Improvement of baculovirus expression system and purification of IL-6 protein expressed in insect cells / 生物工程学报

Based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (Bacmid) which propagated in Escherichia coli, the Bac-to-Bac System provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins. And the efficiency of recombinant baculovirus infecting cells plays an important role on the protein expression. In this study, we introduced an EGFP expression cassette driven by polyhedrin promoter into the p74 locus of Bacmid by homologous recombination. The target Bacmid-egfp was then transformed into E. coli DH10B containing the transposition helper plasmid to gain a new transposition receipt strain E. coli DH10Bac-egfp. Because of the intact attTn7 sites and lacZ', target gene cloned in a pFastBac vector can be transposed into the Bacmid-egfp shutter vector to construct recombinant baculovirus, which would allow the tracing of the target protein expression and the recombinant Bacmid transfection or recombinant baculoviral infection under fluorescence microscopes. Recombinant virus Bac-egfp-DsRed was constructed by transposing DsRed into the Bacmid-egfp in E. coliDHl0Bac-egfp, and the Sf9 cells infected with the recombinant virus expressed DsRed and EGFP efficiently. Another protein IL-6 fused with 6 x his tag was expressed and purified sucessfully from Sf9 cells infected with recombinant virus Bac-egfp-6 x his-IL6 constructed by the improved Bac-to-Bac system.

Establishment of stable expression cell lines for HBsAg variants and analysis of antigenicity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the mechanism of hepatitis B virus infected patients who is negative for HbsAg.</p><p><b>METHODS</b>DNA sequences of 46 patients were analyzed. In these patients, HBsAg was negative but HBV DNA was positive and six new HBsAg variants were identified. Four of the six variants were combined point mutants and two were insertion variants. These S genes were subcloned into eukaryotic expression vector EBO-plpp, and the recombinant eukaryotic expression plasmids were transfected into COS7 cells. Cell lines expressing mutant type HBsAg were obtained. The supernatants were detected by ELISA and RIA.</p><p><b>RESULTS</b>Only the two-amino acid-insertion variants could be detected and the others failed to react with polyclonal and monoclonal antibodies against HbsAg.</p><p><b>CONCLUSION</b>The results indicated that the point mutations and insertions may result in a conformational change of the S gene, which affect HBsAg antigenicity, suggesting a possible relationship between the variants and the negative conversion of HBsAg of the patients.</p>