Heat shock protein 65 (Hsp65) is an important immunodominant antigen against tuberculosis (TB), and interleukin‐2 (IL‐2) plays an important role in the regulation of antimycobacteria immune responses. In order to further increase the immunogenicity of Hsp65 against infection caused by Mycobacterium tuberculosis (MTB), we expressed MTB Hsp65 and human IL‐2 fusion protein, Hsp65‐hIL‐2, in Escherichia coli. The expression of Hsp65‐hIL‐2 was confirmed by Western blotting using anti‐Hsp65 MoAb and anti‐hIL‐2 MoAb, respectively. Hsp65‐IL‐2 and Hsp65 were then purified by Ni‐NTA affinity chromatography. Mice were immunized with purified Hsp65‐hIL‐2 or Hsp65 emulsified in the adjuvant combination dimethyl dioctadecylammonium bromide and monophosphoryl lipid A. Eight weeks after immunization, there was significant proliferation of spleen lymphocytes in response to both Hsp65 and Hsp65‐hIL‐2 proteins. Interestingly, Hsp65‐hIL‐2 fusion protein elicited significantly higher levels of IFN‐γ and IL‐2 in the lymphocytes culture supernatant than that of the BCG (Denmark strain) immunized group and Hsp65 group (P < 0.05). After challenging the immunized mice with MTB, the bacteria loads in the spleens and lungs of mice immunized with the fusion protein were significantly lower than Hsp65 alone group, reaching an equivalent level as BCG immunization group. Our results suggest that the Hsp65 and hIL‐2 fusion protein may serve as an alternative vaccine candidate against MTB infection.

The low production level is one of the limitation factors for decreasing the prices of enzymes and expanding their application in industry as the biocatalysts. This research focuses on developing lipase isozyme co-expression strategies in Pichia pastoris to enhance the expression level of overall lipase isozymes which catalyse the same reaction. The Kex2-mediated co-expression strategy of lipase isozymes could potentially enhance the overall isozyme expression, and isozyme co-expression might provide a new direction for improving the recombinant isozyme expression, and decreasing the production and application prices of these mixed enzymes as biocatalysts.