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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。
Objective To construct recombinant adenovirus expressing human GINS2 gene using AdEasy system and identify it. Methods The pcDNA3.1-GINS2 plasmid was used as a template to amplify the GINS2 gene by PCR and cloned into the shuttle plasmid pAdTrace-TO4 to construct the recombinant adenovirus shuttle plasmid pAdT-GINS2. The correct positive plasmid and backbone plasmid were identified by double enzyme digestion and sequencing pAdEasy-1 was transformed into competent E. coli BJ5183 at the same time. Recombinant adenovirus plasmid pAdE-GINS2 was obtained by homologous recombination and transfected into HEK293 cells. The recombinant adenovirus Ad-GINS2 was packaged and amplified. The virus titer was determined and PCR Identification. Western blot was used to detect the expression of GINS2 protein in HL60 cells infected with recombinant adenovirus and MTT assay was used to detect the effect of recombinant adenovirus on the proliferation of HL60 cells. Results Recombinant adenovirus shuttle plasmid pAdT-GINS2 was verified by double enzyme digestion and sequencing. The recombinant adenovirus plasmid pAdE-GINS2 was constructed correctly by restriction enzyme digestion. The recombinant adenovirus Ad-GINS2 was successfully packaged and identified by PCR. The titer of the recombinant adenovirus was 1.35 × 1012 IU / ml after amplification. Compared with the non-infected and non-infected HL60 cells, the expression of GINS2 and the cell proliferation of HL60 cells were significantly increased (P < 0.05). Conclusions The recombinant adenovirus plasmid carrying human GINS2 gene has been successfully constructed, which can promote the proliferation of human HL-60 cells, which lays a foundation for further study on the role of this gene in the development of acute myeloid leukemia.