Abstract: Objective：To construct recombinant Mycolicibacterium smegmatis（MS） expressing Mycobacterium tuberculosis EsxV/W
fusion protein and analyze the phenotype of the recombinant MS and its effect on the macrophage function. Methods: The EsxV/W
fusion protein was obtained by prokaryotic expression, and polyclonal antibody was prepared by immunizing mice with purified EsxV/W
fusion protein. The recombinant strain MS-EsxV/W and the control strain MS-vec were constructed by electroporation, and the
expression of the fusion protein in MS was detected by using the generated polyclonal antibody. The colony morphology, biofilm
formation, and growth rate of the recombinant MS expressing EsxV/W fusion protein were analyzed. The survival ability of the
recombinant MS under different conditions, such as lysozyme, malachite green, low pH, SDS, H2O2 and NaNO2, was determined using
colony counting method to assess the impact of EsxV/W fusion protein on the phenotype of MS. The recombinant MS was used to infect
ANA-1 macrophages. The release of NO from ANA-1 cells was detected by Griess method. Enzyme-linked immunosorbent assay was
performed to measure the release of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） from ANA-1 cells, and the survival
ability of the recombinant MS in ANA-1 cells was determined using colony counting method. Results：The recombinant strain MSEsxV/
W expressing EsxV/W fusion protein, was successfully constructed. The expression of EsxV/W fusion protein had no significant
effect on the colony morphology, biofilm formation and growth rate of MS. The expression of EsxV/W fusion protein did not change the
tolerance of MS to lysozyme, malachite green or low pH, but decreased the tolerance to SDS and increased the tolerance to H2O2 and
NaNO2. After infecting ANA-1 cells for a certain period of time, the release of NO, TNF-α and IL-6 in ANA-1 cells infected by MSEsxV/
W was significantly increased compared with MS-vec, and the intracellular survival ability of MS-EsxV/W was higher than that
of MS-vec. Conclusion：The EsxV/W fusion protein can change the phenotype of MS and regulate the immune response of ANA-1
macrophages, which is conducive to the subsequent investigation of the function of esxV/W gene

Key words: Mycobacterium tuberculosis, Mycolicibacterium smegmatis, EsxV/W fusion protein, ANA-1 cells