Gene Cloning, Expression, and Immunological Characterization of Kexin in RatPneumocystis carinii

Abstract

Objective: Pneumocystis is an opportunistic pathogen that often causes fatal pneumonia in immunocompromised patients. It remains an important cause of morbidity and mortality in immunocompromised hosts. Therefore, the development of a vaccine is needed. Kexin is a furin-like protease which may be involved in the proteolytic processing of other proteins of Pneumocystis. We focused on the cellular and humoral responses to the kexin antigen fragment of Pneumocystis in our study. Methods: The full-length DNA sequence of the kexin gene was amplified from rat Pneumocystis carinii (P. carinii). The recombinant plasmid pET-kexin was constructed, and the recombinant antigen Hiskexin was purified, respectively. Then the mice were immunized with His-kexin. To ensure the isolated protein used in the next experiments was purified and of the right molecular weight, Western blotting analysis was performed. Then the specific humoral immunity was measured by ELISA. The specific cellular immunity was measured by the proliferation response of spleen lymphocytes. Results: The full-length Pneumocystis kexin gene was cloned successfully. The sera could recognize another recombinant antigen GSTkexin containing the identical amino acid sequence of kexin fragment with His-kexin, as identified by Western blotting and ELISA. The splenocytes collected from the mice immunized with His-kexin had significantly proliferative responses to GST-kexin compared to the control animals. Conclusions: These findings showed that kexin was the potential antigen to induce immune defense against P. carinii infection. The result would provide basis for the further vaccine study in the development of immunoprophylaxis and immunotherapy against P. carinii infection.