大鼠补体C5a蛋白真核、原核表达载体的构建以及体外表达的鉴定、纯化和生物学活性分析

Construction,identification,purification and biological activity of eukaryotic and prokaryotic expression vector of rat complement C5a

目的:构建带His标签的大鼠补体C5a蛋白真核表达载体pIRES2-EGFP-C5a和原核表达载体pET-21a-C5a,分别观察其体外表达情况,将C5a蛋白纯化后分析其生物学活性。方法:以RT-PCR方法从大鼠肝细胞中扩增出大鼠补体C5a基因全长cDNA序列,在序列的N端添加6个组氨酸His标签后,插入含增强绿色荧光蛋白的真核表达质粒pIRES2-EGFP,用脂质体法转染HEK293细胞,免疫印迹法检测C5a蛋白的表达水平;以真核表达质粒pIRES2-EGFP-C5a为基础,将大鼠补体C5a基因亚克隆至原核表达载体pET-21a,体外检测C5a蛋白的表达情况,之后再行Ni2+螯合亲和层析柱纯化C5a蛋白。以C5a刺激中性粒细胞,流式细胞仪检测细胞内活性氧的生成;C5a刺激大鼠肾小球系膜细胞(GMC)不同时间,RT-PCR测定其产生IL-6和TNF-α的水平。结果 :RT-PCR扩增出了大鼠补体C5a基因;并成功构建了pIRES2-EGFP-C5a重组质粒,体外成功转染入HEK293细胞,并在荧光显微镜下可见强绿色荧光蛋白的表达,但免疫印迹法未能检出C5a蛋白的表达;另成功构建了pET-21a-C5a原核表达载体,并在体外用免疫印迹法检测到了C5a蛋白表达。另用亲和层析纯化得到了带His标签的C5a蛋白,证实C5a能促进中性粒细胞呼吸氧爆发。结论:成功构建了大鼠补体C5a真核和原核表达载体。构建的原核表达载体可测到C5a蛋白的表达,且C5a蛋白具有相应的生物学活性。

Objective：To construct eukaryotic expression vector pIRES2-EGFP-CSa,prokaryotic expression vector pET-21a-CSa of rat complement C5a containing histidine tag, and observe its expression and biological activity in vitro. Methods：Total RNA was isolated from rat liver cells, C5a gene was amplified by reverse transcriptional PCR and inserted into pIRES2-EGFP vector. After lipofectamine-mediated transfeetion of HEK293 cells with pIRES2-EGFP plasmid,the expression of rat C5a protein was determined by fluorescence microscope and Western blot. The prokaryotic expression vector of pET-21a-C5a was constructed,and the expression of C5a protein was determined by Western blot. Rat CSa was purified by Ni2. chelating affinity chromatography column. The reactive oxidative species （ROS） generated from neutrophils which were stimulated by C5a was detected by flow analyzer. The mRNA of IL-6 and TNF-α was detected after rat GMCs were stimulated by C5a for different time. Results：The rat complement C5a gene was amplified by reverse transcriptional PCR successfully. The pIRES2-EGFP-C5a plasmid was successfully constructed and tranfeeted into HEK293 cells. The expression of green fluorescent protein was seen under by fluorescence microscopy,but no C5a was detected. Meantime,the pET-21a-C5a plasmid was also constructed,and the expression of C5a could be detected using Western blot. And the histidine tagged C5a protein was purified by Ni2＋ chelating affinity chromatography column. C5a can stimulate the nertrophils to generate ROS. Conclusion：The pIRES2-EGFP-C5a plasmid and pET-21a-C5aplasmid were successfully constructed,and the expression of C5a in prokaryotic expression vector pET-21a-CSa could be detected and the product was biological active.