[Expression of human IL-12 in mammalian cell and study on its biological activities].

OBJECTIVE: To obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis. METHODS: Bicistronic expression vector P35-IRES-P40 was constructed for the simultaneous translation of IL-12 p35 and p40 cDNA subunit through internal ribosomal entry sites (IRES). pCI-dhfr-P35-IRES-P40 vector was constructed for expression in CHO-DHFR- cells. Positively cloned cells were screened by means of ELISA. Pools of clones with increased expression of IL-12 could be generated by selection in methotrexate. To determine the biological activities of rhIL-12, PHA-activated lymphoblasts proliferation assay and IFN-gamma induction assay were used in this study. RESULTS: Genetically engineered cells expressing hIl-12 were obtained and all the cell lines showed the stabile expression of rhIL-12 in high efficiency and good growth properties. CONCLUSION: rhIL-12 have good biological activities, it can stimulate activation and proliferation of T cells and induce production of IFN-gamma.

[Influence of the mutants of hepatitis B surface antigen on the cell immunity].

OBJECTIVE: To study the influence of the mutants of hepatitis B surface antigen on the cell immunity. METHODS: The recombinant plasmids of NS2Swt, NS2S126, NS2S133, NS2S141 and NS2S145 were transfected into Chinese hamster ovary (CHO) cells and the expressed proteins were detected by means of ELISA. Following PHA-activated lymphoblasts proliferation assay and IFN-gamma, IL-2, IL-10 induction assay were done with these proteins. RESULTS: It was identified that these proteins of HBsAg could stimulate human lymphoblasts proliferation. Besides, there were no significant difference between the mutants and the wild. It was deserved to point out that the HBsAg with T126S mutation could increase the expression of IFN-gamma in the culture medium while the HBsAg with M133T mutation induced more expression of IL-10. CONCLUSION: The results suggested that the cellular immune response to mutants of HBV might not be strengthened or weakened. But it should not be ignored that HBV T126S or M133T mutation may assert a potential impact on the cell immunity.

[Baculovirus expression of two human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus].

OBJECTIVE: To develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system. METHODS: The heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized. RESULTS: The baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16. CONCLUSION: The recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

[Antigenic analysis of the common recombinant mutant types of HBsAg].

BACKGROUND: To study the difference between the mutant types and the wild type of HBsAg on the binding efficiency with antibodies in order to find some methods to detect the mutants. METHODS: The recombinant wild type of HBsAg and the mutants including 145 R, 133 T, 126 S, 141 E, 126 S+133 T, 126 S+133 T+145 R and 126 S+133 T+141 E were cloned, respectively. Then the expression vector containing the wild or mutant gene was transfected into COS7 cells, and the recombinant proteins were expressed. The proteins were detected with the routine HBsAg kits. RESULTS: The binding efficiency with monoclonal antibodies of HBsAg expressed by 126 S was higher than that of the wild type, while the results of 145 R, 141 E, 126 S+133 T+145 R and 126 S+133 T+141 E were lower than that of the wild, and 133 T was similar to the wild. But most of the mutants had the same reactivity with the polyclonal antibody. CONCLUSIONS: The effect of mutations on antibody binding differs depending on the amino acid involved and on the location within the "a" loop. So it is necessary to use polyclonal antibody or to find new kits to detect the mutants of HBsAg.

[Antigencity identification of recombinant hepatitis E virus ORF2 protein expressed in Pichia pastoris].

BACKGROUND: To determine the antigenicity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P. pastoris). METHODS: By using the rHEV ORF2 protein from E.coli as control, an indirect ELISA was adopted to identify the sensitivity, specificity and stability of rHEV ORF2 protein from P. pastoris in detection of HEV IgM and IgG antibody in sera from patients with hepatitis E. The reactivity of the rHEV ORF2 against 5 HEV ORF2 monoclonal antibodies (McAbs) was also tested. RESULTS: The minimum concentration of coated antigen with which HEV IgG could be detected was 12.5 ng/ml, while the highest serum dilution to detect both IgM and IgG antibodies against HEV was 1:5 120. No cross-reaction was found with sera from patients with any other types of hepatitis. The 37 degree C acceleration test showed that the rORF2 was highly stable within 12 months at 4 degrees C. The 5 HEV ORF2 McAbs showed better reaction with the rORF2 from P. pastoris, especially that 4B2, 2E2, whose reaction against the rORF2 were 125 and 25 times respectively higher than that of rORF2 from E.Coli. CONCLUSION: There may be more extensive conformational epitopes in the rHEV ORF2 from P. pastoris. The excellent antigenicity, sensitivity and stability suggest that it can be served as a new candidate antigen for the development of diagnostic reagents of hepatitis E.

[Expression and antigenicity analysis of hepatitis G virus NS5 gene].

BACKGROUND: To determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli. METHODS: HGV NS5a,NS5b and core/NS5b fusion genes were cloned into pThioC vector. Three expression plasmids were transformed into JM109(DE3) competent cells then expressed with induction by IPTG. Western blot and ELISA were used to determine the antigenicity after the three recombinant proteins were purified. RESULTS: After identification by restriction enzyme and sequencing, it was confirmed that the expressed was target proteins espected. Purified expression proteins were found strongly immunoreactive among anti HGV positive sera by Western blot and ELISA. Compared with mixed recombinant antigen (including core, NS5a synthetic peptide and NS3 recombinant proteins), in the 22 positive sera detected with mixed antigen, 68%(15/22), 90%(20/22) and 73%(16/22) were positive by P5a,P5b and Pc?5b antigens; In the 70 negative samples with mixed antigen, 7%(5/70), 1%(1/70) and 6%(4/70) were positive by P5a, P5b and Pc?5b antigens. The positive alone was found among RTPCR positive specimen using these recombinant antigens. CONCLUSIONS: NS5 gene expressed in E.coli?which couldn't be covered with other regions of antigens was one of the essential epitopes to HGV immunologic diagnosis.

[Expression of thermal stable, soluble hepatitis E virus recombinant antigen].

BACKGROUND: To obtain thermal stable, soluble, biologically active hepatitis E virus recombinant antigen using thioredoxin fusion expression system. METHODS: HEV ORF2 gene fragment (6964-7126 nt) was inserted into thioredoxin fusion expression vector pThioHisA. The recombinant plasmid was transformed into E. coli BL21 strain. After induction with IPTG, cells were lysed and the supernatant was subjected to 80 degree treatment for 10 minutes. After centrifugation, the supernatant was tested by ELISA. RESULTS: SDS-PAGE analysis showed the thioredoxin. HEV fusion protein was highly expressed and was thermally stable, soluble. HEV specific ELISA confirmed this fusion protein possessing HEV specific antigenicity. CONCLUSIONS: Using thioredoxin fusion expression system, a soluble, thermal stable, biologically active HEV recombinant antigen was successfully expressed.

[Immunogenicity of recombinant HEV ORF2 protein expressed in pichia pastoris].

BACKGROUND: To study to immunogenicity of recombinant HEV ORF2 protein expressed in pichia pastoris. METHODS: BALB/c mice were immunized with the recombinant HEV ORF2 protein. The ability of antiserum to bind HEV was tested using affinity-capture reverse transcription polymerase chain reaction (RT-PCR). Moreover, the recombinant protein was used to immunize BALB/c mice by different routes with different adjuvants. Serum conversion rate of anti-HEV antibody and the ELISA titer were detected. RESULTS: The antiserum could capture native HEV for RT-PCR. As to the immunization effect, the immune response by intramuscular route was better than that of the intraperitoneal route. The protein with alum and CpG adjuvant could elicits more significant immune responses than using the alum adjuvant alone. The best way was to immunize with the protein with alum and CpG adjuvant by intramuscular route with a boosted injection on the 4th week after the first immunization. The ED50 was 0.023 microgram. This is the first report that the antibody elicited by recombinant HEV ORF2 protein expressed in pichia pastoris recognizes native HEV. High immunogenicity of this kind of ORF2 was also demonstrated by inducing strong immune response in mice with good ED50 result. CONCLUSIONS: The high immunogenicity of this kind of HEV ORF2 may make a foundation for the development of new type of hepatitis E vaccine.

The coexpression of the preS1 (1-42) and the core (1-144) antigen of HBV in E. coli.

OBJECTIVE: To study the therapeutic T cell vaccine for the treatment of chronic hepatitis B by improving the cellular immunization of HBsAg vaccine with the coexpression of the preS1 (1-42) and the Core (1-144) antigen of HBV in E. coli. METHODS: The genes of HBcAg (1-144) and preS1 (1-42) were amplified and fused by PCR. This fused gene was inserted in the prokaryotic expression vector pET-11d and expressed in E. coli. RESULTS: It was showed by SDS-PAGE that the protein molecular weight of the coexpression product was about 20 kD, 20% of all bacteria protein. The monoclonal antibodies against core and preS1 antibody could react with this fused protein by Western-blot technique respectively. The fused gene was verified by sequencing. Under the immune electron microscopy, this fused protein is typical particle of HBcAg but in an aggregated form. CONCLUSION: The results might aid for studying T cell immunotherapeutic vaccine for chronic hepatitis B.