肠道病毒71型外壳蛋白VP2基因重组表达及活性鉴定

Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71

目的 克隆表达肠道病毒71型（EV71）外壳蛋白VP2,鉴定重组蛋白免疫活性,为EV71血清学检测试剂和疫苗研究提供依据.方法 利用PCR技术从EV71/Henan/106/2009河南分离株基因组扩增VP2基因,经酶切连接到表达载体pMAL-c2X中,转化大肠杆菌（E.coli TB1）;用异丙基硫代半乳糖苷（IPTG）诱导目的基因表达,并对重组蛋白进行纯化,SDS-PAGE和Western blotting方法对重组蛋白分析鉴定其免疫活性;建立ELISA检测EV71 IgM抗体方法,检测手足口病患儿急性期中和抗体阳性血清60份,其中阳性52份,阴性8份;检测临床诊断手足口病患儿急性期血清标本88份.结果 PCR方法扩增的VP2基因长度约为762 bp;经酶切鉴定插入到表达载体的基因片段与预期目的片段相一致;SDS-PAGE结果显示表达产物相对分子质量约为71 500;经大肠杆菌高效表达和纯化获得了重组蛋白EV71-VP2,通过Western blotting分析,该重组蛋白与手足口病患者血清反应产生特异杂交带;建立ELISA检测方法的灵敏度为87％,特异度为83％.在临床诊断的88份手足口病患儿急性期血清标本中,抗EV71-IgM阳性48份,阳性率为55％.结论 本研究成功克隆并构建了EV71 VP2基因高效原核表达系统,初步结果显示重组蛋白具有较好的抗原性,为EV71诊断试剂的研究奠定了基础.

Objective To clone and express the recombinant capsid protein VP2 of enterovirus type 71 （EV71） and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.Methods VP2 gene of EV71 was amplified by PCR,and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X.The positive recombinants were transferred into E.coli TB1,the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside （IPTG）,and the expression products were analyzed by SDS-PAGE and western blotting method.EV71 IgM antibody detection method by ELISA was set up,and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease （HFMD） patients were determinted,of which 52 samples were positive and 8 samples were negative ; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected.Results VP2 gene of 762 bp was obtained by PCR,the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion.The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE.The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting.The sensitivity and specificity of ELISA was 87% and 83%,respectively.Of the 88 acute phase serum samples from children with HFMD,48 samples （55%） were positive by the ELISA assay.Conclusions VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study.The recombination EV71-VP2 has well antigenicity,which could be useful for developing diagnose reagent or vaccine of EV71.