日本血吸虫TOM34基因的克隆和表达分析  

Cloning,expression and characterization of a gene encoding translocase of the outer mitochondrial membrane 34(TOM34) in Schistosoma japonicum

作　　者：洪炀[1] 李学珍[1,2] 傅志强[1] 石耀军[1] 陆珂[1] 杨健美[1] 朱传刚[1] 苑纯秀[1] 李浩[1] 李祥瑞[2] 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 [2]南京农业大学动物医学院,南京210095

出　　处：《中国人兽共患病学报》2013年第2期122-128,共7页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.31172315);公益性行业(农业)专项(No.200903036);中国博士后科学基金(No.2012M510630)联合资助~~

摘　　要：目的克隆、表达日本血吸虫线粒体外膜转运酶34基因(SjTOM34),并对其生物特性进行初步研究。方法应用RT-PCR方法从42d日本血吸虫成虫cDNA中扩增SjTOM34基因,并进行生物信息学分析。应用实时定量RT-PCR方法分析了SjTOM34基因在日本血吸虫不同发育阶段虫体中的表达状况。构建了SjTOM34基因重组原核表达质粒,并在大肠杆菌中进行表达,Western blotting分析了重组抗原的免疫原性及该蛋白在不同发育阶段虫体中的表达情况,利用免疫组化方法观察了该蛋白在虫体内的分布情况。结果获得了SjTOM34的基因序列,其开放阅读框(ORF)为1 083bp。该基因在所检测的不同发育阶段童虫和成虫中均有表达,其中在35d虫体中的表达量略高于其他时期的虫体。成功构建了重组的原核表达质粒并且在大肠杆菌中获得表达,重组蛋白在上清和沉淀中都有存在,并且具有较好的免疫原性,在所检测的各时期虫体中均可检测到该蛋白的存在,并且主要分布在虫体的实质组织中。结论获得了日本血吸虫SjTOM34基因,初步明确了该基因在日本血吸虫童虫和成虫中的表达情况,及在成虫中的分布。A full-length cDNA encoding the Schistosoma japonicum translocase of outer mitochondrial membrane 34 （SjTOM34） was first isolated from 42-day schistosome cDNAs. The cDNA had an open reading frame （ORF） of 1 083 bp and encoded 360 amino acids. Real time quantitative RT PCR analysis revealed that the expression of SjTOM34 was a little higher in 35-day schistosomes than worms in other stage. The SjTOM34 was subcloned into pET28（＋） and expressed as both inclu sion bodies and supernatant in Escherichia coli BL21 （DE,？,） cells. Western blotting showed that the recombinant SjTOM34 （rSjTOM34） had good immunogenicity. Immunolocalization analysis found that SjTOM34 was distributed all over the worms of S. japonicum. It＇s suggested that SjTOM34 may be important in schistosome, and further investigations are required to fully understand the function of this molecule.

关 键 词：日本血吸虫 线粒体外膜转运酶34 克隆 表达 免疫定位 

分 类 号：R383.2[医药卫生—医学寄生虫学]