Objective To construct prokaryotic recombinant expression plasmid carrying Pneumocystis carinii Mr 55 000 antigen(p55) gene fragment and express the recombinant protein. Methods P. carinii pneumonia(PcP) rat models were established by subcutaneous injection of dexamethasone for 14 weeks. Total RNA was extracted from lung of P. carinii rat and p55 antigen gene fragment was cloned by RT-PCR,which was identified by sequencing. The 690 bp fragment was cloned to pGEX-4T-1,the recombinant plasmid was screened and identified by restriction analysis and PCR. The recombinant plasmid was finally induced with IPTG to express a new fusion protein,and the products were analyzed by SDS-PAGE and Western blot. Results A fragment of 690 bp was obtained by RT-PCR. The recombinant pGEX-4T-1/690 was constructed. SDS-PAGE revealed that the molecular weight of the recombinant protein was approximately Mr 62 000,the maximum amount of the fusion protein produced was 11.6% of the total protein. The recombinant protein can be recognized by GST antibody and by the sera from P. carinii infected rats using Western blotting. Conclusion Prokaryotic expression plasmid pGEX-4T-1/690 has been constructed and the recombinant fusion protein shows antigenicity.

Objective To evaluate immunogenicity of the recombinant protein GST-p55/570 of Pneumocystis carinii.Methods The fusion protein GST-p55/570 was expressed from the prokaryotic expression plasmid pGEX-570,and purified by using glutathione-agarose.The expressed product was analyzed by SDS-PAGE.Thirty-three mice were randomly divided into three groups,immunized with GST-p55/570,GST and PBS,respectively.Each group was immunized for four times at 2 week intervals.At the 7th day after final immunization,spleen was removed to obtain single cell suspension.Proliferation ability of lymphocytes was determined by MTT.Serum samples were collected at pre-immunizaton and two weeks after each immunization.Antibody level in sera of mice was determined by ELISA.The immune response to the recombinant GST-p55/570 recognized by sera of immunized mice was examined by Western blotting.Results The expressed fusion protein GST-p55/570 showed a Mr 47 000.Compared with GST group(1.134 5?0.073 5) or PBS group(1.124 8?0.041 6),a higher stimulation index(2.063 0?0.160 2) was revealed in GST-p55/570-immunized mice(P

To construct the eukaryotic expression plasmid containing the p55 gene fragment of Pneumocystis and to investigate the efficient expression in COS-7 cells, the gene fragment conaining the whole length of p55 gene was used as template to amplify this fragment with PCR and the amplified fragment was then cloned to vector pGEM-T. After enzyme digestion, p55 gene was cloned to the eukaryotic expression vector pcDNA3.1(+) to construct the plasmid pcDNA3.1(+)-582. This plasmid was then transfected to the eukaryotic expression cells COS-7 and PCR and SDS-PAGE assays were used to confirm the presence of target protein in these cells. In these ways, the eukaryotic expression vector for the p55 gene of Pneumocystis of rats was successfully constructed and expressed in COS-7 cells, thus providing the basis for further studies on the nucleic acid vaccine.