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目的:制备抗人源核受体h LRH-1的多克隆抗体,为进一步研究其功能奠定基础。方法:构建含有h LRH-1基因全长克隆的原核表达载体p ET507a-h LRH-1并用IPTG诱导其在Rosseta2菌株中表达重组蛋白His-h LRH-1,经亲和层析纯化后按常规方法免疫新西兰兔制备多克隆抗体,并用Western Blot对其特异性进行鉴定。结果:原核表达载体p ET507a-h LRH-1经测序证实构建成功,将其转化大肠杆菌Rosseta2菌株后成功诱导表达重组蛋白His-h LRH-1,经纯化免疫新西兰兔后得到抗h LRH-1多克隆抗体,Western blot证实抗体具有高度特异性。结论:成功表达His-h LRH-1重组蛋白并制备出多克隆抗体,为进一步用于h LRH-1的免疫学检测及其功能研究奠定了基础。
OBJECTIVE: To prepare polyclonal antibody against h LRH-1, a human nuclear receptor, and to lay the foundation for further study of its function. Methods: The prokaryotic expression vector p ET507a-h LRH-1 containing the full-length h LRH-1 gene was constructed and expressed in Rosseta2 strain by IPTG. The recombinant protein His-h LRH-1 was purified by affinity chromatography. Methods New Zealand rabbits were immunized with polyclonal antibody and identified by Western Blot. Results: The prokaryotic expression vector p ET507a-h LRH-1 was successfully constructed and confirmed by sequencing. The recombinant plasmid pET507a-h LRH-1 was successfully transformed into E. coli Rosseta2 and successfully induced recombinant protein His-h LRH-1. Polyclonal antibodies, Western blot confirmed that the antibodies are highly specific. Conclusion: The recombinant protein of His-h LRH-1 was successfully expressed and polyclonal antibodies were prepared, which laid the foundation for the further immunological detection of h LRH-1 and its function.