利用慢病毒包装小窝蛋白-1过表达稳定细胞株的构建  

Construction of cell line for stable overexpression of Caveolin-1 by lentivirus packaging

作　　者：刘杨 许淑娟 李琼毅[1,2] 刘翊忠 LIU Yang;XU Shu-juan;LI Qiong-yi;LIU Yi-zhong(Key Bio-engineering and Technology Laboratory,Biomedical Research Center of the Northwest Minzu University,Lanzhou 730030,Gansu Province,China)

机构地区：[1]西北民族大学生物医学研究中心生物工程与技术国家民委重点实验室,甘肃兰州730030 [2]西北民族大学生命科学与工程学院,甘肃兰州730030

出　　处：《中国生物制品学杂志》2022年第2期164-169,共6页Chinese Journal of Biologicals

基　　金：甘肃省教育厅项目(2018B-018);中央高校基本科研业务项目(31920180123);西北民族大学引进人才科研启动项目(xbmuyjrc-201627).

摘　　要：目的利用慢病毒包装构建过表达小窝蛋白-1(Caveolin-1)的稳定细胞株。方法PCR扩增仓鼠肾细胞BHK-21中Caveolin-1基因,克隆至pTRIP-CMV载体中,构建重组表达质粒pTRIP-Cav1,将其与对照质粒pTRIP-EGFP分别经LipofectamineTM 2000介导转染HEK-293T细胞,收集包装的慢病毒并转导至BHK-21细胞,构建过表达Caveolin-1的细胞株BHK-Cav1及过表达绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的对照细胞株BHK-EGFP。Western blot、qRT-PCR、免疫荧光法验证细胞株的稳定性及过表达特性,同时检测细胞活力。结果经双酶切及测序鉴定,重组表达质粒pTRIP-Cav1构建正确。与BHK-21细胞比较,各代次BHK-EGFP细胞中EGFP荧光明显,BHKCav1细胞中Caveolin-1蛋白表达水平增加,mRNA水平显著增加(P<0.01),且表达水平不随细胞代次的增长而改变。BHK-Cav1和BHK-EGFP细胞活力与BHK-21细胞比较,差异均无统计学意义(P均>0.05)。结论成功构建了Caveolin-1过表达细胞株BHK-Cav1,其具有较好的稳定性,为下一步研究病毒通过Caveolin依赖型内吞途径感染BHK-21细胞奠定了基础。Objective To construct a cell line stably expressing Caveolin-1 by lentivirus packaging.Methods The Caveolin-1 gene was amplified by PCR from BHK-21 cells and cloned into vector pTRIP-CMV.The constructed recombinant plasmid pTRIP-Cav1 and control vector pTRIP-EGFP were transfected to HEK-293T cells in mediation of LipofectamineTM 2000 respectively.The packaged lentivirus was collected and transduced into BHK-21 cells to construct the cell line BHK-Cav1 for stably overexpressing Caveolin-1 and control cell line BHK-EGFP for overexpressing enhanced green fluorescent protein(EGFP).The cell line was verified for stability and overexpression characteristics by Western blot,qRT-PCR and immunofluorescence assay(IFA),and determined for viability.Results Restriction analysis and sequencing proved that recombinant plasmid pTRIP-Cav1 was constructed correctly.Compared with that in BHK-21 cells,obvious expression of EGFP was observed in BHK-EGFP cells of various passages.The expression levels of Caveolin-1 mRNA and protein in BHK-Cav1 cells increased significantly(P<0.01),while showed no change with the increasing passages.However,the viabilities of BHK-Cav1 and BHK-EGFP cells showed no significant difference with that of BHK-21 cells(each P>0.05).Conclusion BHK-Cav1 cell line for overexpressing Caveolin-1 was constructed,which showed high stability and laid a foundation of further study on virus infection of BHK-21 cells through Caveolin-dependent endocytic pathway.

关 键 词：小窝蛋白-1 慢病毒 仓鼠肾细胞BHK-21 细胞株 稳定性 

分 类 号：R392[医药卫生—免疫学]