靶细胞表面HCV受体分子排列顺序对病毒入侵影响的研究

摘要

Objective To study the effect of HCV receptors′sequence on virus entry based on the two-dimensional structure and via tandem expression of HCV receptors on mouse hepatocytes.Methods The construced recombinant expression vectors pCDH-hLDLR-hSR-BⅠ-hCD81-GFP, pCDH-hLDLR-hCD81-hSR-BⅠ and pCDH-hCLDN-1-hOCLN-DsRed were cotransfected into 293FT cells with package vectors.The collected recombinant lentivirus expressing hCLDN-1-hOCLN was concentrated and attacked mouse hepatocytes.The transgenic mouse hepatocytes with tandem overexpression of CLDN-1 and OCLN were established after G418-selection.The transduced cells LSCCO/Hepa1-6 and LCSCO/Hepa1-6 were sorted via flow cytometry and puro-G418-selection after recombinant lentivirus expressing hLDLR-hSR-BⅠ-hCD81 and hLDLR-hCD81-hSR-BⅠattacked Hepa1-6 respectively.The infectivity of transduced mouse hepatocytes LSCCO/Hepa1-6 and LCSCO/Hepa1-6 to HCV was analyzed via direct-infection of serum-derived virus.Furthermore, the effect of HCV receptors′sequence on virus entry was studied.Results Both LSCCO/Hepa1-6 and LCSCO/Hepa1-6 enhanced HCV-cell binding.The transduced mouse hepatocytes LSCCO/Hepa1-6 had more HCV endocytosis.Conclusion SR-BⅠhas priority over CD81 in HCV entry in the early stage.%目的：通过在小鼠肝癌细胞表面串联表达HCV受体分子，在二维结构的基础上研究HCV受体分子的排列顺序对病毒入侵的影响，为研究HCV的早期感染机制奠定基础。方法将已构建的慢病毒表达载体pCDH-hLDLR-hSR-BⅠ-hCD81-GFP、pCDH-hLDLR-hCD81-hSR-BⅠ和pCDH-hCLDN-1-hOCLN-DsRed与包装质粒共转染包装细胞293FT，包装慢病毒，用收集浓缩的慢病毒攻击小鼠肝癌细胞系Hepa1-6，抗生素G418加压筛选得到串联表达人紧密链接区域分子CLDN-1和OCLN的转基因细胞株hCLDN-1-OCLN／Hepa1-6（CO／Hepa1-6）；重组慢病毒pCDH-hLDLR-hSR-BⅠ-hCD81-GFP和pCDH-hLDLR-hCD81-hSR-BⅠ攻击转基因细胞株CO／Hepa1-6，通过嘌罗霉素puro和G418双抗加压及流式细胞术，最终筛选获得5种分子hLDLR、hSR-BⅠ、hCD81、hCLDN-1和hOCLN共表达且具有不同受体排列顺序的转基因人源化小鼠肝癌细胞株LSCCO／Hepa1-6和LCSCO／Hepa1-6；利用HCV阳性血清直接感染方法分析LSCCO／Hepa1-6和LCSCO／Hepa1-6对HCV的易感性，研究HCV受体分子的排列顺序对病毒入侵的影响。结果两种细胞株LSCCO／Hepa1-6和LCSCO／Hepa1-6对HCV结合均有增加，但LSCCO／Hepa1-6细胞株细胞表面的HCV受体排列顺序更有利于HCV入侵。结论在二维结构的基础上，HCV在感染早期要优先与B族Ⅰ型清道夫受体SR-BⅠ作用。