Chin J Schisto Control

   

Preliminary study on pro?apoptotic gene BAD of Schistosoma japonicum

MA Qian-qian1|2|HONG Yang2|HAN Hong-xiao2|LU Kan2|MA Shuai2|Wang Tao2|LIU Yan- tao2|HAN Qian2|FU Zhi-qiang2|LU Ke2|LI Hao2|LI Xiang?rui1*|LIN Jiao-jiao2|3*   

  1. 1 School of Veterinary Medicine|Nanjing Agricultural University|Nanjing 210095|China;2 Shanghai Veterinary Research Institute|Chinese Academy of Agricultural Science;Key Laboratory of Animal Parasitology|Ministry of Agriculture|China;3 Jiangsu Co?innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses|China
  • Received:2014-11-12 Online:2015-03-28 Published:2015-03-28

日本血吸虫促凋亡基因SjBAD的初步研究

马茜茜1|2|洪炀2|韩宏晓2|陆看2|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*   

  1. 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
  • 通讯作者: 林矫矫
  • 作者简介:马茜茜|女|硕士研究生。研究方向:兽医公共卫生
  • 基金资助:

    国家自然科学基金﹙81271871﹚

Abstract:

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   SjBAD was amplified by PCR and subcloned into a pET?28a(+)vector,and the recombinant plasmid was transformed into competent E. coli BL21 for producing recombinant protein. The expressions of SjBAD 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 SjBAD. Results   SjBAD was successfully cloned,the recombinant plasmid pET?28a(+)?SjBAD was successfully expressed in E. coli,and the molecular weight of the recombinant protein was around 22kDa. Western blotting showed that the recombinant protein had good immunogenicity. The recombinant protein could induce high level of specific IgG antibodies in the BALB/c mice. SjBAD 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 rSjBAD vaccinated group compared with those of the blank control group(both P < 0.05). Conclusions   The pro?apoptotic gene SjBAD is successfully cloned and expressed. The gene is expressed in different development stages of S. japonicum. The rSjBAD vaccinated BALB/c mice can obtain a partial protective immunity against S. japonicum infection.

Key words: Schistosoma japonicum, Apoptosis, BAD

摘要:

目的  了解日本血吸虫促凋亡基因SjBAD的生物学、免疫学和转录表达特征,评估SjBAD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SjBAD的参考序列设计引物,应用PCR技术扩增SjBAD基因,构建重组表达质粒pET?28a(+)?SjBAD。采用实时定量PCR检测SjBAD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SjBAD重组蛋白的抗原性和免疫原性。将重组抗原SjBAD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SjBAD基因,构建了重组表达质粒pET?28a(+)?SjBAD,并在大肠杆菌中成功表达,重组蛋白分子量约为22 kDa。Western blotting表明SjBAD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IgG。实时定量PCR检测发现SjBAD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SjBAD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SjBAD,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SjBAD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。

关键词: 日本血吸虫, 细胞凋亡, BAD

CLC Number: