牛分枝杆菌ESAT-6-CFP-10融合基因重组腺病毒载体的构建及表达

目的构建表达牛分枝杆菌的ESAT-6-CFP-10融合基因的重组腺病毒载体,为研制能有效防治牛结核病的重组腺病毒活载体疫苗打下基础。方法以牛分枝杆菌ValleeⅢ株基因组DNA为模板,应用PCR扩增获得ESAT-6和CFP-10两个目的基因片段,采用重叠延伸剪接技术(SOE)将ESAT-6和CFP-10基因通过(G1y4Ser)3接头连接,得到融合基因。将融合基因T/A克隆后,亚克隆至腺病毒转移载体pShuttle-CMV中,构建重组穿梭质粒pShuttle-CMV-ESAT-6-CFP-10,限制性内切酶消化、菌液PCR、序列分析验证无误后,PmeⅠ酶切,转化含有腺病毒骨架质粒pAdeasy-1的BJ5183-AD-1感受态细胞,经同源重组获得复制缺陷型重组腺病毒载体pAd-CMV-ESAT-6-CFP-10。PacI酶切线性化的重组质粒pAd-CMV-ES-AT-6-CFP-10转染AD-293细胞进行病毒包装和扩增。PCR、Western blot检测包装病毒的融合基因及其表达情况。结果成功构建携带牛分枝杆菌的ESAT-6-CFP-10融合基因的重组腺病毒载体,包装病毒用PCR、Western blot检测到包装病毒的融合基因及其表达。结论本研究成功地构建携带牛分枝杆菌的ESAT-6-CFP-10融合基因的重组腺病毒,为下一步在动物体内进行免疫效果研究打下基础。

Construction and expression of a recombinant adenovirus vector expressing the EAST-6-CFP-10 gene of Mycobacterium bovis

A recombinant adenovirus vector expressing gene encoding the EAST-6 CFP-10 fusion protein of Mycobacterium boris was constructed firstly by the amplification of the genomie DNA from M. boris Vallee Ⅲ strain with PCR by gene SOEing method, then the EAST-6 and CFP 10 genes were linked with the （Gly4Ser）3 linker. The linked gene was inserted ini tially into vector pMD18-T and then subcloned to the adenovirus shuttle plasmid pShuttle-CMV to construct the recombinant shuttle plasmid pShuttle-CMV EAST-6 CFP-10 . It was confirmed correct by restriction endonuclease digestion, PCR and sc quencing, lineared by Pine I enzyme digestion and transformed into E. colt BJ5183 AI~I containing plasrnid pAdeasy-1 to form the homogeneous recombinant adenovirus plasmid vector. After the vector was identified by Pac I digestion and PCR, the lineared homogeneous recombinant plasmid was transfeeted to AD-293 cells using lipofectamineTM 2000. The fusion gene and its expression in virus were detected by PCR and Western blot. In these ways, the recombinant virus carrying the gene encoding the EAST-6-CFP-10 fusion protein was successively constructed ,thus provide basis for the study on the immune effects in animals.