【摘要】 目的:构建含小鼠GITRL基因原核表达载体,经转化E.coli以表达GITRL融合蛋白,纯化蛋白并制备相应的抗血清。方法:将GITRL-pMD18-T重组质粒经EcoRⅠ和SalⅠ双酶切回收目的基因,与原核表达载体PET32a连接,应用电转法转化BL21(DE3)菌。阳性克隆经鉴定后用IPTG诱导GITRL蛋白表达,经Ni+-NTA层析柱纯化融合蛋白,通过SDS-PAGE、West-ernblot进行特异性鉴定,CD4+CD25+T细胞免疫抑制试验验证其生物学功能。采用弗氏完全佐剂常规免疫方案,制备抗GITRL抗血清,抗血清的效价和特异性测定采用双向琼脂免疫扩散法。结果:经酶切鉴定和测序分析证实原核表达质粒构建正确,SDS-PAGE和Westernblot结果显示,在40kD处呈现单一蛋白条带,该蛋白具有阻断CD4+CD25+T细胞免疫抑制功能的作用,双向免疫扩散结果显示兔抗小鼠GITRL抗血清效价为1∶16。结论:成功构建GITRL原核表达质粒,制备和纯化的GITRL融合蛋白具有较好的纯度和生物学功能,制备并获得特异性抗血清,可用于GITRL融合蛋白生物学活性的进一步研究。更多还原

【Abstract】 Objective:To construct the prokaryotic expression vector and to express soluble GITRL fusing protein.Methods:The GITRL gene was digested by EcoRⅠ and SalⅠ,recovered from 1% agarose and ligated into a prokaryotic expression vector named PET32a to construct reconstruction plasmid GITRL-PET32a,positive clones were analyzed and transformed into BL21(DE3).The GITRL-expressing bacteria were cultured in different concentration of IPTG.The fusion protein was purified by Ni+-NTA column and detected by SDS-PAGE and Western blot.In addition,the purified GITRL protein was mixed with Freund’s complete adjuvant to immunize rabbit and the anti-mouse GITRL antibody was produced.Results:Digestion and sequence analysis confirmed that the identification of prokaryotic expression plasmid was correct.The SDS-PAGE and Western blot results showed that the molecular weight of GITRL fusion protein was about 40 kD.mGITRL protein could reverse suppression of CD4+CD25+ T cells.The titer of anti GITRL andibody was 1∶16,detected by two-way immuno-diffusion.Conclusion:The prokaryotic expression vector is successfully constructed,recombinant GITRL protein is produced,which induces high titer of anti GITRL antibody.All of these results provide a foundation for further studying the biological function of GITRL.更多还原