靶向Bi-1基因的重组慢病毒载体构建及其在NIH3T3细胞中的表达

Construction of recombinant lentiviral expression vector targeting human Bax inhibitor-1 gene and its expression in NIH3T3 cells

目的慢病毒载体技术已被广泛应用于基因功能分析、信号转导通路和基因治疗的研究。文中应用慢病毒系统构建凋亡抑制基因Bi-1的慢病毒表达载体并检测其在NIH3T3细胞中的表达,为后续的以Bi-1基因为靶点的肿瘤基因治疗研究奠定基础。方法根据慢病毒表达载体酶切位点的特征序列设计PCR扩增引物并扩增人Bi-1 cDNA,并采用DNA重组技术定向克隆至pLCMV-IG质粒中,构建重组慢病毒载体质粒pLCMV-IG-Bi-1,经PCR、双酶切和测序鉴定后,包装慢病毒感染至小鼠成纤维细胞株NIH3T3中,RT-PCR及Western-blot分别检测NIH3T3细胞中Bi-1基因和蛋白的表达。结果 PCR、酶切及测序结果表明重组慢病毒载体构建成功;NIH3T3细胞感染重组载体包装的慢病毒后出现明显的Bi-1基因高表达。结论成功构建靶向Bi-1基因的重组慢病毒载体,为进一步研究Bi-1基因对细胞生长转化的影响提供了实验依据。

Objective The technigne of lentivirus vector has been widely used in gene functional analysis,studies on signal transduction pathway and gene therapy.The aim of the work was to construct the lentiviral expression vector targeting anti-apoptosis gene Bax inhibitor-1 gene（Bi-1）using the lentivirus-mediated expression system and express Bi-1 in NIH3T3 cells so as to pave a way for Bi-1 gene-targeted gene therapy of tumor.Methods The human Bi-1 gene was amplified by PCR with specific primers,which were designed according to the characteristic sequences of restriction enzyme cutting site in lentivirus expression system,and then cloned into the vector pLCMV-IG using DNA recombinant technique.After the inserted sequences in the recombinant plasmids were identificated by PCR,double enzyme digestion and DNA sequencing analysis,the recombinant lentivirus were packaged and injected into NIH3T3 cells,the Bi-1 mRNA and protein expression were examined by RT-PCR and Western blot.Results PCR,double enzyme digestion analysis and DNA sequencing confirmed that the Bi-1 DNA sequences were successfully inserted into the lentiviral vectors.After transfection with the recombinant lentivirus,Bi-1 expression in NIH3T3 cells was significantly increased at both mRNA and protein levels compared with that in non-transfected and control vector transfected NIH3T3 cells.Conclusion The lentivial expression vector of Bi-1 gene have been successfully constructed,which will be useful to further investigate the role of Bi-1 in cell growth and transformation.