抗SSA表位特异性单链噬菌体抗体的可溶性表达及鉴定

The soluble expression and identification of single-chain fragment V antibodies against SSA antigen epitopes from the pHEN2 phagemid library

目的 获得可溶性抗人SSA抗原表位特异性单链抗体（ScFv）.方法 以赖氨酸为支架八倍体合成3条相对分子质量为60 000的SSA抗原氨基酸多肽链（MAP）：MAP 482～493（P1表位）、MAP 310～323（P2表位）和MAP 230～241（P3表位）.以P1～P3表位为包被抗原,富集筛选相应的噬菌体单克隆抗体（McAb）;阳性克隆特异性、亲和力和序列分析鉴定;表位特异性McAb可溶性表达和鉴定.结果 5轮筛选后,获得了抗3种表位抗体的含完整插入片断的克隆株,具有较高亲和力和特异性.抗P1～P3抗体融合蛋白与相应表位反应的410 nm吸光度值分别为1.43±0.23、0.82±0.31、0.80±0.25,组间交叉反应差异有统计学意义（P值均小于0.01）.其中3个克隆表位特异性McAb-ScFv克隆成功,可溶性表达并纯化.以Hep-2细胞为底物,间接免疫荧光法（ⅡF）检测显示,可溶性ScFv抗体具有在体活性.结论 筛选获得的阳性克隆成功,可溶性表达并具有较好的亲和力、特异性和在体活性,可胜任靶器官的表位表达情况的检测.

Objective To obtain the soluble single-chain fragment V （ScFv）monoclonal antibodies （McAbs） against the SSA antigen epitopes.Methods Three octapeptides （60 000 SSA antigen residues 482-493 termed as P1 epitope, residues 310-323 termed as P2 epitope and residues 230-241 termed as P3 epitope） were synthesized on the lysine frame.The McAbs were panned by coating the corresponding as targets.The specificity, affinity and gene squences of the positive clones were assessed.Soluble single-chain fragment V antibodies special for SSA antigen epitopes were expressed and then identificated.Results After 5 rounds of panning, reactive scFv clones contained full-length scFv antibodies coding regions were obtained,with sufficient affinity and specificity for respective antigen peptides.The absorbance values at 410 nm of the fusion protein of anti-P1-P3 activity with the corresponding peptides were 1.43 ± 0.23, 0.82 ±0.31 and 0.80 ± 0.25, and there was also statistically significant difference in the cross reactions （ P 〈 0.01 ）.Three clones were successfully expressed and then purified by His-bind resin.The activity in vivo of soluble ScFv antibodies was identified to be positive by the indirect immune-fluorescence assay on Hep-2 cells.Conclusion Souble ScFv McAbs against corresponding SSA antigen peptides with high affinity,specificity and activity in vivo were obtained, which are to be competent enough for epitopes expression on the target organs.