人L-ficolin基因重组蛋白的表达及功能研究

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。

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

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.coli.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(M_(r)).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.