人GINS2基因重组腺病毒表达质粒的构建及鉴定

目的利用AdEasy系统构建携带人GINS2基因的重组腺病毒表达质粒,并进行鉴定。方法以pcDNA3.1-GINS2质粒为模板,PCR扩增GINS2基因,克隆至穿梭质粒pAdTrace-TO4中,构建重组腺病毒穿梭质粒pAdT-GINS2,经双酶切及测序鉴定正确的阳性质粒与骨架质粒pAdEasy-1同时转化感受态大肠杆菌BJ5183,经同源重组获得重组腺病毒质粒pAdE-GINS2,转染HEK293细胞,包装出重组腺病毒Ad-GINS2,大量扩增后,测定病毒滴度,进行PCR鉴定。Western blot法检测重组腺病毒感染的人HL60细胞中GINS2蛋白的表达,MTT法检测重组腺病毒感染对HL60细胞增殖的影响。结果重组腺病毒穿梭质粒pAdT-GINS2经双酶切及测序证明构建正确;经酶切鉴定证明重组腺病毒质粒pAdE-GINS2构建正确;经PCR鉴定证明重组腺病毒Ad-GINS2包装成功,经3轮扩增后病毒滴度可达1.35×1012IU/ml;与空载体感染及未感染的HL60细胞相比,重组腺病毒感染的HL60细胞内GINS2蛋白的表达及细胞增殖水平均明显升高(P<0.05)。结论已成功构建携带人GINS2基因的重组腺病毒表达质粒,感染人HL60细胞后可促进其增殖,为进一步研究该基因在急性髓细胞白血病发生发展中的作用奠定了基础。

Construction and identification of recombinant adenovirus vector carrying human GINS2 gene

Objective To construct and identify a recombinant adenovirus vector carrying human GINS2 gene by using AdEasy system.Methods GINS2 was amplified by PCR using plasmid pcDNA3.1-GINS2 as a template,and cloned into shuttle plasmid pAdTrace-TO4.The constructed recombinant adenovirus shuttle plasmid pADT-GINS2 was identified by restriction analysis and sequencing,then co-transformed to competent E.coli BJ5183 with backbone plasmid pAdEasy-1. The recombinant adenovirus plasmid pAdE-GINS2 obtained by homologous recombination was transfected to HEK293 cells for packaging.The obtained recombinant adenovirus Ad-GINS2 was propagated in a large quantity,determined for virus titer and identified by PCR.The expression of GINS2 in HL60 cells infected with Ad-GINS2 was determined by Western blot,while the effect of recombinant adenovirus infection on proliferation of HL60 cells by MTT method.Results Restriction analysis and sequencing proved that recombinant adenovirus shuttle plasmid pAdT-GINS2 was constructed correctly.Restriction analysis proved that recombinant adenovirus plasmid pAdE-GINS2 was constructed correctly.Recombinant adenovirus Ad-GINS2 was packaged successfully as proved by PCR,which reached a titer of 1.35×10 12 IU/ml after three cycles of propagation.The GINS2 expression and proliferation levels of HL60 cells infected with Ad-GINS2 increased significantly as compared with those infected with empty vector and those uninfected(P < 0.05).Conclusion Recombinant adenovirus vector carrying human GINS2 gene was successfully constructed,which promoted the proliferation of HL60 cells.It laid a foundation of further study on the role of GINS2 gene in onset and progress of acute promyelocytic leukemia.