the former reached over 90% and that of the latter was lower. Purified protein products was able to bind to CEA with an affinity constant of 5.4x10(8)/mol.L(-1) and 2.3x10(8)/mol.L(-1), slightly lower than its parental monoclonal antibody E(7)B(10) which had 2.7x10(9)/mol.L(-1).

The biotinylated annexin V was generated by genetic engineering method. The encoding region of annexin V was fused with a gene coding for the carboxyl terminal 87 residues of Escherichia coli biotin carboxyl carrier protein. The fused gene was expressed in E.coli and the annexin V was biotinylated in vivo. The biotinylation efficiency was about 60% as detected by HPLC. The biotinylated annexin V was purified by avidin affinity chromatography and used to detect the apoptosis of the neurons induced by morphine.

The cDNA encoding human manganese-superoxide dismutase was amplified from the human liver cell (L02) total RNA by RT-PCR and sequenced. The human Mn-SOD cDNA was ligated into expression vector pET-24a(+) under T7 promoter. After 5 h induction with 1 mmol/L IPTG 800 mol/L Mn(2+) human Mn-SOD was highly expressed in E.coli BL21(DE3). The protein product was up to 50% of the bacteria total protein in soluble form and had specific SOD activity.

Synthetic oligonucleotides were used to amplify phospholipase A(2) (PLA(2)) gene by RT-PCR from total RNA of snake Agkistrodon acutus venom gland. The PCR products were subcloned and positive clones were screened with acidic PLA(2) gene from Agkistrodon halys Pallas. Finally, four cDNAs of PLA(2) isoenzymes were isolated. Their complete sequences were determined by bidirectional sequencing and their amino acid sequences were deduced. They were designated as A.aAPLA(2)I A.aAPLA(2)II A.aBPLA(2) and A.aLys(49)-PLA(2) according to their isoelectric points calculated by computer and special structure characteristics respectively. The amino acid sequence of 1 10 residues of A.aAPLA(2)I deduced from the cDNA is identical to that of acidic PLA(2) which had been isolated from Agkistrodon acutus. A.aLys(49)-PLA(2) is unique because of the usual Asp(49) is replaced by Lys(49), which may lower its enzymatic activity. Their similarity scores were calculated and compared by computer. The successful cloning of these isoenzymes genes may provide more information for the study on structure-function relationship of PLA(2) family.

Helicobacter pylori is the causative agent of gastritis and peptic ulcer in human. The heat-shock protein A (HspA)may stimulate the immunoresponse protecting human body against challenge of H.pylori. The gene encoding the structural A subunit of H.pylori heat-shock protein was amplified from H.pylori chromosomal DNA by PCR, and was inserted in the prokaryotic expression vector pET-22b(+), and then was transformed into the BL-21(DE3)E.coli strain to express the HspA recombinant protein. HspA gene was measured to be 354 base pairs, and the recombinant protein gene encoded polypeptides of 118 amino acid residues, corres-ponding to calculated molecular weight of 15 kD. Western blot analysis of HspA recombinant protein was confirmed that it could be specifically recognized by the serum of H.pylori-infected patients, and could also be re-cognized by the serum of immunized Balb/c mice with HspA itself. This result suggests that HspA may be an effective protein vaccine for prevention and treatment of the infection of H.pylori.

Superoxide dismutases scavenge superoxide radicals and protect cells from oxidative stress. SOD gene of Bombyx mori nuclear polyhedrosis virus has been cloned by PCR, and is expressed in E.coli with the activity of SOD being 576.13 u/mL. DNA sequence analysis shows that SOD gene of BmNPV encodes 151 amino acids. The homology between BmNPV and AcNPV of the nucleotide sequences of SOD gene is 97.2%, and 56% between BmNPV and human SOD1.

We have modeled three-dimensional structures of basic-acidic hybrid phospholipase A(2)-II and neutral phospholipase A(2) from venom of snake Agkistrodon halys Pallas, based on the known structures of basic and acidic phospholipase A(2)'s from the same source. We have compared these structures of phospholipase A(2)'s, explained the results of fluorescent spectrum study on the phospholipase A(2)'s and calculated the electrostatic potential maps on the catalytic active site. We suggest that the electrostatic potential around the catalytic active site of PLA(2) containing a calcium ion favors the binding of the PLA(2) to its substrate with negative charge.

Signal and leading peptide sequences of rat PAM was inserted into the baculovirus transfer vector, and secretion expression plasmids pBACPAG2 and pBacPAI for the fusion gene PABC-hGRF and PABC-IGF-I were constructed, respectively. By cotransfection with linear genomic DNA of modified Autographa californica nuclear polyhedrosis virus (BacPAK6) and homologous recombination, the recombinant AcNPV, BacPAG and BacPAI, were obtained and identified. Fusion proteins PABC-hGRF and PABC-IGF-I were secreted efficiently from Sf21 cells infected with BacPAG and BacPAI, respectively, and those fusion protein could be purified efficiently by IgG affinity column.

Total RNAs were extracted from the venom gland of the Agkistrodon halys Pallas snake. The thrombin-like enzyme gene was amplified by RT-PCR, cloned, and its nucleotide sequences have been determined. The enzyme called pallase cDNA encodes 708 nucleotides, namely 236 amino acids. Based on the homology, the catalytic residues and disulfide bridges of pallase were deduced as follows catalytic residues, His(41), Asp(86) and Ser(182) and disulfide bridges, Cys(7)-Cys(139), Cys(26)-Cys(42), Cys(74)-Cys(234), Cys(118)-Cys(188), Cys(150)-Cys(167) and Cys(178)-Cys(203). The pallase expression plasmids under the control of the T7 promoter was constructed and the pallase was expressed in E. coli.

The cDNA encoding the mature human glial cell derived neurotrophic factor(GDNF) was isolated by using RT-PCR method from total RNA extracted of fetal human brain. The expression plasmid pET-GDNF was constructed by inserting GDNF cDNA into plasmid pET-28a(+) containing T7 promoter and transformed into E.coli BL21(DE3). An expression strain BLGDNF was selected. SDS-PAGE analysis revealed that the human GDNF protein was highly expressed and accumulated up to above 30% of the total bacterial proteins in the form of inclusion body after the induction. The antibody to GDNF was prepared by immunization of rat using purified GDNF protein. Purified and refolded GDNF protein could significantly promote the survival of dopaminergic neurons.

The cysteine protease (CP) gene of Hyphantria cunea nuclear polyhedrosis virus (HcNPV) has been located at the Hind III--3.5 kb fragment. The nucleotide sequence has been determined. The results indicated that its open reading frame of 975 nt encoded the protein of 324 amino acids. The baculovirus late gene promoter TAAG motif was identified at -24 nt upstream from the start codon ATG; The typical poly (A) signal sequence AATAAA was detected at 33 nt downstream from the stop codon TAA. The HcNPV CP exhibited 81%, 76% and 76% homology at the nucleotide level, and 85%, 78%, 76% homology at the amino acid level to that of CfNPV (Choristoneura fumiferana nuclear polyhedrosis virus), AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV (Bombyx mori nuclear polyhedrosis virus), respectively. HcNPV CP was a memeber of the papain superfamily and showed high homology to other cysteine protease, particularly, to those from Trypanosoma brucei and Dictyostelium discoideumwith 31.8%, 34.5% identity at the amino acid level, respectively. Of 36 residues conserved among cathepsins B, H, L and S and papain, 32 were identical in HcNPV CP. The ERFNIN interspersed amino acid motif was found within the propeptide region of the HcNPV CP. The Gly-Cys-Asn-Gly-Gly motif conserved among all cysteine protease was also detected in HcNPV CP.

The coding region of BmvPK-1 gene of Bombyx mori NPV (Strain ZJ8) is 828 nt long and encodes a 276 aa polypeptide with predicted molecular mass of 32 kD. Dot blot analysis showed its mRNA to be gene is first detectable at 18 h p.i. and reaching the highest transcriptional level at 48 h p.i. The result suggested that BmvPK-1 gene is a late or very late gene. The most conserved 365 bp of the BmvPK-1 gene was deleted in a transfer vector (pUCPK-lac), and a report gene (lacZ) was inserted in the deleted position. Cotransfection of BmN cells with pUCPK-lac DNA and BmNPV DNA resulted in the recombinant virus which expressed detectable product of lacZ gene. But the virus with the deleted BmvPK-1 gene could not be isolated from the wild BmNPV by plaque purification method. The result showed that the BmvPK-1 gene deleted virus can multiply only with the help of the product of this gene from the wild type virus, and the gene is necessary for the virus to finish its life cycle in the cultured cells.

A 600 bp DNA fragment was amplified by PCR, from an adult Schistosoma japonicum cDNA library. Sequence analysis revealed that this fragment containedthe S. japonicum Chinese Mainland strain fatty acid binding protein (Sj-14FABPc) gene. This gene was then cloned into the expression vector pGEX-2T, and subsequently expressed in Escherichia coli. The recombinant GST-fusion protein could be purified by glutathione agarose affinity chromatography. Its molecular weight was about 41 kD. The yield of expression was around 25 mg/L E. coli culture. The immunological test suggested that the recombinant protein had good antigenicity, and could be developed into a new vaccine molecule of S. japonicum.

By using site-directed mutagenesis, we created a unique Xho I site in the CDR3 of the heavy-chain variable domain (V(H)). Two antibody molecules, one carrying one or and the other two repeats of an immunodominant epitope AA32-45 (GVYLLPRRGPRLGV) of the hepatitis C virus core protein in CDR3 of V(H) were engineered and designated Ig-E1, Ig-E2 respectively. We found that both antigenized antibodies lost the HBsAg-binding ability and the insertion of one repeat of GVYLLPRRGPRLGV epitope into the CDR3 of the V(H) domain did not appreciably affect the H chain to assemble with L chain to form a stable H(2)L(2) tetramer. Ig-E1 was able to be recognized by the polyclonal antibody against AA1-58 of the core protein as characterized by Western blot. However, Ig-E2 could not be assembled into H(2)L(2) tetramer.

We used degenerate primers to amplify phospholipase A(2)(PLA(2)) gene by RT-PCR from venom total RNA of Agkistrodon halys Pallas, and screened with B-PLA(2) gene as probe. Finally we isolated three cDNAs containing A-PLA(2) and two other genes showing similar characteristic structure--Asn(49)-PLA(2) and BA-PLA(2). Their complete sequence was determined by bidirectional sequencing and their amino acid sequence was deduced. The amino acid sequence of A-PLA(2) deduced from the cDNA agreed with that determined by protein sequencing except for four residues; Asn(49 )-PLA(2) and B-PLA(2) are very alike (up to 95%), but the Asp(49)-PLA(2) of B-PLA(2) involved in the enzyme reaction is replaced by Asn(49) of Asn(49)-PLA(2). Therefore, it possibly affects the enzyme reaction of Asn(49)-PLA(2). The N-terminus sequence of BA-PLA(2) is highly homologous to B-PLA(2), but its C-terminus sequence is almost the same as A-PLA(2). The successful cloning of these isoenzyme genes not only discloses the structure diversity of PLA(2)(namely DNA modification and recombination), but also provides excellent material for the study of structure-function relationship in PLA(2).

A transfer plasmid p8hLFc harboring human lactoferrin cDNA was constructed and co-transfected with Autographa californica nuclear polyhedrosis virus ( AcNPV BacPAK6 ) DNA into Spodoptera frugiperda (Sf) cells. The recombinant virus AcNPV-hLFc was identified and purified three times by dot hybridization and plaques screening. The expression of human lactoferrin cDNA in insect cells was detected by Western blotting. The expression level of lactoferrin is about 2% of insect cellular total soluble proteins, and an amount of 9.5 mg/L lactoferrin is secreted into culture medium. Recombinant lactoferrin purified with affinity chromatography comigrates with human milk lactoferrin on SDS-polyacrylamide gels. In addition, the ration of A value at ultraviolet A(280)/A(465) shows that the recombinant protein possesses iron-binding capacity.

The heavy and light chain variable region (V(H) and V(K)) genes encoding the murine monoclonal antibody E(7)B(10) to carcinoembryonic antigen were linked into single-chain antibody gene with 36 oligonucleotide (Gly(3)Ser)(3) by using recombinant PCR. The construct was highly expressed in E. coli as inclusion bodies (IB), accounting for 20% of the total bacteria proteins, and was characterized by SDS-PAGE and Western blot. When the content of inclusion bodies was denatured and followed by renaturation, it was shown to possess abiliby to kind to its specific antigen CEA by using RIA and competitive ELISA.

The pga gene was amplified by PCR from Bacillus megaterium CA4098, cloned into pKK223-3 vector and expressed in E. coli HB101. The pga sequence was also determined. The deduced amino acids sequence was compared with those of PGAs from other bacteria and showed that they were similar, especially the active sites were strongly conserved.

We have isolated the cDNA encoding the mature tissue inhibitor of metalloproteinase-3 (TIMP-3) by using RT-PCR method with total RNA extracted from human fresh placenta. The result of sequencing indicated that the mature TIMP-3 polypeptide contained 188 aa residues, including 12 conserved cysteinyl residues in the TIMP family. The expression plasmid pET-TIMP3 was constructed by inserting TIMP-3 cDNA into plasmid pET-24 (a(+)) containing T7 promoter and transformed into E. coli BL21 (DE3). An expression strain BLTIMP3 was selected. SDS-PAGE analysis revealed that the human TIMP-3 protein was highly expressed and accumulated up to above 30% of the total bacterial proteins in the form of inclusion body after induced by 1 mmol/L IPTG for 3--5 h. The partially purified and renatured TIMP-3 can significantly inhibit angiogenesis as shown by CAM assay.

The APLA(2) gene from Agkistrodon halys Pallas has been cloned into the expression plasmid pBLMVL2 and expressed in E. coli RR1. The molecular weight of the expressed product is approximately 14 kD as shown by SDS-PAGE, its expression level is about 30% of the total cellular proteins. The protein was produced as insoluble inclusion bodies. After partially purified by washing the inclusion bodies, the product was denatured and refolded into active form. Then, the expressed APLA(2) was purified by FPLC Superose (TM) 12 and was a single band as shown by SDS-PAGE. The purified expressed protein had specific activity as the native enzyme and cross-reacted with antisera prepared against the native enzyme. The successful expression of the APLA(2) gene from Agkistrodon halys Pallas provides a good basis for further structure-function studies.

Triose-phosphate isomerase is an important candidate for schistosoma antigens. An 800 bp DNA fragment was amplified by RT-PCR from adult Schistosoma japonicum mRNA. Sequence analysis revealed that this fragment contained S. japonicum (Chinese strain) triose-phosphate isomerase gene. Then this gene was cloned into the expression vector pGEX-4T and subsequently expressed in E. coli. The recombinant GST-fusion protein was purified by glutathione agarose affinity chromatography. Its molecular weight was determined to be 54 kD. The yield of expression was around 30 mg/L E. coli culture. Western blotting showed that the recombinant protein had good antigenicity which could be helpful for the making of anti-S. japonicum multi-valent recombinant vaccine.