牛分支杆菌哺乳动物细胞侵入蛋白4E（mce4E）的原核表达与二级结构分析
Expression and secondary structural analysis of Mammalian cell-entry proteins 4 E (mce4 E) in Mycobacterium bovis
为了阐明牛分支杆菌侵入宿主细胞和在肺泡巨噬细胞长期存活的机理，进一步研究牛分支杆菌的致病机理，以牛分支杆菌的DNA为模板，通过PCR的方法扩增克隆牛分支杆菌哺乳动物细胞侵入蛋白4E（Mammalian cell-entry protein 4E，mce4 E）基因,将所扩增基因克隆于原核表达载体pET30a(+)并进行测序,结果显示该基因与GenBank上所公布的牛分支杆菌和人分支杆菌哺乳动物细胞侵入蛋白mce4 E基因同源性为100%。将重组表达载体转入宿主菌BL21进行诱导表达,表达蛋白经SDS -PAGE分析和Western－blot免疫印迹鉴定,结果证明目的蛋白获得高效表达并以包涵体的形式存在, 表达量占菌体总量的53.3%，大小约为45kDa； 利用Ni-NTA琼脂糖柱对表达蛋白进行纯化，纯化率在95%以上；纯化的蛋白经过透析复性、圆二色谱(CD)测定，结果表明重组蛋白为典型的α螺旋型结构，经Jascow32软件分析计算，mce4E蛋白含有39.1%的α螺旋，60.9%的无规卷曲， 无β-折叠和转角，为进一步研究开展牛分支杆菌致病机理的研究和寻找新的药物作用靶位点奠定了基础。
Mammalian cell-entry proteins 4 E (mce4 E) gene was cloned and expressed in pET30a(+) vector, after sequencing, it was high homogeneity (100%) with mammalian cell-entry proteins 4E in human and bovine. Positive plasmid was transformed into BL21 of E.coli. The expressed truncated His-tagged proteins accumulated in inclusion bodies. Purification was performed on a nitrilotriacetic acid (Ni-NTA) agarose column, and renaturation was performed by dialysis. Final renatured protein was identified by SDS-PAGE and western blotting assay. The protein conformation was analyzed by circular dichroism (CD) analysis. The content of secondary structure was α-helix (39.1%) and freedom curl (60.9%), without β-sheet and turn. Our research give some insights into the pathology for mammalian, which will provide good approach for further research for the target site of medicine action.
杨建民、周向梅、赵德明、徐广贤、尹晓敏
基础医学分子生物学微生物学
牛分支杆菌，哺乳动物细胞侵入蛋白，原核表达，圆二色谱（CD）
Mycobacterium bovis Mammalian cell-entry proteins 4 E (mce4 E)，Porkaryotic expression，circular dichroism (CD)
杨建民,周向梅,赵德明,徐广贤,尹晓敏.牛分支杆菌哺乳动物细胞侵入蛋白4E（mce4E）的原核表达与二级结构分析[EB/OL].(2006-02-27)[2025-05-17].http://www.paper.edu.cn/releasepaper/content/200602-259.点此复制
评论