鼠源性抗EHEC O157:H7 Stx2噬菌体Fab抗体库的构建及筛选

Construction and Screening of Mouse Fab Phage Antibody Library against Stx2 of EHEC O157∶H7

目的构建鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,并从中筛选特异性的抗体。方法用EHEC O157∶H7 Stx2类毒素免疫BALB/c小鼠,取脾分离淋巴细胞,提取总RNA,RT-PCR分别扩增抗体轻、重链(к和Fd)基因,经双酶切依次克隆入噬粒载体pComb3X中,电转化大肠杆菌XL1-Blue,以辅助噬菌体M13K07进行超感染,构建抗EHEC O157∶H7 Stx2的Fab噬菌体抗体库。以纯化的Stx2为抗原进行筛选,获得抗EHECO157∶H7Stx2的特异性Fab抗体,Western blot法检测噬菌体抗体与毒素抗原的结合活性,并对所得阳性克隆进行基因序列分析。结果构建了一个库容为1.56×107的Fab抗体库,筛选出3株特异性较强的阳性克隆,其中2个可与Stx2A1亚单位抗原反应,1个可与Stx2B亚单位抗原反应。基因序列分析显示,轻、重链可变区氨基酸序列与GenBank中已注册的鼠免疫球蛋白可变区氨基酸序列同源性分别为98.5%和99.6%。结论已成功构建了鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,为进一步制备抗EHECO157∶H7Stx2的治疗性人源化抗体奠定了基础。

Objective To construct and screen specific mouse Fab phage antibody library against EHEC O157：H7 Stx2. Methods BALB/c mice were immunized with inactivated Stx2 of EHEC O157：H7, and splenic lymphocytes were isolated for extraction of total RNA from which antibody light chain k gene and heavy chain Fd gene were amplified by RT-PCR, identified by restriction analysis and inserted into phagemid pComb3X successively. The constructed recombinant vector was transformed to E. coli XL1- Blue by electroporation, based on which the specific Fab phage antibody library against Stx2 of EHEC O157：H7 was constructed by superinfection with helper phage M13K07. The specific Fab antibody against Stx2 of EHEC O157：H7 was screened using purified Stx2 as antigen. The binding activity of phage antibody to toxin antigen was determined by Western blot, and the obtained positive clones were sequenced. Results The Fab phage antibody library with a capacity of 1. 56 × 10^7 was constructed, and three strong positive clones recognizing Stx2 specifically were screened, of which two reacted with Stx2A1, and the other one with Stx2B. The homologies of amino acid sequences of variable heavy （VH） and light （VL） chain domains were 98. 5% and 99. 6% respectively to those of murine Ig reported in GenBank. Conclusion The specific mouse Fab phage antibody library against EHEC O157：H7 Stx2 was successfully constructed, which laid a foundation of preparation of therapeutic humanized antibody against EHEC O157：H7 Stx2.