大片吸虫Ras家族蛋白基因的克隆表达及免疫反应性研究  

Cloning,expression and immune-reactivity of Ras-related protein of Fasciola gigantica

作　　者：田艾灵 张念章[1] 张芙恺 王萌[1] 黄思扬[1] 朱兴全[1] TIAN Ai-ling, ZHANG Nian-zhang, ZHANG Fu-kai, WANG Meng, HUANG Si-yang, ZHU Xing-quan(State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/ Lanzhou Veterinary Research h~stitute ,Chinese Academy of Agricultural Sciences ,Lanzhou 730046 ,Chin)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室

出　　处：《中国兽医科学》2018年第4期412-418,共7页Chinese Veterinary Science

基　　金：国家重点基础研究发展计划(973计划)项目(2015CB150300)

摘　　要：Ras家族相关蛋白是多种信号转导途径的关键组成蛋白，在寄生虫的生长发育过程中发挥着重要作用，但犬片吸虫的Ras家族蛋白基因（FgRap1）尚未见报道。本研究旨在对FgRapl进行克隆、表达、序列分析及免疫反应性评价。根据肝片吸虫的基因组及转录组数据．设计肝片吸虫Ras家族蛋白基因引物．以大片吸虫总RNA为模板，通过RT—PCR获得Fg尺apl基因；对其进行生物信息学分析及鉴定；然后构建pET—SUMO-FgRap1重组表达栽体，在大肠杆菌Transetta（DE3）感受态细胞中进行诱导表达；对纯化的目的蛋白进行SDS—PAGE和Western—blot分析。结果表明．大片吸虫FgRapi基因大小为648bp，编码215个氨基酸残基。生物信息学分析表明，该蛋白含有9个亲水区，但无跨膜区，有6个α螺旋、8个口折叠、2个较长的口转角、1个较长的无规则卷曲和8个主要的线性B细胞抗原表位。FgRapl蛋白为包涵体表达，用大片吸虫排泄分泌抗原免疫家兔的阳性血清进行Western—blot分析，表明其具有良好的免疫反应性。本研究首次克隆并鉴定了大片吸虫Ras家族蛋白基因，表达的重组蛋白具有良好的免疫反应性，该蛋白是潜在的大片吸虫诊断候选抗原。Ras-related proteins are key molecules in many signal transduction pathways and func- tion in the growth and development of parasites. To identify Ras-related protein of Fasciola gigantica （FgRap1） and analyze its feasibility as a diagnostic antigen,the specific primers for amplifying FgRap1 were designed according to the genome and transcriptome data of Fasciola hepatica, and the complete ORF of FgRap1 was amplified from the total RNA by RT-PCR. The fragment including the FgRap1 complete ORF was cloned into the pET-SUMO expression vector. The Ras-related protein was expressed in Escherichia coli Transetta（DE3） chemically competent cell and immune-reactivity was tested by Western-blot. The results showed that FgRapl gene sequence contained an ORF of 648 bp, encoding a protein of 215 amino acids. Bioinformatics analysis showed that FgRap1 contained 9 hydrophobicity regions but without transmem- brane area. It has 6 alpha regions,8 beta sheets,2 beta turns,1 random coils and 8 B cell epitopes.FgRapl was expressed as inclusion body,and Western-blot analysis using rabbit antibody against F. gigantica excretory and secretory products indicated good immune-reactivity. This study cloned and expressed the FgRap1 gene for the first time,and the recombinant protein has good immune-reactivity,in- dicating that FgRap1 may represent a candidate antigen for the diagnosis of F. gigantica infection.

关 键 词：大片吸虫 Ras家族蛋白基因 鉴定 表达 诊断候选抗原 

分 类 号：S852.735[农业科学—基础兽医学]