肺炎支原体OsmC蛋白的原核表达及活性分析  

Prokaryotic expression,purification,and determination of the peroxidase activity of the OsmC protein from Mycoplasma pneumoniae

作　　者：唐愈菲[1,2] 朱翠明[2] 黄泽智[1] 赵爽[1] 

机构地区：[1]邵阳医学高等专科学校,湖南邵阳422000 [2]南华大学病原生物学研究所,湖南衡阳421001

出　　处：《中国病原生物学杂志》2016年第5期393-396,400,共5页Journal of Pathogen Biology

基　　金：湖南省科技计划项目(No.2013FJ3067)

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。Objectives The aims of this study were to express the osmotically inducible protein C（OsmC）of Mycoplasma pneumoniae in a prokaryote,to purify that protein,and to evaluate its antioxidant capabilities. Methods M.pneumoniae was searched for the gene sequence coding for OsmC and PCR primers were designed to amplify the OsmC of M.pneumoniae.The resulting gene sequence coding for OsmC was double digested with the restriction endonucleases EcoRI and XhoI and the sequence was then ligated into pET-28 ato generate the recombinant plasmid pET28a-MpOsmC.pET28a-MpOsmC was transformed into E.coli BL21（DE3）.Expression of recombinant 6＊His-tagged OsmC protein was induced using IPTG and purified using Ni 2＋-affinity chromatography.Recombinant OsmC was tested for its peroxidase activity on H_2O_2 using a ferrous oxidation xylenol orange（FOX）assay. Results The Mpn625 gene of M.pneumoniae codes for the OsmC protein,and this gene had 426 bp.After amplification with PCR,OsmC of M.pneumoniae was inserted into pET28 a,resulting in the recombinant plasmid pET28a-MpOsmC.After its expression was induced with IPTG,E.coli BL21（DE3）cells harboring the plasmid pET28a-MpOsmC expressed a protein with a molecular mass of19.4ku,which is identical to the theoretical molecular mass of His-tagged OsmC.OsmC was successfully purified.FOX results indicated that the OsmC protein of M.pneumoniae decomposed H_2O_2 in vitro. Conclusion This study has indicated that purified OsmC protein from M.pneumoniae has peroxidase activity,and this finding will help to interpret the mechanisms by which M.pneumoniae withstands oxidative stress.

关 键 词：肺炎支原体 渗透压诱导蛋白C 克隆 表达 

分 类 号：R375[医药卫生—病原生物学]