小鼠11β- HSD1基因真核表达载体的构建及应用

Construction and role of mouse PLJM1-11β-HSD1-GFP plasmid

摘要:

构建小鼠11β-羟类固醇脱氢酶（11β- HSD1）基因的慢病毒真核表达载体PLJM1-11β-HSD1-GFP，建立小鼠前体脂肪细胞（3T3-L1）高表达11β-HSD 1 基因的稳定感染细胞株，为11β- HSD1基因的功能研究奠定基础。方法利用逆转录聚合酶链反应（RT-PCR）从小鼠肝脏cDNA 中扩增11β-HSD1开放阅读框ORF，构建针对11β-HSD 1 基因的真核表达载体，转化感受态DH5α菌株，抽提质粒并进行测序鉴定。用测序成功的质粒和慢病毒包装质粒共同转染293T细胞，产生慢病毒并转染3T3-L1 细胞。根据绿色荧光效率挑取单克隆细胞团筛选出稳定细胞株，通过Western blot鉴定PLJM1-11β-HSD1-GFP转染成功。结果经酶切、PCR 及测序验证，PLJM1-11β-HSD1-GFP 真核表达载体构建成功，并包装慢病毒，绿色荧光效率90%以上，并利用其慢病毒悬液成功感染3T3-L1 细胞，筛选出的稳定感染细胞株的3T3-L1细胞成功高表达11β-HSD1 蛋白。结论成功构建了11β-HSD 1 基因的真核表达载体，并建立高表达11β-HSD 1 的稳定感染细胞株PLJM1-11β-HSD1-GFP-3T3-L1，为进一步研究11β-HSD 1 基因的功能，特别是在肥胖中的研究奠定了基础。

Abstract:

To construct a lentiviral vector of PLJM1-11β-HSD1-GFP and establish a stable cell line of 3T3-L1 with high expression of mouse gene, and lay the foundation for the research of gene. Methods Open reading frame (ORF) fragment of mouse gene was amplified from mouse liver tissues by RT-PCR, then inserted into the PLJM1-NRG1-GFP vector and transformed into competent DH5α strain of Escherichia coli. The plasmid was extracted and used for sequencing. The successfully -sequenced plasmid PLJM1-11β-HSD1-GFP and the packaging plasmid were transfected to 293T cell line to produce recombinant lentivirus. 3T3-L1 cell line was transfected by recombinant virus and the stable cell line was isolated by selecting the single clone with GFP, and high expression of 11β-HSD1 was identified by Western blot. Results The eukaryotic expression vector of PLJM1-11β-HSD1-GFP was constructed and confirmed by DNA sequencing. The lentivirus vector of named PLJM1-11β-HSD1-GFP was successfully constructed and the virus was packaged in 293T cells. 3T3-L1 cells were transfected by recombinant virus and the stable cell line was selected by green fluorescence efficiency, which showed that the lentivirus vector transfection rate was over 90%. A dramatically elevated protein level of 11β-HSDl was expressed in the positive clones of 3T3-L1 cells compared with the control group. Conclusions Mouse PLJM1-11β-HSD1-GFP plasmid has been successfully constructed. The 3T3-L1 cellstransfected by PLJM1-11β-HSD1-GFP could effectively elevate the expression of gene, and can be used in the functional researches of mouse gene, especially those related to obesity.