Cloning Of Kexin Gene Of Rat Pneumocystis Carinii And Immunological Characterization Of Kexin Fusion Protein

Objective: To clone the full-length kexin gene of Pneumocystis carinii, its sequence was amplified by PCR. To estimate immunological characterization of the fusion protein, the prokaryotic expression plasmids were constructed and the fusion proteins which were expressed in E.coli were used as antigen to immunize animals.Methods: The full-length DNA sequence of kexin gene was amplified from rat Pneumocystis carinii. To choose a fragment which would be capable of eliciting the immune responses in the host, the complete kexin gene was analyzed by the method of bioinformatics. Then the fragment was used to construct two plasmids (pGEX-kexin and pET-kexin). The recombinant plasmids were transformed into E.coil BL21 for expression with IPTG inducing, respectively. A great quantity of the recombinant proteins (GST-kexin and His-kexin) were expressed and purified by bag filter.His-kexin was used as the antigen to immunize the mice. GST-kexin was used to detect the anti-His-kexin antibody. Then the level of the anti-His-kexin antibody was measured by ELISA. The proliferation response of splenocytes was measured by lymphocyte proliferation assays. To determine whether the kexin protein has the immunogenicity, western blot analysis was performed. Results: The full-length DNA sequence of 2605bp was obtained by PCR and submitted into GenBank (EU711053). The chosen fragment was inserted into pGEX-4T-1 and pET-28a(+) to construct pGEX-kexin and pET-kexin, respectively. These plasmids were identified by PCR, restriction enzyme digestion and DNA sequencing. The molecular weight of recombinant protein GST-kexin was 43kDa after induced by IPTG, while the molecular weight of His-kexin was 23kDa. As identified by western blot and ELISA, the sera from mice immunized with the recombinant His-kexin had a specific immune reaction with the purified recombinant GST-kexin antigen. The splenocytes collected from the mice immunized with His-kexin had significantly proliferative responses to GST-kexin compared to the control animals (1.763±0.164 and 1.01±0.051 , respectively).Conclusions: The full-length DNA sequence of kexin gene was cloned successfully. We also constructed the recombinant plasmids pGEX-kexin and pET-kexin, expressed fusion protein GST-kexin and His-kexin, respectively. The specific cellular and humoral immunity were generated when the mice were immunized with the protein His-kexin.