携带人OCT4A基因的慢病毒载体的构建及其稳定转染K562白血病细胞株的建立

本研究旨在构建共表达人OCT4A与绿色荧光蛋白（GFP）慢病毒载体pLVX-OCT4A-ZsGreen1,并通过感染获得稳定表达重组质粒的白血病K562细胞系,观察OCT4A在其中的表达。根据Gen Bank数据库提供的OCT4A mRNA序列,合成编码mRNA基因的特异性引物序列,通过RT-PCR技术扩增出OCT4A全长片段,克隆至pCR-Blunt载体。将OCT4A DNA片段酶切回收后克隆至经EcoR1酶切线性化的慢病毒载体pLVX-IRES-ZsGreen1,构建pLVX-OCT4A-ZsGreen1慢病毒重组质粒,利用测序鉴定所构建的重组载体是否正确。经三质粒包装系统包装后获得高滴度慢病毒颗粒并感染人白血病细胞系K562,通过流式细胞仪分选获得稳定表达的白血病细胞系,应用Real-time PCR和Western blot方法分别检测稳定转染细胞系的OCT4A mRNA和OCT4A蛋白表达。应用CCK-8法和平板细胞克隆形成试验测定OCT4A对K562细胞增殖能力的影响。结果表明,经限制性内切酶检测及基因测序证实成功构建了携带OCT4A基因的重组慢病毒;包装病毒颗粒超速离心后病毒滴度达（1.43±0.25）×10^8U/ml;病毒感染K562细胞后经流式细胞仪分选获得GFP＋细胞,实时定量PCR及Western blot证实病毒感染后OCT4A基因及蛋白的表达可有效上调;CCK-8法及平板集落形成试验显示病毒感染的K562细胞体外增殖活性明显升高,与对照组相比均有统计学差异（P〈0.05）。结论：成功构建了表达OCT4A基因的慢病毒载体,并且获得可稳定上调OCT4A表达的K562细胞株。

Construction of Lentiviral Vector Carrying Human OCT4A Gene and Establishment of Leukemic Cell Line K562 Stably Expressing OCT4A

This study was purposed to construct a lentiviral vector carrying human OCT4 A gene and green fluorescent protein（GFP）,and infect the leukemic cell line K562 observe the expression of OCT4 A in K562 cells.According to the sequence of OCT4 A mRNA which was found in GenBankthe special primer sequences were synthesized.The OCT4 A gene was amplified by RT-PCRand then cloned into the pCR-Blunt vector.The OCT4 A DNA fragment was subcloned into the lentiviral vector pLVX-IRES-ZsGreen1 which was restricted by EcoR1 to generate a lentiviral vector pLVX-OCT4AZsGreen1.The sequence of the recombinant plasmid was identified by DNA sequencing.Recombinant lentivirus was generated by co-transfection of three-plasmids into 293 FT cells using lipofectamine 2000 and transfected into K562 cells.Real-time PCR and Western blot were applied to detect the expression of OCT4 A mRNA and proteinCCK-8 and colony formation assay were performed to evaluate the effects of OCT4 A on proliferation of K562 cells.The results showed that the recombinant lentiviral vector pLVX-OCT4AZsGreen1 was successfully constructed.The virus titers were（ 1.43 ± 0.25）× 10^8 U /ml.After infection of K562 cells with the lentivirusthe recombinant plasmid could stably up-regulate the expression of OCT4 A gene and protein according the real-time PCR and Western blot detection results.CCK-8 and colony formation assay showed that exogenous OCT4 A gene could significantly promote cell growth and the colony formation of K562 cells.It is concluded that the recombinant lentiviral vector pLVX-OCT4AZsGreen1 carrying human OCT4 A gene is successfully constructedK562 cells which stably up-regulates the expression of OCT4 A mRNA are obtainedthe results of this study provide fundamental basis for further study on mechanism of OCT4 A in human leukemia development.