大片吸虫磷酸丙糖异构酶基因的克隆表达和免疫原性分析  

Cloning,expression and immunogenicity of triosephosphate isomerase of Fasciola gigantica

作　　者：姚文浩 吴兴隆 秦志博 李文豪 朱惠丽 王磊[1] 胡建和[1] 韩艳辉 YAO Wenhao;WU Xinglong;QIN Zhibo;LI Wenhao;ZHU Huili;WANG Lei;HU Jianhe;HAN Yanhui(Henan University of Science and Technology,Xinxiang 453003,China)

机构地区：[1]河南科技学院,河南新乡453003

出　　处：《畜牧与兽医》2024年第1期39-45,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划项目(2021YFD1301200);中原科技创新领军人才项目(224200510024)。

摘　　要：磷酸丙糖异构酶(TPI)是生物体糖酵解的一种关键酶,对大片吸虫获取能量而赖以生存有着十分重要的作用。本试验根据肝片吸虫TPI(GenBank:KC164346.1)的基因序列,设计带有BamHⅠ和XhoⅠ作为酶切位点的引物,以大片吸虫的cDNA为模板,扩增大片吸虫磷酸丙糖异构酶(FgTPI)基因。结果:FgTPI基因开放阅读框为762 bp,编码253个氨基酸,理论等电点pI为8.07;构建的重组表达质粒FgTPI-pET-28a(+)成功在大肠杆菌中表达,重组蛋白的分子量约为32 kDa;重组蛋白免疫BALB/c小鼠,收取多克隆抗体,ELISA检测结果显示,rFgTPI能诱导较高的IgG抗体水平;用感染大片吸虫动物的血清检测重组蛋白的免疫原性,Western blot分析表明,重组蛋白rFgTPI具有良好的免疫原性;生物信息学预测显示,FgTPI主要以α螺旋和β折叠为主,β转角较少,有3个较长的无规则卷曲;通过α-磷酸甘油脱氢酶偶联法测定其活性,发现FgTPI酶促反应最佳pH值为7.5,最适温度为35℃。本试验结果为深入研究FgTPI的生物学功能、评价其作为抗大片吸虫病疫苗候选分子奠定了基础。Triosephosphate isomerase(TPI)is a key glycolytic enzyme that exists in the organisms and plays an important role in obtaining energy for the life of Fasciola gigantica.In this study,the full-length cDNA encoding FgTPI was cloned according to the gene sequence of TPI from Fasciola gigantica,and the primer with BamHⅠand XhoⅠas enzyme digestion sites was designed.The cDNA from Fasciola gigantica was used as a template.The results were as follows:The open reading frame(ORF)of FgTPI was 762 bp,encoding 253 amino acids with an isoelectric point of 8.07.The ORF of was sub-cloned into pET-28a(+)vectors and expressed in Escherichia coli with a molecular weight of 32 kDa.The BALB/c mouse was injected with the purified recombinant of FgTPI,and the polyclonal antibodies were prepared.ELISA assay analysis showed that rFgTPI could induce high levels of anti-rFgTPI-specific IgG antibodies.The result of western blotting showed that recombinant FgTPI had good antigenicity by using the serum from the sheep infected with Fasciola gigantica.The recombinant protein showed the optimal activity of TPI at pH 7.5 and temperature of 35℃,according toα-glycerol phosphate dehydrogenase coupling method.The above results indicated that FgTPI could be a potential candidate for vaccine against Fascioliasis,which would provide basis for further investigation into the biological function of FgTPI.

关 键 词：大片吸虫 磷酸丙糖异构酶 克隆 表达 酶活性分析 

分 类 号：S855[农业科学—临床兽医学]