用噬菌体表面表达技术筛选及表达抗重组人促红细胞生成素单链抗体ScFv.docVIP

???? 用噬菌体表面表达技术筛选及表达抗重组人促红细胞生成素单链抗体ScFv ???? ????【摘要】　目的　获得抗重组人红细胞生成素(rhEPO)的单链抗体(ScFv)，为得到纯度高、活力强的重组人红细胞生成素(rhEPO)产品和制备红细胞生成素(EPO)检测试剂盒奠定基础。方法　采用噬菌体表面表达和重组抗体技术，从已建立的鼠抗rhEPO杂交瘤细胞株D3中克隆出抗rhEPO单链抗体ScFv基因片段，经噬菌体表面呈现，与固相抗原rhEPO结合，“吸附—洗脱—富集”，酶联免疫吸附法(ELISA)检测，酶切鉴定及PCR扩增鉴定，将阳性克隆重组质粒转化非抑制型E.coli HB2151,诱导表达抗rhEPO单链抗体，Western blot和Dot-blot杂交检测其抗原特异性。最后对阳性克隆进行序列测定，进行序列分析。结果　挑选出了噬菌体表面表达抗rhEPO单链抗体的阳性克隆，可溶性表达的单链抗体主要分布于细菌培养上清中，并且保留了亲本单抗对rhEPO的抗原特异性和免疫活性。表达产物分子量约为32kD左右。结论　本实验成功地获得了抗rhEPO抗体的可变区VH、VL基因，并表达出单链抗体ScFv片段。　　【主题词】　　噬菌体　　促红细胞生成素　　重组抗体 Generation of anti-rhEPO ScFv by using phage display technology Zhong Lingwen, Wu Shuhua, Yang Xiang, et al. Institute of Virology, Chinese Academy of Preventive Medicine, Beijing　100052 　　Abstract　In this paper, the recombinant phage antibody techniques was used to construct, clone, screen, and express anti-rhEPO single chain antibody ScFv. The variable region genes of antibody were amplified by PCR from a hybridoma cell line D3 which secreted monoclonal antibody to rhEPO. The ScFv gene fragments were successfully cloned into phagemid vector pCANTAB5E. The recombinant phages were panned by rhEPO which was coated on a microtiter plate. After three rounds of panning, 8 clones were determined specifically binding to rhEPO antigen. The positive recombinant phagemids were extracted and transformed into non-suppressed E.coli HB2151. The soluble single chain antibody was expressed, and the specificity of the expressed ScFv was determined by ELISA, Western blot and Dot-blot. The result of SDSindicated that the apparent molecular weight of the target peptide which is mainly in culture supernatant is 32kD. The DNA sequence data showed that the ScFv gene included 783bp, encoding 261 amino acids.　　Key words：　Phage　　Human erythropoietin　　Recombinant antibody 　　人类促红细胞生成素(Human erythropoietin, huEPO)作用于骨髓红系造血前体细胞，维持其存活，促进其增殖分化，防止晚期红系祖细胞的程序化死亡(Apoptosis)〔1〕。目前EPO作为一种治疗贫血的多肽药物，已取得了良好的临床效果。　　噬菌体表面表达技术(Phage display technology, PDT)是一项利用丝状噬菌体体外表达外源基因的新型