小鼠resistin基因慢病毒表达载体构建及在KMB17细胞中的表达

Construction of recombinant mice resistin gene lentivirus and its expression in KMB17 cells

[目的]构建携带小鼠resistin基因的慢病毒表达载体,并高效转导靶细胞。[方法]从小鼠脂肪组织提取总RNA,经PCR扩增、酶切、连接、转化后构建重组质粒;通过293T细胞包装为重组慢病毒颗粒,然后感染KMB17细胞,检测小鼠resistin基因在靶细胞中的表达。[结果]PCR扩增到预期的342 bp大小的resistin基因目的片段;构建的重组质粒经酶切得到了342 bp大小的目的片段,测序鉴定为小鼠resistin基因序列;重组慢病毒包装可观察到很强的绿色荧光,说明具有较高的质粒转染效率;重组慢病毒感染KMB17后可检测到小鼠resistin基因转录水平和蛋白水平的表达,证明resistin基因得到有效转导并可在靶细胞中高水平表达。[结论]成功构建了携带小鼠resistin基因的慢病毒表达载体并可在KMB17靶细胞中获得高效表达。

[Objective]To construct recombinant lentiviral vector-mediated delivery of mice resistin gene and to efficiently transduce the plasmid to target cells. [Methods]Total RNA was extracted from mice＇s adipose tissues and converted into complementary DNA. Recombinant lentiviral vector was conducted after PCR amplification,enzyme digestion,plasmid connection and transformation. The recombinant lentiviral vector was transfected in 293 T cells to package virus. And then the packaged virus was infected KMB17 cells and mice resistin gene level and protein level were detected in cells. [Results]The expected fragment of resistin gene about 342 bp was obtained by PCR amplification. Recombinant plasmids were identified by enzyme digestion and sequencing. The high plasmid transfection efficiency was illustrated by detecting a strong green fluorescence of e GEP in the recombinant lentiviral packaging process. The expression of resistin gene and its protein could be detected in infected KMB17 cells to illustrate that resistin gene was transduced and transcribed efficiently in target cells. [Conclusion]Recombinant lentiviral vector expressing mice resistin gene was constructed successfully and transfected the target cells efficiently to express resistin protein.