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目的克隆结核分枝杆菌的ESAT-6和CFP-10基因,构建能同时表达两基因的重组腺病毒活载体,为研制能有效防治结核病的重组腺病毒活载体疫苗打下基础。方法首先利用PCR技术克隆结核分枝杆菌的ESAT-6和CFP-10基因,将两者通过IRES串连插入腺病毒转移载体中,构建重组穿梭质粒pShuttle-CMV-ESAT-6-IRES-CFP-10。该穿梭质粒转化含有腺病毒骨架质粒pAdeasy-1的BJ5183感受态细胞,经同源重组获得复制缺陷型重组腺病毒载体pAdeasy-1-pShuttle-CMV-ESAT-6-IRES-CFP-10。PacI酶切线性化该质粒,转染AD293细胞进行病毒包装。结果ESAT-6和CFP-10基因成功克隆,其序列与GenBank公布的一致。两者通过IRES串连插入腺病毒转移载体,获得的重组穿梭质粒经同源重组,成功获得复制缺陷型重组腺病毒质粒Adeasy-1-pShuttle-CMV-ESAT-6-IRES-CFP-10,转染该质粒的AD293细胞出现细胞病变效应。结论成功克隆了分枝杆菌的ESAT-6和CFP-10基因,构建了含IRES串连的ESAT-6和CFP-10基因的重组腺病毒,为重组腺病毒活载体疫苗的研制奠定了基础。
Objective To clone the ESAT-6 and CFP-10 genes of Mycobacterium tuberculosis and construct a recombinant adenoviral vector that can express both genes simultaneously, which lays a foundation for the development of a recombinant adenoviral live vector vaccine that can effectively prevent and treat tuberculosis. Methods The genes of ESAT-6 and CFP-10 of Mycobacterium tuberculosis were cloned by PCR and inserted into the adenovirus transfer vector through IRES in series to construct recombinant shuttle plasmid pShuttle-CMV-ESAT-6-IRES-CFP- 10. The shuttle plasmid was transformed into BJ5183 competent cells containing the adenovirus backbone plasmid pAdeasy-1, and the recombinant adenovirus vector pAdeasy-1-pShuttle-CMV-ESAT-6-IRES-CFP-10 was obtained by homologous recombination. This plasmid was linearized with PacI and transfected into AD293 cells for viral packaging. Results The ESAT-6 and CFP-10 genes were successfully cloned and their sequences were identical to those published in GenBank. The two were inserted into the adenovirus transfer vector by IRES in series. The recombinant shuttle plasmid obtained by homologous recombination successfully obtained Adeasy-1-pShuttle-CMV-ESAT-6-IRES-CFP-10 replication-defective recombinant adenovirus plasmid AD293 cells stained with the plasmid showed cytopathic effect. Conclusion The mycobacterium ESAT-6 and CFP-10 genes were successfully cloned and the recombinant adenovirus containing ESES-6 and CFP-10 genes linked in series with IRES was constructed, which laid the foundation for the development of recombinant adenoviral live vector vaccine.