title: Recombinant Mycobacterium smegmatis expressing Hsp65-hIL-2 fusion protein and its influence on lymphocyte function in mice.
abstract: 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-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 anti-Hsp65 specific IgG. Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity by MTT assay. IFN-γ- and 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-γ-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.
authors.affiliation: Animal Medical College, Gansu Agricultural University, Lanzhou, Gansu Province, China.
authors.forename: Xiao-Qing
authors.initials: XQ
authors.lastname: Guo
authors.forename: Yan-Ming
authors.initials: YM
authors.lastname: Wei
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D049109:Cell Proliferation; D018834:Chaperonin 60 / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D005260:Female; D007074:Immunoglobulin G / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism*; D008214:Lymphocytes / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D020102:Mycobacterium smegmatis / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins; D013154:Spleen / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D016207:Cytokines; D007074:Immunoglobulin G; D007376:Interleukin-2; D011994:Recombinant Proteins; C074119:heat-shock protein 65, Mycobacterium