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目的 筛选、鉴定抗丙型肝炎病毒 (HCV)非结构蛋白NS5A的人源单链可变区抗体(ScFv)的编码基因 ,为HCVNS5A蛋白质生物学功能的研究及抗HCV的基因治疗研究开辟新途径。方法 采用噬菌体表面展示技术 ,以重组纯化的HCV非结构蛋白NS5A为固相抗原 ,从噬菌体单链可变区抗体半合成库中经过 5轮“吸附 洗脱 扩增”筛选过程 ,获得抗原结合活性和特异性较强的HCVNS5A人源单链可变区抗体基因片段的阳性克隆 ,并对其进行免疫检测及序列测定。结果 筛选得到的ScFv片段基因核苷酸序列为 789nt,编码由 2 62个氨基酸残基组成的多肽 ,具有典型的免疫球蛋白轻链和重链可变区的结构特点。基因编码产物具有HCVNS5A蛋白反应的免疫学活性和特异性。结论 利用噬菌体抗体库技术 ,成功获得了HCVNS5A人单链可变区抗体ScFv编码基因 ,并获得了可溶性单链抗体的表达
Objective To screen and identify the coding sequence of human single chain variable region antibody (ScFv) against the nonstructural protein NS5A of hepatitis C virus (HCV), and to explore new ways of studying the biological function of HCV NS5A protein and the gene therapy of HCV . Methods The phage display technique was used to detect the antigen binding activity after 5 rounds of “elution-elution amplification” screening from the semi-synthetic library of phage single chain variable region antibody using the recombinant non-structural HCV NS5A as solid phase antigen And positive clones of HCV NS5A human single-chain variable region antibody gene fragment, and the immunoassay and sequencing were performed. Results The nucleotide sequence of the ScFv fragment was 789nt and encoded a polypeptide of 62 amino acid residues. The results showed that the typical immunoglobulin light and heavy chain variable domains were structurally characterized. The gene encoding product has the immunological activity and specificity of HCVNS5A protein reaction. Conclusion The scFv gene of HCV NS5A human single chain variable region antibody was successfully obtained by phage antibody library technology and the expression of soluble single chain antibody was obtained