人ICOSIg的真核表达及其对MLR反应的影响

Expression of ICOSIg and its effects on MLR

目的构建人ICOSIg基因的真核表达载体,表达并鉴定ICOSIg蛋白,初步研究其对MLR中细胞增殖以及对IL-10分泌的影响。方法从活化的人T细胞cDNA文库中以PCR方法扩增ICOS胞外区cDNA编码基因,并和人IgG1Fc基因一起装入真核表达载体pcDNA3中,采用脂质体转染COS-7细胞,G418筛选,用ELISA检测其表达情况,经蛋白A亲和纯化,用SDS-PAGE和Westernblotting进行鉴定。进一步通过3H-TdR掺入法和夹心ELSIA法探讨其对双向MLR反应中细胞增殖以及对IL-10分泌的影响。结果酶切和测序证实构建的ICOS膜外区和人IgG1Fc段基因序列正确、阅读框完整;ELISA、SDS-PAGE、Westernblot验证ICOSIg的正确表达;功能实验提示ICOSIg能抑制MLR中的细胞增殖和IL-10的分泌。结论成功构建并表达了ICOSIg,而获得的ICOSIg能抑制MLR。

Objective To construct and express the eukaryotic expression plasmid of hICOSIg gene, and to study the effects of ICOSIg on MLR. Methods The extramembrane encoding region of ICOS was cloned from an activated human T cell cDNA library by PCR. After (digestion) with restriction enzyme, the correct region together with human IgG1-Fc cDNA was inserted into the eukaryotic expression plasmid (pcDNA3). The right recombinant was transfected into COS-7 cells with DOTAP. After purified with protein A affinity column chromatography, ICOSIg protein was identified by sandwich ELISA, SDS-PAGE, and Western blotting. And then, the effects of ICOSIg protein on cell proliferation and IL-10 secretion in MLR was investigate by (~3H)-TdR and sandwich ELISA. Results The ORF of ICOSIg gene was coinci dent with what we had expected and protein was correctly expressed and could inhibit cell proliferation and IL-10 secretion in MLR. Conclusion ICOSIg vector is constructed successfully and the recombinated ICOSIg plays a inhibiting role in MLR.[