人型支原体毒力相关蛋白D的原核表达与纯化  

Prokaryotic expression and purification of virulence-associated protein D from Mycoplasmahominis

作　　者：董巍檑[1] 马艳[1] 刘杰[1] 刘良专[2] 李冉辉[2] 何璐[1] 

机构地区：[1]南华大学附属第一医院妇产科,湖南衡阳421001 [2]南华大学医学院病原生物学研究所,特殊病原体防控湖南省重点实验室

出　　处：《中国病原生物学杂志》2016年第6期509-512,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31500156);湖南省分子靶标新药研究协同创新中心项目(湘教通2014-405号);特殊病原体防控湖南省重点实验室资助项目[湘科计字(2014)5号;湘教通(2012)312号]

摘　　要：目的原核表达并纯化人型支原体(Mycoplasma hominis,Mh)毒力相关蛋白D(Virulence-associated protein,VapD)。方法从GenBank查找Mh VapD基因序列,设计引物并进行优化,合成VapD片段并以此为模板,PCR扩增VapD,然后用EcoRI和XhoI进行双酶切并连接表达载体pET28a,构建原核重组质粒pET28a-VapD,转化大肠埃希菌BL21(DE3),IPTG诱导目的蛋白表达,重组产物经镍柱亲和层析纯化。结果合成的VapD基因全长为303bp,连接原核表达载体pET28a得到重组质粒pET28a-VapD。重组质粒转化大肠埃希菌BL21(DE3),IPTG诱导表达分子质量标准为15ku的重组Mh VapD蛋白,与理论分子质量大小相符合。VapD蛋白具有可溶性,纯化后的目的蛋白电泳检测为单一条带。结论成功构建原核重组载体pET28a-VapD并纯化获得VapD蛋白,为进一步研究VapD蛋白的致病机制奠定了基础。Objectives To express virulence-associated protein D （VapD） of Mycoplasma hominis in a prokaryote and purify that protein. Methods The DNA sequence of the M. hominis VapD gene was obtained from GenBank and transformed in E. coli. VapD was synthesized and amplified with PCR using specific primers. VapD was double-digested with EcoRI and XhoI and the gene was ligated into pET-28a to generate the prokaryotic expression vector pET28a-VapD. pET28a-VapD was transformed into E. coli BL21 （DE3） and protein expression was induced with IPTG. Recombinant VapD protein was purified with Ni2＋-affinity chromatography. Results The VapD gene was 303 bp in length. The plasmid pET28a-VapD, which encodes the N-terminal His-tagged VapD protein, was successfully constructed. Upon in duction of protein expression with IPTG, E. coli BL21 （DE3） cells harboring the plasmid pET28a-VapD expressed a soluble protein with the expected molecular mass of 15 ku, which was identical to the theoretical molecular mass of the His- tagged VapD protein. The VapD protein was purified to an electrophoretically homogeneous state. Conclusion The prokaryotic expression vector pET28a-VapD was constructed and the VapD protein was purified. This study has helped to investigate the potential role of the VapD protein in the pathogenetic mechanisms of M. hominis infection.

关 键 词：人型支原体 毒力相关蛋白D 克隆 蛋白纯化 

分 类 号：R375[医药卫生—病原生物学]