猪囊尾蚴头节单链抗体-PE40重组载体的构建及表达

Expression and construction of Cysticercosis cellulosae scolex ScFv and PE40 recombinant vector

从绿脓杆菌ATCC27853菌株中提取基因组,PCR扩增PE40基因,与pMD18-T载体连接并转化感受态大肠杆菌JM109中,构建重组质粒pMD-PE40;经鉴定正确后将PE40基因插入抗猪囊尾蚴头节单链抗体基因重组质粒pMD-ScFv中,构建重组质粒pMD-ScFv-PE40,经酶切、电泳回收目的片段,并亚克隆到表达载体pET-28a中,构建重组表达载体pET-ScFv-PE40;鉴定后转化E.coliBL21,IPTG诱导后进行SDS-PAGE和Western-blotting验证。结果表明,试验成功构建了重组表达载体pET-ScFv-PE40,目的片段约为1 800 bp;Western blotting证明重组蛋白具有一定的反应原性,为重组免疫毒素的制备及动物保护性试验奠定了基础。

To construct recombinant vector pET-ScFv-PE40,the single-chain antibody gene of against Cysticercus cellulosae scolex and Pseudomonas aeruginosa exotoxin PE40 gene was connected,and subcloned into expression vector pET-28a.Then BL21/pET-ScFv-PE40 was induced for protein expression and analysis.The results showed the recombinant expression vector pET-ScFv-PE40 was constructed successfully.The expressed protein was about 68 000.Western-blotting indicated that recombinant protein has a significant reactionogenicity.The product could serve as a basis for preparating of recombinant immunotoxin and animal protection experiment.