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.

Subjective To acquire and analyze adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags and new genes from an adult S. japonicum cDNA library, and to search new vaccine candidates and drug targets. Methods A cDNA library was constructed from adult stage S. japonicum. Clones were selected randomly from the cDNA library and were se-quenced. ESTs and new genes were acquired after analysis in GenBank databases by BLAST and other programs. All ESTs and new genes were submitted to GenBank and received accession numbers. Results 149 ESTs were acquired from a total 382 clones that were randomly selected from the adult S. japonicum cDNA library. All ESTs were successfully submitted to the dbEST at Genbank. Some of them were homologous with sequences of male, female, egg, schistosomula, cercaria and miracidia of S. japonicum. 18 new genes of adult S. japonicum were acquired. Some genes were housekeeping genes and some genes might be interesting as vaccine candidates or drugs targets. Conclusions The EST straltegy is a rapid, efficient and economical method to acquire ESTs and to discover new genes of adult stage S. japonicum from cDNA libraries.[

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.

Objective To clone and sequence the partial gene of Schistosoma japonicum phosphoglycerate kinase (SjPGK). Methods A pair of primers were designed and synthesized according to the cDNA sequence of Schistosoma mansoni phosphoglycerate kinase (SmPGK) gene. The gene fragment of SjPGK was amplified and isolated from the total RNA of S.japonicum by reverse-transcription polymerase chain reaction (RT-PCR). The gene fragment was cloned into the cloning vector of pMD18-T, The positive clones were acquired and identified with restrictive enzymes and PCR amplification. After being sequenced with DNA auto-sequence analysis instrument,the cDNA sequence of SjPGK was searched for homologue identity with NCBI BLAST program. Results The gene encoding SjPGK was obtained and isolated by RT-PCR .The fragment of SjPGK was about 830 bp.The cDNA sequence of the phosphoglycerate kinase was highly homologous between Schistosoma mansoni and Schistosoma japonicum. The identity of nucleotide sequence was 85% and score 672, and the identity of amino sequence was 94% and score 473. Conclusion The partial gene of encoding SjPGK is cloned into the cloning vector of pMD18-T, which gives the basis for discovering new candidate vaccine molecular for schistosomiasis.

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 specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.