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目的:克隆抗角蛋白抗体的可变区(V区)基因,构建抗角蛋白单链抗体(scFv)表达载体并表达。方法:根据人源性抗角蛋白Fab段的基因序列设计引物,用PCR方法克隆抗角蛋白抗体的V区基因,并重组到scFv的表达载体中,在大肠杆菌进行表达,表达产物经体外变性复性后进行SDS-PAGE鉴定。用ELISA检测其特异性结合活性。结果:成功地克隆抗角蛋白抗体的V区基因,构建了scFv的表达载体,并在大肠杆菌中表达。ELISA检测证实,表达的scFv保留了亲本Fab抗体的特异结合活性。结论:获得功能性抗角蛋白的scFv,可用于进一步的临床应用研究。
OBJECTIVE: To clone and express the variable region (V region) of anti-keratin antibody and construct the expression vector of anti-keratin scFv. Methods: Primers were designed according to the gene sequence of human anti-keratin Fab fragment. The V region gene of anti-keratin antibody was cloned by PCR and recombined into the scFv expression vector and expressed in Escherichia coli. The expressed product was denatured in vitro Refolding SDS-PAGE after identification. Specific binding activity was detected by ELISA. Results: The V region gene of anti-keratin antibody was successfully cloned and the expression vector of scFv was constructed and expressed in E. coli. ELISA testing confirmed that the expressed scFv retained the specific binding activity of the parental Fab antibody. Conclusion: Functional anti-keratin scFv can be used for further clinical research.