Abstract: The genome DNA of Mycobacterium bovis(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.bovis 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.bovis 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.