大鼠HSG基因重组腺病毒载体的构建及鉴定

摘要

目的 构建大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)的腺病毒载体,观察其在转染大鼠C6胶质瘤细胞后的表达.方法 由前期构建的pGM-T-rHSG质粒中酶切rHSG基因片段,亚克隆入 pShuttle-CMV-EGFP重组穿梭载体中,而后与腺病毒骨架质粒pAdxsi酶切连接,经过抗性筛选及酶切鉴定得到阳性的pAdxsi-GFP-rHSG重组质粒；pAdxsi-GFP-rHSG质粒经PacI线性化后转染293细胞,包装重组腺病毒,进行PCR鉴定和PCR产物测序鉴定,Westem-blot法检测C6胶质瘤细胞转染rHSG后的表达效果.结果 酶切鉴定证实rHSG基因正确插入穿梭载体pShuttle-CMV-EGFP,收获病毒后的PCR及测序鉴定pAdxsi-GFP-rHSG重组成功,并能在C6胶质瘤细胞内高效表达.结论 成功构建出了介导大鼠增殖抑制基因的腺病毒载体pAdxsi-GFP-rHSG,能在C6胶质瘤细胞内高效表达.%Objective To construct a recombinant adenovirs encoding rat hyperplasia suppressor gene (rHSG) and to investigate the the expression of it on rat C6 brain glioma cells.Methods The rHSG gene was extracted by enzymaticcuting the used pGM-T-rHSG and was identified in previous experiment.It was subcloned in the pShuttle-CMV-EGFP plasmid.The rHSG gene was transferred from pShuttle-rHSG to pAdxsi viral DNA by double enzymatcuting obtaining the recombinant plasmid pAdxsi-GFP-rHSG.The recombinant pAdxsi-GFP-rHSG was selected by kansmycin resistanc and restriction endonuclease; After linearized by Pac Ⅰ,the recombinant adenovirus DNA pAdxsi-GFP-rHSG was transfected into 293 cells for packaging and amplification.Adxsi-GFP-rHSG was further identified by PCR analysis and PCR products sequencing analysis.The expression was detected by Western-blot analyses with rat C6 brain glioma.Results Restriction enzyme digestion confirmed that rHSG gene was correctly inserted into pShuttle-CMV-EGFP.After being packaged in 293 cells,the recombinant adenovirus Adxsi-GFP-rHSG was identified by PCR analysis and DNA sequencing analysis.The cells transfected with Adxsi-GFP-rHSG resulted in positive expressing the rHSG protein.Conclusion Recombinant adenovirus encoding rat hyperplasia suppressor gene (rHSG) Adxsi-GFP-rHSG was successfully constructed.The expression on rat C5 brain glioma cells was more effective.It wil be helpful to use it in the research of the role rHSG specific therapy in the spongioblastoma tissue.