猪补体C5a蛋白表达、纯化和生物学活性检测  

Prokaryotic expression,purification and bioactivity detection of swine complement C5a protein

作　　者：陈漫[1] 李志萍[1] 王习文[1] 王园园[1] 董晓琳[1] 高玮村 李博[1] 李乾学[1] 夏志平[1] 

机构地区：[1]军事医学科学院军事兽医研究所/吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国兽医学报》2016年第4期630-634,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目"猪源C5a适配体抑制炎症的机理研究"(31402167);武汉军科博源生物股份有限公司合作项目"广谱抗炎猪C5a适配体药物研发"资助项目

摘　　要：参照NCBI中公布的猪补体C5a蛋白氨基酸序列及猪C5蛋白的全基因序列,通过生物信息学分析获得猪C5a蛋白基因全长,为222 bp,该序列定向插入原核表达载体p ET-28a中,转化E.coli BL21(DE3),IPTG诱导表达,SDSPAGE和Western-blotting检测分析表明,C5a基因获得表达,重组蛋白大小为10 000。利用镍柱亲和层析进行蛋白纯化后,体外细胞试验测定重组C5a蛋白生物学活性。结果显示,成功构建了p ET-28a-C5a重组质粒,纯化获得目的蛋白纯度达到90%以上,质量浓度为2.5 g/L。细胞试验检测到C5a具有趋化和诱导中性粒细胞的活性。本研究为揭示猪C5a参与炎症过程的作用机制及制备C5a拮抗剂奠定了基础。Referring to the amino acid sequence of swine complement C5a and the gene sequence of swine C5 protein which published in NCBI, and by bioinformatic analysis, the full-length gene of porcine protein C5a was 222 bp. This sequence was cloned into prokaryotic expression vector pET- 28a and transformed into E. coli BL21 （DE3） for expression under induction of IPTG. SDS-PAGE and Western-blotting analysis showed that the expressed recombinant protein C5a was about 10 000. The protein was purified by Ni-NTA affinity chromatography and determined biological activity in vitro. The results showed that the pET-28a-C5a recombinant plasmid was constructed successfully,the purity of protein more than 90% at the concentration of 2.5 g/L. C5a had the ac- tivity to chemotactic and induction neutrophil. This study revealed that swine CSa protein involved in the inflammatory process and laid the foundation for the preparation of CSa antagonists.

关 键 词：猪C5a蛋白 原核表达 蛋白纯化 活性检测 

分 类 号：Q955[生物学—动物学]