Recombinant Mycobacterium smegmatis expressing Hsp65-hIL-2 fusion protein and its influence on lymphocyte function in mice

摘要

ABSTRACT Objective:To construct a strain of recombinantMycobacterium smegmatis expressing the heat shock protein65(Hsp65) and human interleukin2 (IL-2) fusion protein (rMS-Hsp65/IL-2) and to explore the effect of this construct on lymphocyte function in mice.Methods:The fusion gene encodingHsp65-hIL-2 was cloned into shuttle vector pSMT3. The recombinant plasmid pSMT3-Hsp65-hIL-2 was transferred toMycobacterium smegmatis by electroporation. Positive clones were selected by hygromycin and identified by PCR. The expression of fusion protein Hsp65-hIL-2 was verified using indirect immunofluorescence staining. Mice were immunized for two times by subcutaneously injection with1í106CFU rMS-Hsp65/IL-2 at a three-week interval. Two weeks after the second immunization, mice were sacrificed and the serum samples were collected for determination of anti-Hsp65 specific IgG. Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity byMTT assay.IFN-γ andIL-2 in the medium of the treated cells were also determined byELISA.Results:Successful construction of rMS-Hsp65/IL-2 was verified byPCR and immunofluorescence staining. Compared to the splenic lymphocytes isolated from mice immunized with Bacille Calmette-Guerin or mice immunized with Mycobacterium smegmatis alone, the splenic lymphocytes isolated from mice immunized with rMS-Hsp65/IL-2 showed a marked increase in the proliferation of lymphocytes, together with an increased production of important cytokines such asIFN-γandIL-2. Conclusions: rMS-Hsp65/IL-2markedly enhances lymphocyte function. Therefore, the fusion protein generated by rMS-Hsp65/IL-2 may be of potential value in generating an effective vaccine against tuberculosis.