肺炎支原体OsmC蛋白的原核表达及活性分析

发布时间：2018-02-25 02:15

  本文关键词： 肺炎支原体 渗透压诱导蛋白C 克隆 表达　出处：《中国病原生物学杂志》2016年05期 　论文类型：期刊论文

【摘要】：目的表达肺炎支原体(Mycoplasma pneumoniae)渗透压诱导蛋白C(OsmC)并测定其活性。方法在Mp中找出编码OsmC蛋白的基因,根据Mp OsmC基因序列设计特定引物,PCR扩增OsmC基因。利用EcoRI和XhoI对其进行双酶切,然后连接到表达载体pET28a,构建重组质粒pET28a-MpOsmC,转化大肠埃希菌BL21(DE3),IPTG诱导目的蛋白表达,采用镍柱亲和层析对重组OsmC蛋白进行纯化,利用氧化铁二甲酚橙试剂(FOX)测定OsmC蛋白的活性。结果肺炎支原体Mpn625基因编码OsmC蛋白。PCR扩增OsmC基因片段大小为426bp,连接到原核表达载体pET28a得到重组质粒pET28a-MpOsmC。重组质粒转化大肠埃希菌BL21(DE3),IPTG诱导表达分子质量单位为19.4ku的重组Mp OsmC蛋白,与理论值相符。通过亲和层析,得到高纯度的目的蛋白。FOX试剂法测定Mp OsmC具有分解H_2O_2活性。结论重组Mp OsmC蛋白具有氧化酶活性,为探讨肺炎支原体的抗氧化机制提供了理论依据。
[Abstract]:Objective the expression of Mycoplasma pneumoniae (Mycoplasma pneumoniae) osmotic pressure induced protein C (OsmC) and determine its activity. Methods to find the encoding OsmC protein in Mp gene, the specific primers designed according to the Mp sequence of OsmC gene, PCR gene was amplified by OsmC. The double enzyme digestion by EcoRI and XhoI, and then connected to the expression vector pET28a. The recombinant plasmid pET28a-MpOsmC was transformed into E.coli BL21 (DE3), the expression of target protein was induced by IPTG, using nickel affinity chromatography of recombinant OsmC protein was purified using ferric oxide two reagent xylenol orange (FOX) determination of the activity of the OsmC protein. The results of Mycoplasma pneumoniae Mpn625 gene encoding OsmC protein.PCR amplified OsmC gene fragment size is 426bp. Connected to the prokaryotic expression vector pET28a to obtain recombinant plasmid pET28a-MpOsmC. recombinant plasmid was transformed into Escherichia coli BL21 (DE3), IPTG induced expression of a molecular mass of recombinant 19.4ku Mp Osm The C protein is consistent with the theoretical value. By affinity chromatography, a high purity target protein is obtained. The.FOX reagent method for the determination of Mp OsmC has the ability to decompose H_2O_2 activity. Conclusion the recombinant Mp OsmC protein has the activity of oxidase, which provides a theoretical basis for exploring the antioxidant mechanism of Mycoplasma pneumoniae.

【作者单位】： 邵阳医学高等专科学校;南华大学病原生物学研究所;
【基金】：湖南省科技计划项目(No.2013FJ3067)
【分类号】：R375.2
，

本文编号：1532628