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Objective:To coastruct 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- Hsp65-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 immunofluorescence staining.Mice were immunized for two times by subcutaneously injection with 1×10~6 CFU 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 anli-Hsp65 specific IgG.Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity by MTT assay.IFN-γand IL-2 in the medium of the treated cells were also determined by ELISA.Results:Successful construction of 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- 7 and IL-2. Conclusions:rMS-Hsp65/IL-2 markedly enhances lymphocyte function.Therefore,the fusion protein generated by rMS-Hsp65/lL-2 may be of potential value in generating an effective vaccine against tuberculosis.
Objective: To coastruct 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-Hsp65-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 immunofluorescence staining. Mice were immunized for two times by subcutaneously injection with 1 × 10 -6 CFU 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 anli-Hsp65 specific IgG .plenic lymphocytes were isolated and treated with the rMS-Hsp65 / IL-2 to determine lymphocytic proliferation activity by MTT assay. I FN-γand IL-2 in the medium of the treated cells were also determined by ELISA. Results: Successful construction of 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-7 and IL-2. Conclusions: rMS-Hsp65 / IL-2 markedly enhances lymphocyte function. Before, the fusion protein generated by rMS-Hsp65 / lL-2 may be potential value in generating an effective vaccine against tuberculosis.