【摘要】 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。更多还原

【Abstract】 A recombinant adenovirus vector expressing gene encoding the EAST-6-CFP-10 fusion protein of Mycobacterium bovis was constructed firstly by the amplification of the genomic DNA from M.bovis 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 initially 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 sequencing,lineared by Pme Ⅰ enzyme digestion and transformed into E.coli BJ5183-AD-1 containing plasmid 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 transfected 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.更多还原