Biomedical subjects

Xiang-Fu Wu

Publications and source records attributed to Xiang-Fu Wu.

At least 37 records · Page 2Linked to original sources

PubMed2002

[Coexpression of caiB and caiE with two plasmids in E. coli].

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.

Acyltransferases↗

PubMed2002

[Cloning and expression of Tachypleus tridentatus factor C].

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.

Amino Acid Sequence↗

PubMed2002

[Cloning and expression of Buthus martensii Karsch scorpion toxin gene (BmK IT3) in Escherichia coli].

According to the reported sequence of Buthus martensii Karsch scorpion toxin gene (BmK IT3), we synthesized two primers, which were complementary in a region. By the means of PCR, we got the gene. The gene was fused in expression vector pET-28a, which gave rise to a recombinant plasmid pET(IT3R). Then it was transformed into E. coli BL21 (DE3). With IPTG induction, the gene was efficiently expressed. And the fusion product was soluble.

Animals↗

PubMed2002

[Couple production of human calcitonin and rat peptidylglycine alpha-amidation monooxygenase in insect cells].

Human calcitonin (hCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. Calcitonin has important physiological function in vivo. We describe the couple expression of a synthesized modified human calcitonin(hmCT) gene fused with glutathione-S-transferase and rat peptidylglycine alpha-amidation monooxygenase (PAM) in insect cells infected by recombinant baculovirus GSTCT/PAM. Using Western blotting against hmCT or rat PAM, the GSThmCT fusion protein had been identified as well as the PAM. Following affinity chromatography with glutathione agarose column, the GSThmCT fusion protein produced by insect cells was purified. The purified fusion protein was also interacted with antibody against hmCT. The couple expression of a modification enzyme and its substrate in eucaryotic expression system may be used for producing other biological activity peptides.

Animals↗

PubMed2002

[Identification of functional region of helicase gene promoter in Bombyx mori nuclear polyhedrosis virus].

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.

Animals↗

PubMed2002

[Cloning and expression of 21.7 kD protein gene of Schistosoma japonicum (Chinese strain)].

A 558 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with a pair of primers that were designed according to published Sj21.7p gene encoding 21.7 kD protein of Schistosoma japonicum(Philippines strain). Sequence analysis indicated that this frame, named Sj21.7 (Ch), with 99% homology to Sj21.7 p, contained a complete open reading fragment (ORF) of 21.7 kD protein gene of Schistosoma japonicum(Chinese strain). The amino acid sequence shared 98% homology with 21.7 kD protein of Schistosoma japonicum. This fragment was cloned into the expression vector pET28a (+) and subsequently expressed in Escherichia coli with IPTG induction. SDS-PAGE analysis revealed that the molecular weight of this expressed product was 25.4 kD. Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.

Amino Acid Sequence↗

PubMed2001

Functional Analysis of Helicase Gene Promoter and Homologous Region 3 Enhancer in Bombyx mori Nuclear Polyhedrosis Virus.

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.

Journal Article↗

PubMed2001

Cloning and Expression of Targeting CuZn-SOD to Central Nervous System.

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.

Journal Article↗

PubMed2001

A Candidate Oral Vaccine to Helicobacter pylori Fusion Protein of HspA and CtxB.

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.

Journal Article↗

PubMed2000

Fused Expression of Anti-CEA Single-chain Antibody Gene with Core-streptavidin Gene in Insect Cells.

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.

Journal Article↗

PubMed2000

Cloning and Expression of Actin Gene of Schistosoma japonicum Chinese Strain.

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).

Journal Article↗

PubMed2000

Cloning and Expression of Tetanus Toxin Fragment C in E.coli.

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.

Journal Article↗

PubMed2000

Cloning, Expression and Tumor Suppression of Human Endostatin.

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.

Journal Article↗

PubMed2000

A Structure-function Analysis of Human GDNF.

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.

Journal Article↗

PubMed2000

High Expression of Human Persephin in Insect Cells.

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.

Journal Article↗

PubMed2000

In Vitro Amidating Processing of Products Expressed by Gene Engineering.

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.

Journal Article↗

PubMed2000

Cloning of the CtxB Gene of Vibrio cholerae and Its Expression in E.coli.

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.

Journal Article↗

PubMed2000

Inactivation Analysis of HcNPV Cysteine Protease Gene and Chitinase Gene.

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.

Journal Article↗