Construction and expression of the eukaryotic expression vector containing the p55 gene fragment of rat Pneumocystis / 中国人兽共患病学报

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.

THE ROLE OF MAPK IN LPS-INDUCED iNOS EXPRESSION IN RAT SCHWANN CELLS / 解剖学报

Objective To explore the role of mitogen-activated protein kinase(MAPK)in lipopolysaccharide(LPS)-induced iNOS expression and NO production in rat Schwann cells by the use of inhibitors PD98059 selective for extracellular signal-regulated kinase 1/2(EPK1/2), SB202190 for P38 MAPK and SP600125 for the c-Jun NH_2-terminal kinase (JNK). Methods Schwann cells were pretreated with PD98059 (30, 50, 70?mol/L), SB202190 (10, 20, 40?mol/L) and SP600125 (10, 30, 50?mol/L) at the indicated concentrations for 1 hour before the stimulation with LPS (10mg/L) for 4 hour. The estimation of iNOS mRNA, IL-6 mRNA and TNF-? mRNA was performed by RT-PCR; the changes of iNOS protein expression were investigated by Western blotting. The NO level was observed with measurement of nitrite in the cell culture medium. Results LPS could significantly activate MAPK signal pathway and lead to the expression of iNOS and NO production. The iNOS expression and NO production induced by LPS stimulation were significantly inhibited by the three highly specific inhibitors of MAPK. In addition, the inhibitors decreased LPS-induced the expression of IL-6 mRNA and TNF-? mRNA. Conclusion Activation of MAPK pathway is involved in iNOS expression and NO production in rat Schwann cells, and the inhibition of the signal transduction pathway can be effective to reduce the production of iNOS and NO, which may be a useful strategy against inflammatory and immune reaction after peripheral nerve injury.

Immunogenicity of p55 Gene Fragment from Pneumocystis carinii / 中国寄生虫学与寄生虫病杂志

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