IgE高亲和力受体蛋白的制备及其生物功能的鉴定

Preparation of IgE high affinity receptor protein and its biological function identification

目的通过基因工程技术制备人IgE抗体结合其高亲和力受体α段(FcεRIα)蛋白并对其生物学功能进行鉴定,为探讨FcεRIα在过敏性疾病中的作用奠定研究基础。方法应用基于PCR的精确合成法(PAS)方法获得人FcεRIα基因,构建原核表达载体pET-FcεRIα,低温诱导表达FcεRIα并采用His标签纯化重组蛋白;通过ELISA法鉴定重组人FcεRIα蛋白的生物功能。结果扩增出大小约为560bp的人FcεRIα基因;pET-FcεRIα质粒经双酶切及测序鉴定正确;诱导表达并纯化出相对分子质量大小约为22 000的人FcεRIα;重组人FcεRIα能有效检测人血清中的抗FcεRIα自身抗体水平,并且能够与人血清中IgE抗体高效结合。结论成功制备人FcεRIα蛋白,并初步具备检测人血清中抗FcεRIα自身抗体及IgE抗体的能力,为后续研究提供了有利的实践基础。

Objective To prepare human alpha segment of high affinity IgE receptor(FcεRIα)protein by genetic engineering technology and to identify its biological function for laying the foundation for further researching the role of FcεRIαin allergic diseases.Methods The human FcεRIαgene was obtained by the PCR based accurate synthesis(PAS)method and the prokaryotic expression vector pET-28a(+)was constructed.The FcεRIαwas expressed at low temperature induction and the recombinant protein was purified by His tag.The biological function of recombinant human FcεRIαprotein was identified by ELISA.Results The human FcεRIαgene was amplified by PAS with a size of approximately 560 bp.The pET-FcεRIαplasmid was correct through the double enzyme digestion and sequencing identification.The human FcεRIαwith a molecular weight of approximately 22 000 was induced and purified.The recombinant human FcεRIαcould effectively detect human serum anti-FcεRIαautoantibody and could combined with serum IgE antibodies with high efficiency.Conclusion Human FcεRIαprotein is successfully prepared,which preliminarily has the ability for detecting the human serum anti-FcεRIαautoantibodies and IgE antibodies,and provides a favorable practical base for further study.