摘要
以日本血吸虫18 d虫体的cDNA为模板,PCR获得Sjmago nashi编码基因的全长cDNA,其开放阅读框为441 bp,将该序列提交至NCBI,登录号为GQ403668;应用荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的表达情况。结果表明:该基因在不同发育阶段均有表达,在13日龄童虫表达量相对最高,32日龄次之;在不同性别成虫虫体中,该基因在雄虫的表达量远高于雌虫,说明该基因为日本血吸虫性别发育差异基因。以pET28a(+)为载体构建重组表达质粒,在大肠杆菌中表达,表达产物分子质量为19.5 ku,并以Ni-NTA HisBind Resin纯化重组蛋白。Western blotting试验显示该重组蛋白具有良好的抗原性,在小鼠免疫试验中,与空白对照组比较,免疫组小鼠获得25.94%的减虫率和30.01%的肝脏减卵率。
PCR technique was applied to amplify a full-length cDNA encoding protein S.japonicum mago nashi（Sj mago nashi） from schistosomula cDNA.The cDNA containing the ORF of Sj magonashi was cloned with the length of 441 bp,which was encoding 146 amino acids.The expression profiles of Sj mago nashi were determined by qPCR-using-the template cDNAs isolated from 7,13,18,23,32,42,42 d female and male parasites.qPCR analysis revealed that the gene was similarily expressed in each development stage,but 10 times higher expressed in-42 d-male adult worm than-42 d-female adult worm,suggesting this gene as male differently expressed.The cDNA containing the Open Reading Frame of mago nashi was then subcloned into a pET28a（＋） vector and the recombinant plasmid was transformed into competent E.coil/BL21 for producing recombinant protein.The recombinant protein was purified,and showed a good antigenicity detected by Western blotting.Animal experiment indicated that the vaccination of recombinant Sj mago nashi protein in mice led to 25.94% worm and 30.01% liver egg burden reduction respectively,when compared with those of the blank control.
出处
《扬州大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2011年第2期11-15,35,共6页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
国家自然科学基金资助项目(30671581)
国家重点基础研究计划项目(973-2007CB513108)
国家高技术研究发展计划项目(863-2006AA10A207)
