小鼠miR-122重组慢病毒载体的构建和鉴定

Construction and identification of miR-122 recombinant slow-virus carrier in mice

目的构建携带miR-122的重组慢病毒表达载体,为研究miR-122的功能和作用机制奠定基础。方法从小鼠基因组DNA采用PCR法扩增出pre-miR-122基因,测序后克隆至pCDH-CMV-MCS-EF1-copGFP逆转录病毒载体上,测序鉴定,病毒包装后,转染NIH3T3细胞,并通过绿色荧光蛋白进行病毒滴度测定。将重组慢病毒感染肝前体细胞(HPCs),利用实时定量PCR检测miR-122的表达。结果重组慢病毒载体pCDH-CMV-miR-122-EF1-copGFP经PCR扩增及测序鉴定正确,并得到了滴度为(1～5)×106IU/mL的病毒液。pCDH-CMV-miR-122-EF1-copGFP感染的肝前体细胞中miR-122表达显著增强。结论成功构建了小鼠miR-122重组慢病毒,并在肝前体细胞中高效表达出miR-122,为进行miR-122的功能和机理奠定了良好的基础。

Objective To construct the restructuring lentiviral expression vector that carries miR-122, and lay a foundation for us to study the functions and mechanisms of miR-122. Methods The pre-miR-122 gene was amplified from mouse genomic DNA by PCR, which was cloned to the retroviral vectors of pCDH-CMV-MCS-EF1-copGFP and sequenced. After viral packaging, the NIH3T3 cells were transfected with recombinant lentiviral vector. The titer of virus was detected by green fluorescent protein determination. Finally the expression of miR-122 of hepatic progenitor cells （HPCs） infected with recombinant lentiviral vector was analyzed by using real-time quantitative PCR. Results PCR and DNA sequencing assays demonstrated that the recombinant lentiviral vector of pCDH-CMV-miR-122-EF1-copGFP was successfully constructed, and the titer of concentrated virus was （1-5）×106 IU/mL. The level of miR-122 expression in HPCs was significantly increased after infection with pCDH-CMV-miR-122-EF1-copGFP. Conclusion Mouse miR-122 recombinant lentiviral vector： pCDH-CMV-miR-122-EF1-copGFP was successfully constructed, which resulted in high miR-122 expression in HPCs. Our results lay a good foundation to explore the functions and mechanisms of miR-122.