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制备嵌合HCV多中和表位及HCV包膜蛋白E2的HBV S抗原病毒样颗粒(virus-like particle,VLP),并进行鉴定、纯化和浓缩。HEK293T细胞用DMEM培养至90%密度时,将构建的重组真核表达载体pCI-MEpS、pCI-E2S用脂质体法转染HEK293T细胞,48h后在培养上清中得到自我装配的嵌合HCV多中和表位及包膜蛋白E2的HBV病毒样颗粒(VLP-MEpS,VLP-M2S)。蔗糖密度梯度离心,透析浓缩后,电化学发光法进行HBsAg定量测定、电镜分析和Western blotting鉴定。制备出的嵌合病毒样颗粒VLP-MEpS和VLP-E2S经浓缩后其HBsAg定量最高达到3×10~4 ng/mL。实验表明我们成功制备出嵌合有HCV多中和抗原表位的HBV VLP,为进一步分析诱导的中和抗体研究奠定基础。
HBV S antigen-like particle (VLP), a chimeric HCV multi-neutralizing epitope and HCV envelope protein E2, was prepared and identified, purified and concentrated. When HEK293T cells were cultured in DMEM to 90% density, the recombinant eukaryotic expression vectors pCI-MEpS and pCI-E2S were transfected into HEK293T cells by lipofectamine. After 48h, self-assembled chimeric HCV Multiple neutralizing epitopes and envelope protein E2 HBV virus-like particles (VLP-MEpS, VLP-M2S). Sucrose density gradient centrifugation, dialysis concentration, the electrochemical luminescence method for quantitative determination of HBsAg, electron microscopy and Western blotting identification. The prepared chimeric virus-like particles VLP-MEpS and VLP-E2S after concentration of HBsAg up to 3 × 10 ~ 4 ng / mL. Experiments show that we successfully prepared HBV VLP chimeric HCV multi-neutralizing epitopes, which lay the foundation for the further analysis of induced neutralizing antibodies.