CLEC4M慢病毒载体的构建及其在K-562细胞中的表达

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...

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Bibliographic Details
Published in临床肝胆病杂志 Vol. 30; no. 6; pp. 518 - 521
Main Author 王媛媛 聂青和 朱婷
Format Journal Article
LanguageChinese
Published 第四军医大学唐都医院 传染科,西安,710038 2014
Subjects
丙型肝炎病毒
慢病毒感染
树突细胞
丙型肝炎病毒
树突细胞
hepertitis C virus
dendritic cells
慢病毒感染
lentivirus infections
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Summary: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。
Bibliography:hepertitis C virus;lentivirus infections;dendritic cells
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 ho
ISSN:1001-5256
DOI:10.3969/j.issn.1001-5256.2014.06.010