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首先对猪囊尾蚴头节特异性单链抗体重链可变区基因(Vh)和轻链可变区基因(Vl)进行酶切鉴定,PCR鉴定以及测序分析,然后采用重叠延伸PCR(SOE PCR)技术,将重链可变区基因和轻链可变区基因,通过linker链连接成Vh-linker-Vl单链抗体基因(ScFv)。将此基因与pMD18-T载体连接,转化感受态大肠杆菌J M109,从而进一步克隆并对重组载体pMD-ScFv进行鉴定。结果:成功构建并克隆了抗猪囊尾蚴头节单链抗体,为重组免疫毒素ScFv-PE40的构建?表达及活性测定奠定了良好的基础。
First of all, the Vh gene and the Vl gene of Vibrio cholelithiae specific scFv were digested with restriction endonucleases, identified by PCR, sequenced and analyzed by overlap extension PCR (SOE PCR ) Technology, the heavy chain variable region gene and the light chain variable region gene are linked into the Vh-linker-Vl single chain antibody gene (ScFv) through a linker. The gene was ligated with pMD18-T vector and transformed into competent E. coli J M109 for further cloning and identification of the recombinant vector pMD-ScFv. Results: The single chain antibody against Cysticercus cellulosae was successfully constructed and cloned, which laid a good foundation for the construction, expression and activity determination of recombinant immunotoxin ScFv-PE40.