猪带绦虫六钩蚴TSOL18真核表达载体pVTSOL18m的构建

Construction of eukaryotic expression vector containing TSOL18 gene of Taenia Solium Oncosphere

根据Kozak原理设计引物,以重组原核表达载体pGEX-TSOL18为模板扩增TSOL18基因,用EcoRI、XhoI酶切后与经相同处理的pVAX1载体连接并转化JM109感受态细胞,经测序证明读码框正确后,再根据PCR点突变的方法设计引物,将目的基因NruⅠ位点中的碱基(第347位)经三次PCR反应突变,最终获得含突变位点的TSOL18m基因,EcoRⅠ、XhoⅠ再次酶切后与pVAX1载体连接,成功构建了含点突变的重组pVAX1表达载体,为TSOL18基因重组犬2型腺病毒活载体疫苗的研制奠定了基础。

According to Kozak rule, primers which were specific to the open reading frame （ORF） of TSOL18 were designed and the complete TSOL18 fragment was amplified by Polymerase Chain Reaction（PCR）. The amplified gene digesed by EcoR Ⅰ and Xho Ⅰ was ligated into pVAX1 vector, and transformed into JM109 competent cells. Then, according to the method of site mutation by PCR, two pairs of primers were designed and synthesized. After three PCRs, the 1 128 bp mutated fragment was obtained and subcloned into the downstream of CMV promoter of pVAX1 vector,providing the basis of development of live virus vaccine based on TSOL18 gene and canine adenovirus type 2.