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
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

国家自然科学基金(81072402;8127333)


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

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    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。

    Abstract:

    Objective:To construct eukaryotic expression vector pIRES2-EGFP-C5a,prokaryotic expression vector pET-21a-C5a 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 transfection 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 C5a 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 tranfected 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-C5a plasmid were successfully constructed,and the expression of C5a in prokaryotic expression vector pET-21a-C5a could be detected and the product was biological active.

    参考文献
    相似文献
    引证文献
引用本文

季明德,单 锴,庞蓉蓉,赵 聃,王迎伟.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[J].南京医科大学学报(自然科学版),2013,(10):1344-1350

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2013-06-06
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2013-10-23
  • 出版日期:
通知关闭
郑重声明