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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 the eukaryotic expression vector pES2-EGFP-C5a with His-tagged rat complement C5a and the prokaryotic expression vector pET-21a-C5a and to observe its in vitro expression. The C5a protein was purified and analyzed for biological activity. Methods: The complete cDNA sequence of rat C5a gene was amplified by RT-PCR from rat hepatocytes. Six histidine His tags were added to the N-terminus of the sequence and inserted into the eukaryotic expression vector containing the enhanced green fluorescent protein The plasmid pIRES2-EGFP was transfected into HEK293 cells by lipofectamine 2000. The expression of C5a protein was detected by Western blotting. The C5a gene of rat complement was subcloned into prokaryotic expression based on the eukaryotic expression plasmid pIRES2-EGFP-C5a The vector pET-21a was used to detect the expression of C5a protein in vitro. Then C5a protein was purified by Ni2 + chelating affinity chromatography. C5a-stimulated neutrophils, flow cytometry detection of intracellular reactive oxygen species; C5a stimulation of rat glomerular mesangial cells (GMC) at different times, measured by RT-PCR to produce IL-6 and TNF-α Level. Results: The rat C5a gene was amplified by RT-PCR. The recombinant plasmid pIRES2-EGFP-C5a was successfully constructed and transfected into HEK293 cells in vitro. The expression of strong green fluorescent protein (EGFP) was observed under fluorescence microscope. However, The expression of C5a protein was not detected by Western blotting method. The prokaryotic expression vector pET-21a-C5a was successfully constructed and the C5a protein expression was detected by Western blot. In addition, the His-tagged C5a protein was purified by affinity chromatography and confirmed that C5a can promote neutrophil respiratory burst. Conclusion: The rat C5a eukaryotic and prokaryotic expression vector was successfully constructed. The constructed prokaryotic expression vector can detect the expression of C5a protein, and the C5a protein has the corresponding biological activity.