噬菌体呈现抗体库的构建及抗Fas-Fab抗体的研究

Construction of phage antibody library and screening of anti-FasFab antibody

目的构建噬菌体抗体库,获得具有功能的抗FasFab噬菌体抗体。方法以Fas重组蛋白为抗原免疫Balb/c小鼠。取其脾细胞提取mRNA采用RTPCR方法扩增抗体基因,构建重链和κ链基因库,用重组Fas抗原对所构建的抗体库进行4轮筛选,并以ELISA法鉴定其功能。结果获得抗体重链Fd基因和κ链基因长度约700bp。构建的重链Fd基因为3.5×106的抗体重链基因库。构建的重链和κ链基因库的容量均为3.1×106。经VCSM13感染得到噬菌体的滴度为8.9×1016cFu/L的噬菌体抗体库,含有抗体重链和κ链基因的噬菌体占27%。用重组人Fas抗原进行4轮筛选,得到100%的富集,说明Fas重组抗原富集了抗FasFab噬菌体抗体,经ELISA检测均有抗Fas抗体的特异性。结论制备的可溶性抗FasFab抗体具有抗Fas抗体的特异性,为进一步的研究奠定了基础。

Aim The phage display antibody library was constructed to obtain functional anti Fas Fab. Methods Balb/c mice were immunized with recombinant Fas protein. mRNA was isolated from splenocytes and antibody heavy chain genes Fd and κ chain genes were amplified by RT PCR, and combinatorial Fab antibody library. This iuelicated that the screening of antibody library was done with recombinont Fas antigen and the immunifacient function of the antibody was determined by ELISA. Results The amplified products were the desired fragments and contained 700 bp. The amplified antibody heavy chain Fd genes were cloned into pcomb3 vector at first, and transformed into E.coli XL1 blue to construct an antibody heavy chain library with a size of 3.5×106 members. The κ chain genes were then randomly combined with heavy chain genes to generate a combinatorial vector encoding both chains and capable of generating Fab fragments. These combinatorial vectors were transformed to E.coli XL1 Blue,and the combinatorial Fab antibody library was 3.7×106. The phage antibody library was further prepared by infection of helper phage VCSM13 to display the antibody Fab fragments on the surface of phage. The titer of phage in the library was 8.9×1015 cfu/L. The phage displaying antibody fragments were subjected to four rounds of panning with recombinant Fas antigen at solid phase. The eluted phage was enriched for nearly 100 fold and the percent of recombinant clones increased from 27% to 100% after four rounds of panning. Anti Fas Fab phage antibody displaying on phage surface and soluble anti Fas Fab showed specific binding activity to Fas by ELISA. Conclusion The successful preparation of anti Fas Fab antibody paved a way for further study and application.