抗人肺癌噬菌体展示可溶性ScFv的筛选与鉴定及其序列测定

Production and characterization of soluble phage display single chain variable fragment of monoclonal antibody

目的:制备抗人肺癌单链抗体(single chain Fvferment antibody,ScFv),并对抗体生物学特性进行初步研究。方法:以人肺腺癌细胞系A2为抗原,对5F-11杂交瘤细胞噬菌体抗体库进行富集和筛选。以人肺腺癌细胞系A2和正常人淋巴细胞为抗原,进行酶联免疫吸附试验,从富集后的噬菌体抗体库中筛选出只与A2细胞结合的阳性克隆。筛选的噬菌体克隆转染大肠埃希菌HB2151,得到可溶性单链抗体分泌克隆。可溶性抗体分泌克隆测序。应用ELISA、竞争性ELISA、SDS-PAGE及蛋白质印迹法对其中的2A7-1克隆进行初步鉴定。结果:以肺腺癌细胞A2为抗原进行了4轮富集。进一步筛选得到18个仅识别A2细胞而与人淋巴细胞无反应的融合抗体分泌克隆。转染大肠埃希菌HB2151后筛选到能与A2细胞特异结合的可溶性抗体分泌克隆2A7-1。竞争性ELISA结果显示,5F-11能强烈抑制2A7-1与A2细胞的结合。SDS-PAGE蛋白质印迹法显示得到大小约为30×103的单链抗体。结论:通过噬菌体抗体技术成功分离到了鼠单抗5F-11的可溶性ScFv分泌克隆,为进一步的抗体应用研究奠定了基础。

OBJECTIVE：To isolate the soluble single-chain variables fragment （ScFv） specific for human lung adenocarcinoma A2,and to characterize this soluble ScFv. METHODS：A recombination phage display library was established from 5F-11 hybridoma. The phage clone displayed recombinant ScFv antibody was selected by enzyme-linked immunosobent assay （ELISA） from the monoclonal antibody 5F-11 phage display library by using human lung adenocarcinoma A2 cell and human lymphocyte as cell antigen. Each antigen-positive fusion ScFv phage clone was used to infect E.coli HB2151 cell to get soluble ScFv. Nucleotide sequence of all antigen-positive soluble ScFv secreted phage clones was carried out. The specificity and immunologican characteristics of one antigen-positive soluble secreted phage clone 2A7-1 were analysis by ELISA,SDS-PAGE and Western-blot. The relatively affinity of 2A7-1 was detected by completion ELISA. RESULTS：After panning against immoblilzed lung adenocarcinoma cell line A2 four rounds,eighteen phage clones from the enrich library were found being reacted with A2 cell,and did not react with human lymphocyte cells. The original hybridoma antibody 5F-11 could strongly compete with soluble phage clone 2A7-1 binding to the A2 cell. SDS-PAGE revealed an approximate 30×103 protein was expressed from 2A7-1,and could be detected by anti-E-tag. CONCLUSIONS：The soluble phage display ScFv fragment of monoclonal antibody 5F-11 is successfully produced by phage antibody technology. This may be useful to widen the range of application of the antibody.