中文摘要：

为了阐明牛分枝杆菌侵入宿主细胞和在肺泡巨噬细胞长期存活的机理，本研究以牛分枝杆菌的DNA为模板，通过PCR的方法扩增克隆牛分枝杆菌哺乳动物细胞侵袭蛋白4E（Mammalian cell-entry protein 4E，mce4E）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上所公布的牛分枝杆菌和人分枝杆菌mce4E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-blotting免疫印迹鉴定，结果证明目的蛋白获得高效表达，表达量占菌体总量的53．3％，分子量约为45ku；利用Ni-NTA琼脂糖柱对表达蛋白进行纯化，纯化率大于95％；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，为进一步开展牛分枝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。

英文摘要：

The genome DNA of Mycobacterium boris （C68-2） was extracted by CTAB method. A pair of primers was designed and synthesized according to the sequences of mammalian cell-entry proteins 4E（mce4 E） gene of M. boris in GenBank. The PCR product was inserted into pGEM-T vector. Then the expression plasmid was constructed by inserting the target gene fragment into pET30a（＋） vector. By transforming recombinant pET30a-mce4E into BL21, the mce4E protein of M. boris was expressed in 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 refolded 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 new insights into the pathology for mammalian, and provide good approach for further research for the target site of medicine action.