小鼠GITRL融合蛋白的表达、纯化及免疫原性的研究  

Prokaryotic expression and purification of mouse GITRL fusion protein

作　　者：马洁[1] 王胜军[1] 毛朝明[2] 杨敏[1] 王锁英[2] 许小朋[1] 邱谷风[1] 胡正军[1] 姜旭淦[1] 邵启祥[1] 丁庆[1] 许化溪[1] 

机构地区：[1]江苏大学检验医学研究所免疫学研究室,镇江212013 [2]江苏大学附属医院,镇江212001

出　　处：《中国免疫学杂志》2009年第8期675-677,683,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金(30871193);江苏省自然科学基金(BK2004405);江苏大学拔尖人才培养工程和高级人才启动基金(05JDG042)资助

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。Objective： To construct the prokaryotic expression vector and to express soluble GITRL fusing protein. Methods： The GITRL gene was digested by EcoR Ⅰ and Sal Ⅰ , 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 andibedy 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 anti- body. All of these results provide a foundation for further studying the biological function of GITRL.

关 键 词：GITRL 蛋白表达 融合蛋白 小鼠 

分 类 号：Q78[生物学—分子生物学]