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目的了解日本血吸虫促凋亡基因Sj BAD的生物学、免疫学和转录表达特征,评估Sj BAD重组蛋白作为日本血吸虫病疫苗候选分子的潜力。方法根据Sj BAD的参考序列设计引物,应用PCR技术扩增Sj BAD基因,构建重组表达质粒p ET-28a(+)-Sj BAD。采用实时定量PCR检测Sj BAD基因在日本血吸虫不同发育期及42 d雌、雄虫体内的转录水平;应用Western blotting和间接ELISA法分析Sj BAD重组蛋白的抗原性和免疫原性。用重组抗原Sj BAD免疫BALB/c小鼠,通过计算减虫率及肝脏减卵率,评估重组抗原作为血吸虫病候选疫苗分子的潜力。结果成功克隆了日本血吸虫Sj BAD基因,构建了重组表达质粒p ET-28a(+)-Sj BAD,并在大肠杆菌中成功表达,重组蛋白分子量约为22 k Da。Western blotting表明Sj BAD具有较好的抗原性和免疫原性,间接ELISA法检测表明重组蛋白免疫小鼠产生了高水平的特异性抗体Ig G。实时定量PCR检测发现Sj BAD基因在日本血吸虫的各个发育阶段均有转录,以14 d表达量最高,且在雄虫体内的表达量高于雌虫。两次动物免疫试验表明,重组蛋白Sj BAD在BALB/c小鼠体内诱导了30.82%和27.87%的减虫率,及42.52%和45.84%的肝脏虫卵减少率,均显著高于PBS对照组(P均<0.05)。结论成功克隆、表达了日本血吸虫促凋亡相关基因Sj BAD,该基因在日本血吸虫不同发育期虫体内均有表达。纯化的重组Sj BAD蛋白在小鼠体内能诱导产生部分抗血吸虫感染的免疫保护效果。
Objective To understand the biology, immunology and transcriptional expression of the Sj BAD gene of Schistosoma japonicum and to evaluate the potential of Sj BAD recombinant protein as a candidate for vaccine against Schistosoma japonicum. Methods According to the reference sequence of Sj BAD, primers were designed and Sj BAD gene was amplified by PCR to construct recombinant expression plasmid p ET-28a (+) - Sj BAD. Real-time quantitative PCR was used to detect the transcriptional level of Sj BAD gene in different developmental stages of Schistosoma japonicum and 42 d female and male. Western blotting and indirect ELISA were used to analyze the antigenicity and immunogenicity of Sj BAD recombinant protein. BALB / c mice were immunized with the recombinant antigen Sj BAD, and the potential of the recombinant antigen as a candidate vaccine molecule for schistosomiasis was evaluated by calculating the worm reduction rate and the rate of liver evisceration. Results The Sj BAD gene of Schistosoma japonicum was successfully cloned and the recombinant plasmid p ET-28a (+) - Sj BAD was constructed and expressed successfully in E. coli. The molecular weight of the recombinant protein was about 22 kDa. Western blotting showed that Sj BAD had good antigenicity and immunogenicity. Indirect ELISA showed that recombinant protein immunized mice produced a high level of specific IgG. Real-time PCR showed that Sj BAD gene was transcribed at all stages of development in Schistosoma japonicum, and reached the highest level on 14th day and higher than that on female. Two animal immunization tests showed that the recombinant protein SjBAD induced a reduction rate of 30.82% and 27.87% in the BALB / c mice and a reduction rate of 42.52% and 45.84% in the liver, both significantly higher than that of the PBS control group (P <0.05). Conclusion Sj BAD, a gene related to apoptosis, was successfully cloned and expressed in Schistosoma japonicum at different stages of development. Purified recombinant Sj BAD protein was able to induce the immunoprotective effect of producing partial anti-schistosome infection in mice.