Immunoprotective Effects Of Dna Vaccine Of P55 Gene Fragment Against Pneumocystis Carinii

Objective To construct eukaryotic expression plasmid for p55 gene fragment of Pneumocystis carinii from rats and express p55 protein in COS-7 cells. To evaluate the immunogenicity and protective effects of the recombinant plasmid against Pneumocystis carinii.Methods The p55 gene fragment was amplified from rat Pneumocystis carinii genome by PCR , cloned to pGEM-T vector, and then subcloned in eukaryotic expression vector pcDNA3. 1(+). The p55 gene fragment was expressed in COS-7 cells by transfecting with pcDNA3. 1(+)-582. The gene expression was examined by RT-PCR.The expressed protein was analyzed by Western blotting. Female mice were randomly divided into three groups. Mice were injected in quadriceps femoris with recombinant plasmid pcDNA3.1(+)-582 (immunized group) , plasmid pcDNA3.1(+) and PBS(control groups) respectively. Antibody level in sera of mice was determined by ELISA. Lymphoproliferation ability of the lymphocytes was observed by MTT colorimetric assay.To evaluate the protection, SD rats were immunized with the interest plasmids 3 times at 2-week intervals.Two weeks after the final immunization, the rats were immunosuppressed by quantitative subcutaneous injection of dexamethasone twice a week. Two weeks after immunosuppressed, the rats were challenged by Pneumocystis carinii. Quantitation of Pneumocystis carinii was tested by Real-Time PCR.Results The recombinant plasmid pcDNA3.1(+)-582 was identified by restriction endonuclease analysis, PCR and DNA sequence analysis. RT-PCR and Western-blotting analysis showed that the p55 gene fragment was transfected successfully into COS-7 cells and efficiently expressed at mRNA and protein levels. The antibody titers against pcDNA3.1(+) -582 in the immunized group was significantly higher than that of pcDNA3.1(+) or PBS group at the second week and the fourth week after the third immunization. Compared to control groups, the pcDNA3.1(+)-582 immunized group did elicit a vigorous proliferative response in splenocytes,and DNA vaccine immunization resulted in a significant reduction in organism burden.There was significant difference between the immunized group and the control groups(P<0.05).Conclusions The DNA vaccine pcDNA3.1(+)-582 was constructed and expressed successfully. The humoral and cellular immune responses against pcDNA3.1(+) -582 were induced in BALB/c mice after they were administered.It could provide partial protection.