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中文摘要：
目的：构建含小鼠G1TRL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-pMDl8．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ilrrG诱导GITRL蛋白表达，经Ni^＋-NTA层析柱纯化融合蛋白，通过SDS-PAGE、Westem blot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Westemblot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G1TRL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G1TRL融合蛋白生物学活性的进一步研究。
英文摘要：
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.
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