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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× 10
Objective To construct an anti-SSA single-chain antibody library by phage display technology. Methods Peripheral blood mononuclear cells were isolated from patients with positive anti-SSA antibodies, RNA was extracted and cDNA was reverse transcribed. The immunoglobulin heavy chain variable region (VH) and the kappa chain variable region (Vκ) are amplified. VH and VK were ligated in vitro into single chainFv (scFv) genes by overlapping polymerase chain reaction (PCR) with linker fragments. Then digested with Sfi I and Not I and ligated with the phagemid pHEN2; the pHEN2 scFv was electrotransformed into E. coli TG1 to establish an anti-SSAscFv cDNA library. Randomly selected clones were used to amplify the insert to amplify the insertion efficiency. TG1 containing pHEN2 scFv was infected with helper phage VCS M13 to generate scFv phage antibodies. The presence or absence of anti-SSA antibodies in the constructed scFv phage antibody library was tested by enzyme-linked immunosorbent assay (ELISA). Results The primers complementary to the VH and Vκ scaffolds could amplify the VH and Vκ fragments of 30 0-40000 bp in size. The overlap PCR was performed in vitro to form a VH linker Vκ80. The scFv gene was cloned into pHEN2 and the number of clones counted after electroporation TG1 was 30 × 10 7 cfu. Twelve clones were randomly selected. Among them, 11 clones were amplified by PCR, and the insertion efficiency was about 91%. After infection of TG1 containing pHEN2 scFv with helper phage VCS M 13, 1 2 x 10 14 pfu / ml phage antibody particles were generated. The anti-SSA activity of the scFv phage antibody was detected by the anti-SSA ELISA kit, which was 20 to 20 times higher than the A value of the VCS M13 of the same dilution. Conclusion The number of independent clones contained in anti-SSAscFv phage antibody library was 30 × 10 10