The coexpression of the preS1 (1-42) and the core (1-144) antigen of HBV in E. coli / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The results might aid for studying T cell immunotherapeutic vaccine for chronic hepatitis B.</p>

Expression and antigenicity analysis of hepatitis G virus NS5 gene / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Expression of thermal stable, soluble hepatitis E virus recombinant antigen / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To obtain thermal stable, soluble, biologically active hepatitis E virus recombinant antigen using thioredoxin fusion expression system.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>Using thioredoxin fusion expression system, a soluble, thermal stable, biologically active HEV recombinant antigen was successfully expressed.</p>

Immunogenicity of recombinant HEV ORF2 protein expressed in pichia pastoris / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study to immunogenicity of recombinant HEV ORF2 protein expressed in pichia pastoris.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>The high immunogenicity of this kind of HEV ORF2 may make a foundation for the development of new type of hepatitis E vaccine.</p>