Abstract: Objective To construct recombinant plasmid pET28a-Rv0757 with Mycobacterium tuberculosis (Mtb) Rv0757 gene, and to determine the effect of the protein of Rv0757 gene on ANA-1 cells. Methods We recombined the amplified Rv0757 gene into theprokaryotic plasmid pET28a(+). The expressed product was identified by SDS-PAGE and Western blot. Murine macrophages were treated with purified protein PhoP. Survived cells, lactic dehydrogenase (LDH), nitric oxide (NO), and cell apoptosis were detected after the treatment. Results We successfully constructed the recombinant plasmid pET28a-Rv0757. The target protein was confirmed by SDS-PAGE and Western blot. The protein had no significant effect on cell numbers and LDH activities in the culture supernatant, but it inhibited the release of NO and apoptosis of ANA-1 cells. Conclusion pET28a-Rv0757 plasmid with prokaryotic expression was successfully constructed. The targetprotein has no toxicity on macrophages, but it can inhibit NO release and cell apoptosis of ANA-1.