The Immune Protective Effects Of MP65 And Sap2 Recombinant Expression Plasmid On Mouse Systemic Candida Albicans Infection

Objective: Candida albicans is the most common opportunistic fungal pathogen which has become one of the most important pathogen of nosocomial infections (application of immunosuppressive agents and broadspectrug antibiotic from surgical trauga, organ transplanting, cancer-treatment) and immunocompromised patients (primary and secondary immunodeficiencies, for example AIDS) during the last few decades, mortality of caused systemic infection is 40%~50%. Although new antifungal drugs were applicated in clinic, the incidence and mortality of infection still can't be reduced because of the narrow spectrug of anti-fungal medicine and the occurrence of new drug resistance to Candida albicans. Therefore the study of safe and effective immune agents will provide a new promising approach to fungal infection prevention and treatment, in which fungi vaccine research was the focus.Mannan-mannoprotein conjugates are not only an important component of Candida albicans cell wall but also an important adhesin molecule. MP65 is the main mannoprotein be purified from the acidic extraction. The protein core part can indu ce T cell response and have a wide range of protective effects on many kinds of candida infection. And Sap2 is the most important extracellular protease secreted by Candida albicans under certain conditions, which play a crucial role in the nutrition of Candida albicans and is closely related to adhesion, invasion and destruction of host tissue barrier, so it is an important virulence factor of Candida albicans. As a widespread antigen component existed in the majority of Candida albicans, its existence form of serum is soluble. It is found that the Candida patient's serum retain high titer of Sap2 antibodies. Studies had verified that Sap2 extraction of Candida albicans can weaken invasion to mucosal tissue in mice immunized with them.For the reason above, in this research we selected the gene encoding MP65 and Sap2 protein, constructed the recombinant eukaryotic and prokaryotic expression plasmid of them. BALB/c mice were immunized with eukaryotic plasmids, it is aim to analyse immunogenicity of MP65, Sap2 as well as the immunization protective effect of resistance to Candida albicans systemic infection which provided an experimental foundation in further research and production of fungi vaccine.Method: 1. Construction of eukaryotic and prokaryotic expression plasmid: Candida albicans total RNA was extracted by hot phenol method. Then the mp65 and sap2 gene were obtained by RT-PCR and cloned into pMD18-T vector for sequence identification. After receiving the correct sequence results and re-identification by BamHI, EcoR I and EcoR I, XhoI digestion, the target gene were cloned into prokaryotic expression plasmid pGEX4T-2, pET32a and eukaryotic expression plasmids pcDNA3.1 respectively.2. Expression, purification and identification of purpose protein: Recombinant prokaryotic expression plasmids were transfered into E. coli BL21 and induced by IPTG. Then BL-21 cell transformed were crumbled by ultrasonics and the results were analized by SDS-PAGE electrophoresis. The recombinant protein were expressed mainly as the form of inclusion body and were purified by gradient dialysis with low concentrations of urea and nickel chelate column effectively. Utilize Western blot method for preliminary identification of its antigen specificity and reactivity.3. Large quantity acquisition of Eukaryotic expression plasmid: Eukaryotic expression plasmid pcDNA3.1-mp65 and pcDNA3.1-sap2 were extracted by alkaline lysis method separately, and were served after purification and quantitation.4. Animal immunization: Forty 4 week-old BALB/c mice were divided into four groups randomly, among each group the male and female was equally. Four groups were respectively named with pcDNA3.1-mp65 plasmid group, pcDNA3.1-sap2 plasmid group, pcDNA3.1-mp65 + pcDNA3.1-sap2 plasmid group and PBS control group. Quadricepse of leg were injected with eukaryotic expression plasmid, 100μg per mouse, total 4 times at interval two weeks. Serum were harvested from the inn -er canthal vein blood before every time of immunization.4. Detection of the immune effect: anti-MP65 and anti-Sap2 IgG in the serum were detected by indirect ELISA. CD4+, CD8+ lymphocyte phenotypes in the spleen were analyzed by Flow cytometry.5. Candida albicans challenge experiment: 20 days after the last boost, BALB/c mice were injected via tail vein with 1×106 lethal dose of Candida albicans, the survival were observed for 15 days and evaluate the protective effect of the DNA vaccines.Results: 1. Mp65 and sap2 gene were obtained successfully by RT-PCR. The sequence analysis shows that the mp65 (1140bp) and sap2 (1197bp) gene were homologous basically with the ones in GenBank.2. Prokaryotic expression plasmid pGEX4T-2-mp65 and pET32a-sap2, eukaryotic expression plasmid pcDNA3.1-mp65, pcDNA3.1-sap2 had been constructed successfully. Prokaryotic expression plasmid pGEX4T-2-mp65 and pET32a-sap2 were transformed to BL-21. GST and His fusion protein with approximate molecular mass of 66KD were expressed and identified by SDS-PAGE and Western blot. The main expression form of the protein was inclusion body.3. The titers of IgG in serum increased gradually after animals were immunized with pcDNA3.1-mp65 and pcDNA3.1-sap2 following time extending. After the last immunization, the highest titer of anti-MP65 IgG was 1:1600, while the highest titer of anti-Sap2 IgG was 1:3200. Flow cytometry results showed that percentage of CD4+T cell increased in both mp65 and Sap2 plasmid group after immunization of mice, which exists significant difference compared with the PBS control group by analysis of variance (P<0.05). And no statistically significant difference was found in the percentage of CD8+ T cell between immune groups.4. Fifteen days after the Candida albicans impact, the survival rate of mice immunized with pcDNA3.1-mp65 plasmid or pcDNA3.1-sap2 plasmid was 20% respectively, survival rate of mice immunized with pcDNA3.1-mp65 +pcDNA3.1-sap2 plasmid was 40 %, and PBS group survival rate was 0% ,which exists significant difference by Chi-square test (P <0.05).Conclusion:1. Prokaryotic expression plasmid pGEX4T-2-mp65, pET32a-sap2 and eukaryotic expression plasmid pcDNA3.1 -mp65, pcDNA3.1-sap2 of mp65 and sap2 gene of Candida albicans were constructed successfully.2. Prokaryotic expression plasmids of mp65 and sap2 gene were induced and expressed fusionin protein in E. coli. BL-21 successfully.3. Recombinant eukaryotic expression plasmid of mp65 and sap2 gene can stimulate animals producing humoral and cellular immune response.4. Both mp65 and sap2 recombinant eukaryotic expression plasmid have a certain protective effect against systemic Candida albicans infection.