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目的构建噬菌体抗体库,获得具有功能的抗FasFab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RTPCR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FasFab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FasFab抗体具有抗Fas抗体的特异性,为进一步的研究奠定了基础。
Objective To construct a phage antibody library and obtain a functional anti-FasFab phage antibody. Methods Balb / c mice were immunized with Fas recombinant protein. Take the spleen cells to extract mRNA using RT PCR method to amplify antibody genes, construction of heavy chain and κ chain gene library, recombinant Fas antigen library constructed by screening four rounds, and its function was identified by ELISA. Results The length of antibody heavy chain Fd gene and κ chain gene was about 700bp. The constructed heavy chain Fd gene was 3.5 × 106 antibody heavy chain gene library. The capacity of constructed heavy chain and kappa chain gene pool were both 3.1 × 106. The phage antibody library with phage titer of 8.9 × 1016cFu / L was obtained by VCSM13 infection, and the phage containing antibody heavy chain and κ chain gene accounted for 27%. Four rounds of screening with recombinant human Fas antigen yielded 100% enrichment, indicating that the recombinant Fas antigen was enriched in anti-Fas-Fab phage antibody. The anti-Fas antibody was detected by ELISA. Conclusion The prepared anti-Fas-Fab antibody has the specificity of anti-Fas antibody, which lays the foundation for further research.