人L-ficolin基因重组蛋白的表达及功能研究  

Construction,Expression and Functional Studies of Recombinant Human L-ficolin

作　　者：陈明[1] 章晓联[1] 

机构地区：[1]武汉大学医学院免疫学系,湖北武汉430071

出　　处：《武汉大学学报（医学版）》2007年第1期16-20,共5页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:30370310);教育部新世纪人才支持计划(编号:NCET-04-0685)

摘　　要：目的:构建人L-ficolin基因的原核表达载体并在大肠杆菌中表达。方法:用PCR方法从质粒pCDNA3-L-ficolin中扩增L-ficolin cDNA片断,并将该片断插入pGEX-KG原核表达载体中,以进行插入基因的融合表达,表达后再用Thrombin切除GST以得到单纯的L-ficolin蛋白,SDS-PAGE和Western blot对表达产物进行鉴定,并以细菌侵袭试验检测所得蛋白的生物学活性。结果:酶切结果证实,成功地构建了pGEX-KG-L-ficolin原核表达载体,并使之在大肠杆菌中获得稳定的表达,表达产物的分子质量(Mr)与预期值相一致。原核表达的L-ficolin蛋白具有调理吞噬作用,促进吞噬细胞吞噬细菌的能力,L-ficolin调理吞噬能力呈剂量依赖关系,并显著高于对照蛋白。结论:成功地构建了重组表达载体pGEX-KG-L-ficolin,经过Thrombin作用后得到的L-ficolin蛋白具有生物学活性,为进一步研究人L-ficolin的功能奠定了基础。Objective： To construct a procaryotic expression vector of human L-ficolin full lenth cDNA, and to express and purify L-ficolin in E. coli for the further study of its functions. Methods： The L-ficolin full length cDNA fragment was amplified and cloned in-frame into the prokaryotic vector pGEX-KG, and expressed as a fusion protein GST-L-ficolin in E. coll. The expressed GST-L-ficolin fusion protein was purified via GST-Sepharose 4B Column and L-ficolin was further purified by Thrombin digestion and identified by SDS-PAGE and Western blot. The bioactivity of the purified L-ficolin protein was measured. Results： The pGEX-KG-L-ficolin was successfully constructed. Western blot analysis showed that L-ficolin and GST-L-ficolin were expressed in E. coli as the predicated molecular mass （Mr）. The recombinant L-ficolin protein significantly enhanced and stimulated the activities of phagocytosis of monocytes to Salmonella bacteria. The phagocytosis activations stimulated by L-ficolin were in a dose dependent manner, and was increased much higher than that of control GST protein. Conclusion： The expression vector pGEX-KG-L-ficolin has been constructed successfully and expressed as a bioactive protein in E. coli, which is helpful for further studying and understanding the roles and mechanism＇s of L-ficolin in protection against microorganisms infection.

关 键 词：L-FICOLIN 原核表达 GST—L-ficolin融合蛋白 

分 类 号：R392.11[医药卫生—免疫学]