CLEC4M慢病毒载体的构建及其在K-562细胞中的表达  

Construction of lentiviral vector encoding CLEC4M and overexpression of CLEC4M in K- 562 cells

作　　者：王媛媛[1] 聂青和[1] 朱婷[1] 

机构地区：[1]第四军医大学唐都医院传染科,西安710038

出　　处：《临床肝胆病杂志》2014年第6期518-521,共4页Journal of Clinical Hepatology

基　　金：国家自然科学基金资助项目(81170390)

摘　　要：目的本研究拟构建CLEC4M慢病毒表达载体,建立稳定过表达的K-562细胞株。方法应用逆转录PCR克隆出正常人CLEC4M基因序列,将序列插入GV166载体,构建GV166-CLEC4M慢病毒表达载体,之后转染293T细胞进行慢病毒包装。用获得的慢病毒液感染人白血病细胞系K-562,通过实时荧光定量PCR和Western Blot检测K-562细胞中CLEC4M的过表达情况。结果测序结果显示成功构建重组慢病毒表达质粒GV166-CLEC4M;依据实时荧光定量PCR可测定慢病毒能够有效感染K-562细胞;CLEC4M在K-562细胞中的mRNA和蛋白水平都成功的过表达。结论 CLEC4M慢病毒表达载体的构建,为进一步研究CLEC4M基因在HCV入胞的分子机制奠定了基础。Objective To construct the lentiviral vector encoding CLEC4M and prepare K -562 cells with stable overexpression of CLEC4M.Methods The gene sequence of normal CLEC4Mwas cloned by reverse transcription PCR and then inserted into GV166 vector to construct GV166-CLEC4Mlentiviral expression vector,and then lentiviral packaging was performed by transfection of293T cells.The ob-tained lentiviral liquid was used to infect human leukemia cell line K-562.Real-time PCR and Western blot were used to detect the over-expression of CLEC4M in K-562 cells.Results Sequencing showed that the recombinant lentiviral expression plasmid GV166-CLEC4M was successfully constructed.Lentiviruses could efficiently infect K-562 cells,according to real-time PCR.CLEC4Mwas successfully o-ver-expressed in K-562 cells at mRNA and protein levels.Conclusion The construction of lentiviral vector encoding CLEC4M lays a foundation for further study of CLEC4M gene involved in HCV entry into host cells.

关 键 词：丙型肝炎病毒 慢病毒感染 树突细胞 

分 类 号：R512.63[医药卫生—内科学]