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目的:制备青杄FKBP12基因的多克隆抗体,为进一步分析FKBP12的蛋白定位、表达等提供基础。方法:采用PCR方法扩增FKBP12基因得到其全长cDNA序列,并将其克隆至原核表达载体pET-48b中,转化入BL21菌株。经IPTG诱导,表达了分子量约为33kD的重组蛋白,SDS-PAGE和Western blotting检测鉴定表达产物。此蛋白经亲和纯化后,作为抗原注射新西兰兔,进行抗体制备。结果:成功获取了多克隆抗体,制备的FKBP12兔抗血清效价在1∶729 000以上,ELISA结果表明融合蛋白具良好的免疫原性。间接ELISA法检测纯化后抗体效价,表明纯化后抗FKBP蛋白兔多克隆抗体效价高,检测灵敏度为16ng/mL。结论:所制备的抗体能满足后续试验要求的效价值,为进一步研究提供基础。
OBJECTIVE: To prepare polyclonal antibodies against FKBP12 gene of barley and provide basis for further analysis of FKBP12 protein localization and expression. Methods: The full length cDNA of FKBP12 was amplified by PCR and cloned into prokaryotic expression vector pET-48b and transformed into BL21 strain. After induced by IPTG, a recombinant protein with a molecular weight of about 33 kD was expressed. The expressed product was identified by SDS-PAGE and Western blotting. After affinity purification, this protein is injected as an antigen into New Zealand rabbits for antibody preparation. Results: The polyclonal antibody was successfully obtained. The titer of FKBP12 rabbit antisera was above 1: 729 000, and ELISA showed that the fusion protein had good immunogenicity. The titer of purified antibody was detected by indirect ELISA. The results showed that the polyclonal antibody against FKBP was purified and its titer was 16 ng / mL. Conclusion: The prepared antibody can meet the requirements of the follow-up test value, providing the basis for further research.