嵌合有HCV多中和抗原表位的乙肝S抗原病毒样颗粒的制备与鉴定

Preparation and identification of virus-like particle expressing chimerized HBV S antigen with HCV multi-neutralizing epitopes

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。

Identification and purification after preparing virus-like particles chimerized HBV S antigen with HCV multi neutrali zing epitopes and envelop protein E2 were carried out in this study. HEK293T cells cultured in DMEM medium until 90% den sity were transfected with recombinant eukaryotic expressing vector pC1-MEpS and pCI-E2S using lipofectamin. Virus-like par ticles chimerized HBV S and HCV multi-neutralizing epitopes or envelop protein E2 were harvested from the supernatant after 48 hours. After sucrose density gradient centrifugation, dialysis and concentration, the chimeric VLPs were identified by Western blotting, electron microscopy, and quantitative determination of HBsAg by electrochemical luminescence assay. After transfeetion with recombinant pC1-MEpS and pCI-E2S in HEK293T cells, chimerical VLPs were confirmed by Western blotting, the highest value of HBsAg reached 30× 10^3 ng/mL after concentration as measured by electrochemical luminescence assay. Taken together, chimerized VLPs with HBV S and HCV multi-neutralizing epitopes or envelop protein E2 were successively pre pared, which provides the basis for studying the neutralizing antibody induced by chimerized VLPs.