Construction of lentiviral vector encoding CLEC4M and overexpression of CLEC4M in K- 562 cells
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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。Abstract: Objective To construct the lentiviral vector encoding CLEC4M and prepare K- 562 cells with stable overexpression of CLEC4M. Methods The gene sequence of normal CLEC4M was cloned by reverse transcription PCR and then inserted into GV166 vector to construct GV166- CLEC4M lentiviral expression vector, and then lentiviral packaging was performed by transfection of 293T cells. The obtained lentiviral liquid was used to infect human leukemia cell line K- 562. Real- time PCR and Western blot were used to detect the overexpression 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. CLEC4M was successfully over- 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.
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Key words:
- hepertitis C virus /
- lentivirus infections /
- dendritic cells
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