日本血吸虫新基因Sjnanos的克隆、表达及免疫保护效果评估  

Cloning,expression and characterization of a novel gene of Schistosoma japonicum

作　　者：王艳[1] 郭凡吉[1] 彭金彪[1] 李晔[1] 洪炀[1] 陈实[1] 傅志强[1] 石耀军[1] 林矫矫[1] 

机构地区：[1]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开放实验室,国家防治动物血吸虫病专业实验室,上海200241

出　　处：《中国人兽共患病学报》2010年第7期631-637,共7页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(30671581);973课题(2007CB513108);863计划(2006AA10A207)联合资助

摘　　要：目的扩增、表达日本血吸虫Sjnanos编码基因并评估其重组蛋白诱导的免疫保护效果。方法从42d龄的日本血吸虫成虫中扩增了一个日本血吸虫性别差异表达的基因,GenBank登录号为AY814948,并对其进行生物信息学分析。将该基因亚克隆入原核表达载体pET28a,转化至大肠埃希菌(E.coli)BL21,用异丙基-β-D硫代半乳糖苷(IPTG)进行诱导表达,用纯化的重组蛋白免疫小鼠,ELISA检测其血清特异性抗体效价,蛋白印迹分析检测其抗原性,以看家基因NADH为内参,应用荧光定量PCR技术分析该基因在血吸虫各个阶段的表达状况。结果同源性分析表明,该基因为日本血吸虫的一个新基因,其编码序列的开放阅读框(ORF)为525bp,编码174个氨基酸,理论相对分子量为19.9,PI为8.2。荧光定量PCR技术分析显示该基因在7d、13d、18d、23d、32d、42d虫体均有表达,但在18d和13d虫体的表达量明显高于其他天数的虫体,在雄虫的表达量高于雌虫。成功构建了该基因的重组表达质粒pET28a(+)-Sjnanos,并在大肠埃希菌中获得表达,Western-blot分析表明重组蛋白具有较好的免疫原性,动物免疫保护试验获得了31.4%的减虫率和53.8%的肝脏减卵率。结论获得日本血吸虫新的抗原基因Sjnanos,该基因的重组蛋白在小鼠中诱导了部分免疫保护作用。To clone and express the gene（nanos）of Schistosoma japonicum,and study the antigenicity and immunogenicity of the protein,the nanos gene amplified by PCR from worms＇ cDNA was cloned into pronucleus expression vector pET28a（＋）to construct pET28a（＋）-Sjnanos recombinant plasmid and transformed into E.coli BL21（DE3）.In the presence of IPTG,the 21KD protein was expressed and purified under denaturing conditions to immune the BALB/c mice.The gene sequencing data showed that length of the whole ORF was 525bp and encoded 174 amino acids with a prediction of 21KD.Results of Western blot and ELISA revealed that the fusion protein had good immunogenicity.Real-time PCR analysis showed the mRNA transcription level of nanos was much higher in 13d,18d and 23d within Schistosomula than in other stages.After immunizing BALB/c mice with fusion protein,the percentage of worm and liver egg reduction was 31.4% and 53.8%,respectively.The gene of Sjnanos was cloned for the first time,and the successful expression and purification of pET28a（＋）-Sjnanos would be helpful for the further protection in animals.

关 键 词：日本血吸虫 nanos基因 抗原性 免疫保护 

分 类 号：R383.2[医药卫生—医学寄生虫学]