人源性抗结核分枝杆菌噬菌体展示单链抗体文库的构建

目的 构建人源抗结核分枝杆菌噬菌体展示单链抗体文库,为结核特异性单链抗体的筛选奠定基础. 方法 从抗结核分枝杆菌抗体阳性患者淋巴细胞中提取总RNA,逆转录成cDNA,PCR扩增获得抗体的重、轻链可变区基因,然后拼接装配成单链抗体基因,重组于噬菌粒载体pCANTAB5S,转化大肠埃希菌E.coli TG1,以辅助噬菌体拯救后,构建人源性抗结核分枝杆菌噬菌体展示单链抗体库. 结果 成功构建人源性抗结核分枝杆菌噬菌体展示单链抗体文库,库容量达107. 结论 以结核患者淋巴细胞免疫球蛋白可变区基因片段和噬菌粒展示载体pCANTAB5S为基础,成功构建人源性抗结核分枝杆菌噬菌体展示单链抗体库,并达到建库标准,可进一步从中筛选获得结核特异性单链抗体.

Construction of a human phage display ScFv library against mycobacterium tuberculosis

Objective To construct a human phage display single-chain Fv(ScFv)antibody library against mycobacterium tuberculosis(MTB),for specific ScFv antibody cloning.Methods Total RNA was isolated from the lymphocytes of patients with positive serum antibody against MTB and reveree transcribed into cDNA.The heavy chain and light chain variable region gene of human immunoglobulin were amplified individually by PCR and then assembled into ScFv genes.The ScFv genes were Hgated into phagemid pCANTAB5S.The human phage display ScFv library agaiast MTB was constructed by transforming the recombinant phagemid into E.coli TG1 with the presence of helper phage M13K07.Results The human phage display ScFv library containing 107 different clones was constructed successfully.Conclusions A phage display ScFv library against MTB has been constructed based on the variable region gene of immunoglobulin of the lymphocytes of TB patients and phagemid pCANTABSS.The specific ScFv antibodies can be screened from this library.