携带EphrinA1-caspase-3基因的重组腺病毒载体的构建和包装

Construction and package of the recombinant adenovirus vector carrying the EphrinA1-caspase-3 gene

目的:构建携带ephrinA1-caspase-3基因的重组腺病毒载体,通过包装、扩增、鉴定,为后续开展目的基因靶向治疗乳腺癌研究奠定基础。方法:以pMD18T-Casp3、pMD18T-EphrinA1为模板扩增caspase-3、EphrinA1基因,以pET28a为载体,通过双酶切、连接、转化、测序鉴定,构建pET28a-EphrinA1-Casp3。然后以质粒pEC3.1(+)为载体,获得pEC3.1-EphrinA1-Casp3。采用LR体外同源重组,构建pAd-EphrinA1-Casp3,以HEK293包装和放大培养。结果:成功将PCR扩增的EphrinA1胞外端基因和人活性型caspase-3基因定向克隆入质粒pET28a,并将EphrinA1-caspase-3融合基因转移到穿梭质粒pEC3.1(+),获得pEC3.1-EphrinA1-Casp3载体;经过体外同源重组,成功构建携带EphrinA1-caspase-3基因的重组腺病毒载体pAd-EphrinA1-Casp3;并在HEK 293细胞中完成包装和扩增;获得重组腺病毒rAd-EphrinA1-Casp3。结论:利用GatewayTM技术成功构建重组腺病毒载体pAd-EphrinA1-Casp3,经HEK293细胞包装,获得重组腺病毒rAdEphrinA1-Casp3。

Objective:To construct the recombinant adenovirus vector carrying the EphrinA1-caspase-3 gene,and to lay a good experimental foundation for the further research on the targeting treatment of breast cancer with the desired gene after packaging and amplification.Methods:The caspase-3 gene and EphrinA1 gene were amplified by PCR with the clone vector pMD18T-Casp3 containing the human active caspase-3 gene and pMD18T-EphrinA1 as the templates,respectively.The PCR product of these genes and the plasmid pET28a were digested with double restriction enzymes,and then were collected,connected,and transformed into E.coli DH5αcompetent cells.The positive clones with correct gene sequencing were named pET28a-EphrinA1-Casp3.Then the pET28a-EphrinA1-Casp3 and the shuttle vector pEC3.1(+)were connected after double enzyme digestion,and the pEC3.1-EphrinA1-Casp3 was obtained after transformation and sequencing analysis.And then the expression cassette of EphrinA1-Casp3 was transferred to pAd/pl-DEST adenoviral vector in vitro with LR homologous recombinant reaction and the recombinant adenovirus plasmid pAd-EphrinA1-Casp3 was produced.In the end the pAd-EphrinA1-Casp3 was linearized and transfected into HEK293 cells for packing and amplification,and the recombinant adenovirus rAd-EphrinA1-Casp3 was identified by PCR and obtained.Results:The PCR product of the human active caspase-3 gene and that of the EphrinA1 gene were cloned into the adenovirus vector pET28a in the correct direction successfully,and the fused gene EphrinA1-caspase-3 was transferred to the adenovirus shuttle vector pEC3.1(+)to acquire recombinant plasmid pEC3.1-EphrinA1-Casp3.The recombinant adenovirus vector pAd-EphrinA1-Casp3 containing EphrinA1-Casp3 gene was constructed successfully by the application of LR homologous recombinant reaction in vitro.And after the transfection of pAd-EphrinA1-Casp3 into HEK293 cells for packing and amplification,the recombinant adenovirus rAd-EphrinA1-Casp3 was obtained.Conclusion:The recombinant adenovirus vector pAd-EphrinA1-Casp3 expressing EphrinA1-Casp3 gene can be constructed successfully with GatewayTM technology,and the recombinant adenovirus rAd-EphrinA1-Casp3 is prepared through packing and amplification in HEK293 cells.