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

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• 作者：Xiao-Qing Guo^a ; Yan-Ming Wei^a ; ^{weiym@gsau.edu.cn} ; Bo Yu^b ; ^{yub06227@163.com}
• 关键词：Mycobacterium tuberculosis ; Mycobacterium smegmatis ; Hsp65 ; IL-2 ; Fusion protein ; Lymphocytes ; Mice
• 刊名：Asian Pacific Journal of Tropical Medicine
• 出版年：2012
• 期刊代码：pca68_19957645
• 类别：med
• 出版时间：May, 2012
• 卷：5
• 期：5
• 页码：347-351
• 文件大小：371 K

摘要

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脳10⁶ 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-纬- 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-纬-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.