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

Objective: To construct a strain of recombinant Mycobacterium smegmatis expressing the heat shock protein 65(Hsp65) and human interleukin 2 (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 encoding Hsp65-hIL-2 was cloned into shuttle vector pSMT3. The recombinant plasmid pSMT3-11sp65-hIL-2 was transferred to Mycobacterium 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 immunofluoreseence staining. Mice were immunized for two times by subcutaneously injection with 1x10(6) CFC 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 IgC. Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity by MTT assay. IFN-gamma and IL-2 in the medium of the treated cells were also determined by ELISA. Results: Successful construction or rMS-Hsp65/IL-2 was verified by PCR 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 as IFN-gamma and IL-2. Conclusions: rMS-Hsp65/IL-2 markedly 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.