Biomedical subjects

Xiang-Fu Wu

Publications and source records attributed to Xiang-Fu Wu.

At least 73 records · Page 4Linked to original sources

PubMed1999

Efficient Secretion of Proteins Expressed from Insect Cells Directed by PAM Signal Peptide.

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.

Journal Article↗

PubMed1999

Cloning and Expression of cDNA for Thrombin-like Enzyme from Agkistrodon halys Pallas Snake Venom.

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.

Journal Article↗

PubMed1999

CYP6B2 Expression in Insecticide-Resistant and -Suspcitible Strains of Helicoverpa armigera.

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.

Journal Article↗

PubMed1998

Expression of Human Lactoferrin cDNA in Insect Cells.

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.

Journal Article↗

PubMed1998

Cloning of Schistosoma japonicum Chinese Strain TPI Gene and Characterization of Its Expression Product in Escherichia coli.

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.

Journal Article↗

PubMed1998

Construction of Single-chain Antibody to Carcinoembryonic Antigen and Its High Expression in E. coli.

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.

Journal Article↗

PubMed1998

Expression of the APLA(2) Gene from Agkistrodon halys Pallas.

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.

Journal Article↗

PubMed1998

Cloning and Expression of the cDNA Encoding Human Tissue Inhibitor of Metalloproteinase-3 and Its Inhibition on Angiogenesis.

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.

Journal Article↗

PubMed1998

Nucleotide Sequence Analysis of the HcNPV Cysteine Protease Gene.

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.

Journal Article↗

PubMed1998

Cloning and Expression of the pga Gene of Bacillus megaterium in E. coli.

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.

Journal Article↗

PubMed1998

Studies on the Nucleotide Sequence, Transcription and Deletion Analysis of the BmNPV Protein Kinase Gene.

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.

Journal Article↗

PubMed1998

Construction of Antigenized Antibodies Expressing an Immunodominant Epitope AA32-45 of the Hepatitis C Virus Core Protein.

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.

Journal Article↗

PubMed1998

Cloning of Schistosoma japonicum Chinese Strain Fatty Acid Binding Protein (Sj-FABPc) Gene and Its Overproduction in Escherichia coli.

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.

Journal Article↗

PubMed1998

Research on the Diversity of PLA(2) Gene from Agkistrodon halys Pallas.

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).

Journal Article↗

PubMed1997

High Expression in Insect Cells of A Functional Chimeric Antibody with Specificity for HBsAg.

We have constructed a double-recombinant virus BacHL4.2 containing both the chimeric heavy- and light-chain cDNAs from monoclonal antibody of the HBsAg gene. Both murine-human chimeric antibody heavy- and light-chain were expressed in Sf9 cells infected with a double-recombinant virus BacHL4.2 or co-infected with separate heavy- and light-chain recombinant viruses. In both cases, expressed products were correctly assembled into normal H(2)L(2) immunoglobulin monomers. ELISA and functional immunoblot assay showed that the recombinant chimeric antibody exhibited a specificity for HBsAg similar to that of the parental murine monoclonal antibody OH3.

Journal Article↗

PubMed1997

Separate Expression and Characterization of the Heavy- and the Light-chain of A HBsAg Specific Murine-human Chimeric Antibody in Insect Cells.

Both the heavy- and light-chain of a murine-human chimeric antibody with specificity for hepatitis B virus surface antigen have been expressed separately in a baculovirus expression system. The heavy- and light-chain variable region (VH and VL) genes encoding the marine-monoclonal antibody OH3 were combined with human gamma3 and kappa constant region genes to construct the murine-human chimeric genes, respectively. The transfer vectors containing the chimeric genes were cotransfected into Sf9 cells with linearized virus DNA and recombinant viruses were isolated by dot hybridization, PCR and Southern blot analysis. The chimeric heavy- and light-chain was expressed respectively in the recombinant viruses-infected insect cells and the characterization by Western blot and competitive ELISA demonstrated that both of the expressed chimeric heavy- and light-chain have the specificity of HBsAg binding.

Journal Article↗

PubMed1997

Nucleotide Sequence Analysis of HaSNPV Protein Kinase.

The protein kinase gene of Helicoverpa armigera single nucleocapsid nuclear polyhedrosis virus (HaSNPV) has been cloned and sequenced. It is located approximately 1.25 kb downstream of the polyhedrin gene. The predicted molecular mass of this 267 amino acid protein (HavPK) is 31 kD. HavPK shows a 43.0% amino acid homology to vPK from Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) and a 39.0% homology to PK-1 from Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV). Comparison of HavPK with protein kinases from other organisms showed that HavPK was also homologous to the catalytic domains of eukaryotic protein kinases and strongly indicated that HavPK was a serine/threonine protein kinase.

Journal Article↗

PubMed1997

Expression of the Arrowhead Proteinase Inhibitor B (API) Gene in the Silkworm Larvae and Pupae.

The arrowhead proteinase inhibitor B (API) gene was cloned into baculovirus transfer vectors of pBacPAK8 and pOSX, and two recombinant transfer vectors, pBacPAK (API) and pOSX (API), were constructed. Co-infection was accomplished with either one of the recombinant transfer vectors and Bm-BacPAK6 DNA in BmN cells. Then recombinant virus of CrBK9, CrBK10 and BX was selected. API gene was successfully expressed in silkworm larvae and pupae. The recombinant API (rAPI) showed strong inhibitive activity on bovine pancreas trypsin. The inhibiting activity of the expression product was 1.0x10(5) u/ml haemolymph for silkworm larvae and 1.3x10(5)u/ml haemolymph for pupae. In addition, SDS-PAGE of the preliminarily purified rAPI showed its molecular weight to be about 19 kD.

Journal Article↗