小鼠4-1BB-Fc分子的真核表达及其体外抑制T细胞增殖的效应  

The eukaryotic gene expression and suppression effect on T cell proliferation in vitro of mouse recombinant 4-1BB-Fc molecule

作　　者：黄保军[1] 徐军发[1] 熊平[1] 冯玮[1] 徐勇[1] 郑芳[1] 方敏[1] 龚非力[1] 

机构地区：[1]华中科技大学同济医学院免疫学系

出　　处：《中华微生物学和免疫学杂志》2006年第8期675-679,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家重点基础研究发展规划(973)资助项目(No.2001cb500008)

摘　　要：目的克隆并构建含小鼠4-1BB胞外段和人IgG Fc融合基因的真核表达质粒,表达具有生物学活性的4-1BB-Fc融合蛋白,初步探讨其体外生物学效应。方法借助RT-PCR技术,从小鼠脾脏总RNA中扩增4-1BB全长编码基因,并导入克隆载体pGEM-T Easy。经测序证实,用PCR扩增其膜外区cDNA,酶切后与hIgG1 Fc基因一起装入真核表达载体pcDNA3．1中。应用脂质体将重组子pcDNA3．1-4-1BB-Fc转染COS-7及CHO细胞,经G418筛选,获得稳定表达4-1BB-Fc融合蛋白的CHO细胞株。双抗体夹心FLISA检测融合蛋白表达,经蛋白A亲和层析纯化,借助免疫印迹进行鉴定。应用FACS检测融合蛋白与DC细胞系DC2．4表面4-1BBL结合情况。采用MTT及CFSE(羧基荧光素乙酰乙酸)标记法检测4-1BB-Fc融合蛋白对同种异基因小鼠T细胞增殖的抑制作用。结果经测序证实,所克隆和构建的小鼠4-1BB-Fc cDNA阅读框及连接部位序列正确;ELISA与免疫印迹证实4-1BB- Fc蛋白的表达及其对T细胞增殖具有明显抑制作用。结论成功构建4-1BB-Fc融合基因并获稳定表达,可望用于探讨4-1BB参与移植排斥的作用及其机制,并为研究4-1BB-Fc的其他生物学作用奠定初步基础。Objective To clone and construct eukaryotic expression plasmid containing mouse 4-1BB extra-membrane encoding region and human IgG Fc fusion gene, express 4-1BB-Fc fusion protein with high biological activity, and study its biological effect in vitro. Methods Mouse full-length 4-1BB cDNA was cloned from total RNA of the mouse spleen with RT-PCR technique, ligated to the pGEM-T Easy vector and identified by sequence scanning. Its extra-membrane encoding region was cloned, digested by restrictive enzyme and inserted into the eukaryotic expression plasmid pcDNA3.1 together with human IgG1 Fc cDNA. The recombinant plasmid, pcDNA3.1-4-1BB-Fc, was transfected into COS-7 and CHO cells by using Lipofect AMINE^TM2000, and the CHO cell lines stably expressing the fusion protein was obtained through G418 selection. 3he 4-1BB-Fc protein was purified by protein A affinity chromatography column. The expression of 4-1BB-Fc was identified by sandwich ELISA and Western blot. The binding of 4-1BB-Fc protein to 4-1BBL expressed in DC ceil line DC2.4 was determined by flow cytometry （FACS）. The suppression effect of 4-1BB-Fc on T lymphocyte proliferation in vitro was tested by MTF and CFSE-labeling method. Results The ORF and linking sequence of4-1BB-Fc gene was coincident with what was expected. ELISA and Western blot confirmed protein expression in CHO ceils and the purified 4-1BB-Fc protein was proved to suppress T lymphocyte proliferation in vitro. Conclusion The 4-1BB-Fc eukaryotic expression vector was successfully constructed and a purified recombinant 4-1BB-Fc protein with biological activity was obtained. This lays the experimental foundation for further studies on the role of 4-1BB-Fc in transplant rejection and other biological functions.

关 键 词：小鼠4-1BB-Fc 真核表达 稳定转染 混合淋巴细胞反应 

分 类 号：R392[医药卫生—免疫学]