含人p27基因的重组腺病毒的构建及鉴定

Construction and Identification of Recombinant Adenovirus Containing Human p27 Gene

目的:构建并鉴定含有人p27基因和EGFP基因的重组腺病毒载体,并观察其表达。方法利用腺病毒载体系统Adeno-X,通过质粒抽提、电泳、酶切、连接、转化等多种基因工程技术,构建重组腺病毒质粒pAdeno-p27-EGFP;酶切、PCR鉴定重组腺病毒质粒;利用293细胞包装重组腺病毒载体PAd-p27-EGFP,通过荧光倒置显微镜观察EGFP的表达。结果:经酶切和PCR鉴定,成功构建重组腺病毒质粒pAdeno-p27-EGFP,利用脂质体转染人293细胞后,转染腺病毒质粒pAdeno-p27-EGFP可见EGFP表达和细胞致病效应(CPE)。结论:利用腺病毒载体系统Adeno-X成功构建含CMV启动子的Flag-p27和EGFP基因的腺病毒载体,为进一步研究细胞周期调控与慢性移植物失功之间的关系,并为寻找慢性移植物失功的基因治疗的途径奠定基础。

Objective：To construct and identify the recombinant adenovirus plasmid containing exogenous p27 gene and EGFP gene and to detect the gene expression. Methods： Genetic engineering techniques, including plasmid extraction, agarose gel electrophoresis, restriction enzymolysis, connection, transformation of competent cells, were used to construct the recombinant plasmid of pCMV-IRES2-EGFP-p27. The adenovirus plasmid pAdeno-CMV-p27-EGFP was constructed by adeno-x expression system. The plasmids were identified by enzymolysis and PCR. Recombinant adenovirus vector was packaged by cell line 293. The expression of EGFP was detected under the fluorescence microscopy. Resuits： pCMV-IRES2-EGFP-p27 was successfully constructed and identified by enzymolysis and sequencing. Fluorescence microscopy revealed that the expression of EGFP could be detected at 48th h after the plasmid was transfected into astrocytes, and the expression of EGFP was increased to the peak at 48-72 h. The adenovirus plasmids pAdeno-CMV-p27- EGFP was successfully constructed and identified by enzymolysis and PCR. The CPE and the expression of EGFP could be observed after the plasmid pAdeno-CMV-p27-EGFP was transfected. Conclusion： The adenovirus plasmid containing human p27 gene was constructed successfully by adeno-X system. It will establish a foundation for the future research about the relationship of chronic graft dysfunction and immunological tolerance.