关键词: 日本血吸虫, 体表蛋白, 荧光定量PCR, 免疫组化

Abstract: Objective To clone, express and characterize a tegument protein gene of Schistosoma japonicum （Sj29）, and investigate the immune protection of the recombinant protein against S. japonicum in mice. Methods The gene coding for Sj29 protein was amplified by PCR, and the sequence was analyzed by bioinformatics tools. Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c（+）. The recombinant plasmid was transformed into E. coli BL21（DE3）and induced the recombinant with IPTG. The recombinant protein（rSj29）was purified by His-binding-resin affinity chromatography and characterized by Western blotting. Three groups each with 10 BALB/c mice were immunized subcutaneously three times（two weeks interval）respectively with 100 μl recombinant rSj29（0.1 mg/ml）, adjuvant or PBS. At the 15th day after the final inoculation, each mouse was challenged by 40±2 cercariae of S. japonicum. At the 53th day after infection, the mice were sacrificed to obtain the number of adult worms, number of eggs in liver and feces. Serum samples were collected at pre-immunization and certain time after immunization, and were analyzed for IgG by ELISA. The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique. mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR. Results A 576 bp Sj29 gene fragment was obtained. The recombinant protein rSj29 with Mr 22 900 was expressed in the form of inclusion body. The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice. The number of adult worms（15.4±5.9）, number of hepatic eggs（40 143.3±2 995.9）and number of fecal eggs（3 803.9±110.9）in re-combinant protein group were significantly higher than those of PBS control group （20±3.4, 49 318.1±6 648.3, 5 238.1±303.5, respectively）（P<0.05）. There was a high level of specific IgG against rSj29 （maximum dilution 1 ∶ 32 000）in recombinant protein group. Immunohistochemical analysis showed the Sj29 protein expressed on the surface of different stages of S. japonicum. mRNA level of Sj29 was the highest at the 32nd day post-infection. Conclusion The recombinant protein rSj29 induces certain degree of protective immunity in mice.

Key words: Schistosoma japonicum, Tegument profein, Real-time quantitative PCR, Immunohistochemistry