抗SSA噬菌体抗体库的构建及鉴定

Construction of anti-SSA phage single-chain Fv antibody library

目的 用噬菌体展示技术构建抗SSA单链抗体库。方法 分离抗SSA抗体阳性病人外周血单个核细胞 ,提取RNA并反转录cDNA。扩增免疫球蛋白重链可变区 (VH)和κ链可变区(Vκ)。通过重叠聚合酶链反应 (PCR)用连接片段将VH和Vκ体外连接成单链抗体 (single chainFv ,scFv)基因。而后用SfiⅠ和NotⅠ酶切并与噬菌粒 pHEN2 连接 ;将 pHEN2 scFv电转至大肠杆菌TG1,建立抗SSAscFvcDNA库。随机挑选克隆用PCR扩增插入片段了解插入效率。用辅助噬菌体VCS M13感染含 pHEN2 scFv的TG1,产生scFv噬菌体抗体。用酶联免疫吸附 (ELISA)检测构建的scFv噬菌体抗体库中有无抗SSA抗体存在。结果 与VH和Vκ支架结构互补的引物可扩增出 30 0～ 4 0 0bp大小VH和Vκ片断 ;重叠PCR体外连接成约 80 0bp大小scFv基因 (VH linker Vκ)。将scFv基因克隆至 pHEN2 ,电转TG1后计数克隆数为 3 0× 10 7cfu。随机挑选 12个克隆 ,其中 11个克隆PCR扩增出插入片段 ,插入效率约为 91%。用辅助噬菌体VCS M 13感染含 pHEN2 scFv的TG1后 ,产生 1 2× 10 14 pfu/ml噬菌体抗体颗粒。抗SSAELISA试剂盒检测其抗SSA活性 ,scFv噬菌体抗体的A值比相同稀释度的VCS M13的A值高 2 0～ 2 2倍。结论 建立的抗SSAscFv噬菌体抗体库含有的独立克隆数为 3 0×

Objective To construct anti SSA phage single chain Fv antibody library by phage display technology.Methods RNA was isolated from the peripheral blood mononclear cells (PBMC) of patients with anti SSA antibody and reverse transcribed to cDNA,amplified VH and Vκ and linked them together with a linker segment by overlapping PCR to form VH linker Vκ (scFv gene),cut the scFv gene and phagmid vector pHEN 2 by Sfi Ⅰ and Not Ⅰ,ligated them and electrically transformed to TG1.Twelve clones were randomly selected to amplify the insertion fragment by PCR.The TG1 containing pHEN2 scFv was infected by helper phage VCS M13 to produce scFv phage antibody.The anti SSA activity of the phage antibody was tested by ELISA with the plate coated by purified SSA antigen.Results The fragment of VH and Vκ were about 300 to 400 bp.They were linked successfully by overlapping PCR in vitro to form scFv of about 800 bp.After cloning scFv to phagmid pHEN 2 and transforming pHEN 2 scFv to the TG1,3 0×10 7 clones formed.The insertion was amplified in 11 of the 12 randomly selected clones by PCR.After VCS M13 infection,1 2×10 14 pfu/ml scFv phage antibody were produced.In the anti SSA ELISA,the A produced by scFv phage antibody was 2 0 to 2 2 times of that produced by VCS M13.Conclusion The scFv phage antibody library contains about 3 0×10 7 individual clones,and the insertion efficiency is about 91%.This phage antibody library contains scFv antibody with the anti SSA activity.It can be used for the selection of monoclonal anti SSA phage antibody.