Expression of human IL-12 in mammalian cell and study on its biological activities / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>rhIL-12 have good biological activities, it can stimulate activation and proliferation of T cells and induce production of IFN-gamma.</p>

Influence of the mutants of hepatitis B surface antigen on the cell immunity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the influence of the mutants of hepatitis B surface antigen on the cell immunity.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Baculovirus expression of two human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Antigencity identification of recombinant hepatitis E virus ORF2 protein expressed in Pichia pastoris / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To determine the antigenicity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P. pastoris).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Antigenic analysis of the common recombinant mutant types of HBsAg / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>