Prokaryotic Expression and Identification of Outer Membrane Protein 2 of Chlamydia trachomatis

Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylorihpaA

AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then doned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coliDH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine^(TM)2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

Cloning and expression of mouse peroxiredoxin I in 1EC-6 Cells

AIM: To clone and express mouse peroxiredoxin Ⅰ in IEC-6 cells.METHODS: Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced,pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCRand Western blot. RESULTS: A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin Ⅰ. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRI-BamHI fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified,which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin Ⅰ in IEC-6 cells.CONCLUSION: Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.

Primary targeting of recombinant Fv-immunotoxin hscFv_(25)-mTNFα against hepatocellular carcinoma

AIM: To obtain human recombinant Fv-immunotoxin hscFv25-mTNFα (mutant human TNFαfused to human scFv25) against hepatocellular carcinoma (HCC).METHODS: Two relevant sites of enzymatic digestion were added to mTNFα by PCR. mTNFα was linked to the 3' end of hscFv25 in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv25-mTNFα was expressed in Escherichia coliand purified from inclusions. After purified by glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot. And then, the purified recombinant Fvimmunotoxin was injected into nude mice with HCC xenografts through their tail veins, mTNFα protein and PBS were used as control at the same time. After treated for two weeks, nude mice were executed. The bulk and weight of tumors were observed. The tumor tissues were stained by immunohistochemical method with TNFα antibody.RESULTS: The expression ratio of recombinant Fv-immunotoxin hscFv25-mTNFα was 12% of bacterial protein. The result of tumor restraining trials of hscFv25-mTNFα showed 2/5 complete remission and 3/5 partial remission, mTNFα restraining trials showed 5/5 partial remission. The therapeutic result of hscFv25-mTNFα was better than that of mTNFα (F=8.70, P<O.05). The hscFv25-mTNFα remedial tumor tissues were positive for TNFα by immunohistochemical staining. The positive granules mainly existed in the cytoplasm of tumor cell.CONCLUSION: Recombinant Fv-immunotoxin hscFv25-mTNFα has better therapeutic effect than mTNFα. It can inhibit the cellular growth of HCC and has some potential of clinical application.

Diagnosis of Helicobacterpyloriinfection and diseases associated with Helicobacter pylori by Helicobacter pylori outer membrane proteins

AIM:TO examine the serological response of patients with upper gastrointestinal diseases and Helicobocter pylori(H pylon) infection to two Hpyloriouter membrane proteins(OMPs) (M,18 000 and M,26 000)acquired by gene recombinant technique,and to determine the diagnostic significance of serological tests derived from these OMPs. METHODS:Recombinant vectors encoding the two Hpylori OMPs were used to transform and express in BL21(DE3) E.COli.After purification with Ni^(2+)-NTA agarose resin,colloid gold kits were prepared with purified recombinant proteins to detect H pylori infectJon and H pylori-associated diseases by the immunity-marker technology.We selected 150 patients with Hpyloriinfection and digestive symptoms without previous treatment,induding chronic gastritis(n=60),duodenal ulcer (n=30),gastric ulcer(n=30),and gastric cancer(n=30). As controls,33 Hpylori-negative healthy volunteers were also recruited.Serum samples were collected from all subjects,and the antibodies to specific proteins of H pylori were tested with the colloid gold test kits.The sensitivity, specificity and accuracy of the colloid gold tests were evaluated,by using the combination of standard diagnostic methods(^(13)C urea breath test and bacteria culture)and classic enzyme-linked immunosorbent assay(ELISA)as reference. RESULTS:After purification with Ni^(2+)-NTA agarose resin, the purity of recombinant fusion proteins was about 95%. The recombinant fusion proteins were recognized by the specific monodonal antibodies against the two Hpylori OMPs, as demonstrated by the ELISA.Of the 150 serum samples from patients infected with Hpylori 141(94.0%)responded positively to the recombinant protein with M_126 000,while the seropositive rates were 95.0%,96.7%,96.7% and 90.0% for patients with H pylori-associated chronic gastritis, duodenal ulcer,gastric ulcer,and gastric cancer respectively. The sensitivity,specificity,and accuracy of the colloid gold kit with M_1 26 000 protein were 94.0%,97.0%,and 94.5%, respectively.Compared with the classic ELISA,bacteria culture and ^(13)C urea breath test results in detecting Hpylori- infection,there was no significant difference(P>0.05).For the colloid gold kit with M,18 000,the seropositive rates were 52.0%,40.0%,40.0%,53.3% and 86.7%,respectively, in Hpylori-infected patients,and those with Hpylori-assodated chronic gastritis,duodenal ulcer,gastric ulcer,and gastric cancer.There was a significant difference(P<0.05)in seropositivity between patient with gastdc cancer(86.7%) and those with other diseases(43.3%). CONCLUSION:The two colloid gold kits derived from the recombinant OMPs are useful tools either for detecting Hpyloriinfection,or for,predicting Hpylori-assodated gastric malignancy.

Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

Oncosuppressive Effects of Natural and Recombinant Parvoviruses

The infection of animals with certain autonomous parvoviruses has been shown to have an onco-sup-pressive effect, whilst the viruses themselves have a preferred tropism for transformed cells (oncotropism). However, in many instances, the level of this response is not sufficient for complete suppression of tumorigenesis. Our goal is to enhance the antitumor potential by combining both the intrinsic oncosuppressive and oncotropic determinants of the virus with an additional therapeutic gene in a recombinant parvoviral vector.

Expression of reconstructive hF Ⅷ in the hrDNA by using hrDNA targeting vector

Our lab has constructed a new nonviral vec-tor—hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression cassette of reconstructive hF Ⅷ (hFVIII-BDDAK39) to pHrneo to construct targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HT1080 cells, we identified the homologous recombinants by PCR and Southen blotting, and tested the expression of hFVIII- BDDAK39 in the hrDNA locus. The hFⅧ-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific inte-gration was 2.0×10?5. hFⅧ-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently (32±5 ng·106 cells?1·24 h?1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.

Overview of the Basic Research on Vascular Surgery in China

This paper sums up the fundamental research projects on Vascular Surgery supported by National Natural Science Foundation of China from 1996 to 2000, and presents the experimental results and advances in the aspects of ischemia disease, the formation mechanism and prevention of restenosis, the development of abdominal aorta aneurysm and portal hypertention.