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目的:制备高效价、高特异性的抗CD133胞外区新疆双峰驼来源的多克隆抗体,为制备高亲和力的抗CD133纳米抗体做准备。方法:将CD133胞外区基因序列构建到原核表达载体pET28a中,诱导表达及纯化CD133蛋白,免疫新疆双峰驼及新西兰兔。通过酶联免疫吸附实验(ELISA)和Western blot检测多克隆抗体的效价及与CD133特异性结合活性。结果:ELISA测定抗-CD133骆驼源抗体的效价可达到百万以上,通过Western blot检测多克隆抗体可特异性结合CD133蛋白。结论:重组人CD133蛋白可以在骆驼体内激发高滴度抗体反应,为今后构建骆驼免疫单域抗体噬菌体展示文库奠定基础。
OBJECTIVE: To prepare high titer and high specificity polyclonal antibody against Bacillus thuringiensis anti-CD133 from Bactrian camels in preparation for the preparation of high affinity anti-CD133 Nanobody. Methods: The CD133 gene sequence was constructed into prokaryotic expression vector pET28a, CD133 protein was induced and purified, and Xinjiang Bactrian camels and New Zealand rabbits were immunized. Polyclonal antibody titers and specific binding activity to CD133 were detected by enzyme-linked immunosorbent assay (ELISA) and Western blot. Results: The titer of anti-CD133 camel antibody was over 1 million by ELISA, and the polyclonal antibody could specifically bind to CD133 protein by Western blot. CONCLUSION: Recombinant human CD133 protein can stimulate high titer antibody reaction in camels and lay the foundation for the construction of phage display library of camelid immune single-domain antibody in the future.