日本血吸虫促凋亡基因SjBAD的初步研究  

Preliminary study on pro-apoptotic gene BAD of Schistosoma japonicum

作　　者：马茜茜[1,2] 洪炀[2] 韩宏晓[2] 陆看 马帅[2] 王涛[2] 刘艳涛[2] 韩倩[2] 傅志强[2] 陆珂[2] 李浩[2] 李祥瑞[1] 林矫矫[2,3] 

机构地区：[1]南京农业大学动物医学院,南京210095 [2]中国农业科学院上海兽医研究所,农业部动物寄生虫学重点实验室 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心

出　　处：《中国血吸虫病防治杂志》2015年第2期139-145,共7页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金﹙81271871﹚

摘　　要：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 characteristics of pro-apoptotic gene SjBAD of Schistosoma japonicum,such as its biology,immunology,and transcriptional expression,and evaluate its potential of the recombinant protein as a vaccine candidate for schistosomiasis. Methods Sj BAD was amplified by PCR and subcloned into a p ET-28a（＋）vector,and the recombinant plasmid was transformed into competent E. coli BL21 for producing recombinant protein. The expressions of Sj BAD in different development stages of schistosomula and 42-day male and female worms were determined by real-time PCR. The immunogenicity of the recombinant protein was analyzed by Western blotting and ELISA. The potential of this protein as a vaccine candidate molecule was assessed by testing the worm reduction rate and liver egg reduction rate in the BALB/c mice immunized by the recombinant antigen Sj BAD. Results Sj BAD was successfully cloned,the recombinant plasmid p ET-28a（＋）-Sj BAD was successfully expressed in E. coli,and the molecular weight of the recombinant protein was around 22 k Da. Western-blotting showed that the recombinant protein had good immunogenicity. The recombinant protein could induce high level of specific Ig G antibodies in the BALB/c mice. Sj BAD was expressed in all tested 7-,14-,21-,28-,35- and 42-day worms,and was highly expressed in 14-day schistosomula,while the expression level in 42-day male worms was higher than that in 42-day female worms. Two independent animal trials showed that 30.82% and 27.87% worm reduction rates,as well as 42.52% and 45.84% liver eggs reduction rates were obtained in the r Sj BAD vaccinated group compared with those of the blank control group（both P〈0.05）. Con Conclusions The pro-apoptotic gene SjBAD is successfully cloned and expressed. The gene is expressed in different development stages of S. japonicum. The r Sj BAD vaccinated BALB/c mice can obtain a partial protective immunity against S. japonicum infection.

关 键 词：日本血吸虫 细胞凋亡 BAD 

分 类 号：R383.24[医药卫生—医学寄生虫学]