Objective To detect Pneumocystis carinii (Pc) DNA by loop-mediated isothermal amplification (LAMP). Methods After injected with hydrocortisone acetate for 8 weeks, the bronchoalveolar lavage fluid (BALF) of Wistar rats were collected and a portion of BALF were examined for identifying Pc organisms using microscope. Then Pc DNA was extracted by phenol-chloroform extraction. Four primers which recognized 6 distinct regions on the mtrRNA gene of Pc were designed and used for LAMP assay. To evaluate the specificity of the assay, M. tuberculosis, M. pneumoniae, C. pneumoniae, P. gondii and rat leucocyte were used as negative controls. To compare the sensitivity of the LAMP to that of conventional PCR, Pc DNA were 10-fold serially diluted and was amplified by LAMP and PCR. LAMP results were judged by naked eye, electrophoretic analysis and restriction digestion. Results Pc organisms were detected from BALF of rats injected with hydrocortisone acetate. After LAMP reaction, positive signal was observef rats injected with hydrocortisone acetate. By contrast, no positive signal was observed for the negative controls in the study. The amplified product digested by restriction enzyme demonstrated 3 bands (82, 135, 189 lip) upon agarose gel electrophoresis, in good agreement with the predicted sizes. The detection limit of LAMP assay was 1 pg/μl of Pc DNA per reaction and that of PCR was 10 pg/μ1 of Pc DNA per reaction. Conclusion LAMP assay has usefulness for rapid detection of Pc.

Objective To detect Toxoplasma gondii DNA by loop-mediated isothermal amplification(LAMP). Methods DNA was extracted by phenol-chloroform extraction from T. gondii tachyzoites. Four primers which recognized 6 distinct regions on the B1 gene of T. gondii were designed and used for LAMP assay. To evaluate the specificity of the method, Plasmodium vivax, P. falciparum, Pneumocystis carinii, Schistosoma japonicum, and mouse leucocytes were used as controls. The parasite extract (T. gondii) was 10-fold serially diluted for evaluating the sensitivity of LAMP, and was amplified by LAMP. LAMP results were read with naked eye and analyzed by electrophoresis. Results After LAMP reaction, positive amplification was observed with T. gondii, but no positive signal was toted for the negative controls in the study. The sensitivity of LAMP assay reached up to 2-3 T. gondii tachyzoites/ml per reaction. Conclusion LAMP assay shows proper specificity and sensitivity for the detection of T. gondii.

Objective To establish a method for detecting cercaria of Schistosoma japonicum.Methods The loop-mediated isothermal amplification(LAMP)was used.The cercaria DNA of Schistosoma japonicum was extracted by using GeneReleaser.Four primers which recognized 6 distinct regions on the calcium-binding protein gene of cercaria of Schistosoma japonicum were designed and used for LAMP assay and Clonorchis sinensis was used as the negative control for evaluating the specificity and 20,10,5,1 cercariae of Schistosoma japonicum were amplified by LAMP for evaluating the sensitivity.The LAMP results were judged with the naked eyes and electrophoretic analysis.Results After LAMP reaction,the positive signal was observed with cercariae of Schistosoma japonicum.By contrast,no positive signal was observed for Clonorchis sinensis.The detection limit of LAMP assay was 1 cercaria of Schistosoma japonicum per reaction.Conclusion LAMP assay has usefulness for rapid detection of cercariae of Schistosoma japonicum.

Objective To express P30 surface antigen of RH strain of Toxoplasma gondii in E.coli BL21(DE3). Methods The P30 gene from Toxoplasma gondii was cloned to the pET28b vector after PCR, and the recombinant expression plasmid pET28b-P30 was constructed. Then the recombinants were transformed into E.coli BL21(DE3) after identified by the restriction enzyme digestion, PCR and DNA sequence determination annlysis. A single colony of E.coli BL21(DE3) containing the recombinant plasmid, pET28b-P30 was inoculated in LB culture, then diluted 1∶100 into 2 ml LB culture and induced by 0.2 mmol/L IPTG, and the expression product was identified by SDS-PAGE and Western blot. Results The recombinant plasmid of pET28b-P30 was constructed. ② Plasmid pET28b-P30 could express a specific 30 kDa fusion protein in E.coli BL21(DE3). Conclusions The expression plasmid which contains the gene fragment encoding P30 surface antigen of Toxoplasma gondii has been successfully constructed and is highly expressed in E.coli BL21(DE3) as an inclusion body.

Objective To investigate the immune efficacy of nucleic acid vaccination in rabbits against Schistosoma japonicum with pcDNA3 1(+)/MLP(SJP) Methods The 24 New Zealand rabbits were randomly divided into 2 groups. The rabbits of experimental group were vaccinated by each quadriceps muscle of leg with pcDNA3 1(+)/MLP nucleic acid vaccination and the control group rabbits were vaccinated with pcDNA3 1(+). Each rabbit was immunized four times with 2 weeks interval. The rabbits were challenged 2 weeks after final DNA boosting by percutaneous infection with cercariae. Sixty days after infection the rabbits were sacrificed, the livers were investigated, and the worms and eggs in livers were counted. Blood sera were collected from rabbits and investigated. Results In the experimental group,the egg reduction rate was 28 10%. The rabbits of experimental group produced IgA, IgG 1, INF-?. Conclusion DNA vaccination with pcDNA3.1(+)/MLP could induce partial protective immunity against Schistosoma japonicum in rabbits.

The protective effect and mechanism of Schistosoma japonicum cathepsin B (Sjcb2) DNA vaccine in the mouse model of schistosomiasis were studied through construction pcDNA3 .1 (+ ) / Sjcb2 DNA recombinant vector ,which provided effective candidate antigen for anti-schistosome vaccine .The 6-week-old female BALB/c mice were randomly divided into pcDNA3 .1(+ )/Sjcb2 DNA vaccine group ,pcDNA3 .1(+ ) plasmid group and normal saline group ,respectively .Each group was composed of 35 mice ,and 100 μg of S jcb2 plasmid DNA was injected in the hind leg quadriceps of mice once every two weeks .PCR and immunohistochemistry assay were used to detect the expression and stability of Sjcb2 gene in mice .MTT assay was used for testing the specific proliferation response of mice spleen lymphocytes .The level of Sjcb2 antibodies in mouse serum and the IFN-γand IL-4 levels in mice spleen lymphocyte culture supernatant before and after schistosome infection were assayed by ELISA .At last ,we counted load of Schistosome adult worms in mouse and eggs in liver of mouse .The results showed that the Sjcb2 gene was detected in all mice of the Sjcb2 DNA vaccine group ,and Sjcb2 gene expression was positive in the muscle cells in Sjcb2 DNA immunized mice by IHC assay .MTT assay showed that T-cell proliferation rate was in-creased significantly in S jcb2 DNA vaccinated group .ELISA results showed that the IFN-γlevels were increased significantly in the vaccinated group ,while the IL-4 levels were significantly increased after Schistosoma japonicum infection in all mice of every group .The load of worms and eggs in Sjcb2 DNA vaccinated group was reduced significantly than that of control group (P<0 .05) ,the reduction rates of adult worms and eggs were 36 .32% and 60 .61% respectively .In conclusion ,the Sjcb2 gene was stably expressed in muscle cells of mice after injection of S jcb2 recombinant plasmid ,and S jcb2 produced protective effects of anti-schistosoma infection in mice possibly by mean of regulating Th1 cell subgroups through increasing the IFN-γ level and decreasing IL-4 levels .