中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2014年
4期
324-327
,共4页
黄学勇%刘国华%胡小宁%杜燕华%李幸乐%许玉玲%陈豪敏%许汴利
黃學勇%劉國華%鬍小寧%杜燕華%李倖樂%許玉玲%陳豪敏%許汴利
황학용%류국화%호소저%두연화%리행악%허옥령%진호민%허변리
手足口病%肠道病毒属%抗原%DNA,重组
手足口病%腸道病毒屬%抗原%DNA,重組
수족구병%장도병독속%항원%DNA,중조
Hand,foot and mouth disease%Enterovirus%Antigens%DNA,recombinant
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.
目的 剋隆錶達腸道病毒71型(EV71)外殼蛋白VP2,鑒定重組蛋白免疫活性,為EV71血清學檢測試劑和疫苗研究提供依據.方法 利用PCR技術從EV71/Henan/106/2009河南分離株基因組擴增VP2基因,經酶切連接到錶達載體pMAL-c2X中,轉化大腸桿菌(E.coli TB1);用異丙基硫代半乳糖苷(IPTG)誘導目的基因錶達,併對重組蛋白進行純化,SDS-PAGE和Western blotting方法對重組蛋白分析鑒定其免疫活性;建立ELISA檢測EV71 IgM抗體方法,檢測手足口病患兒急性期中和抗體暘性血清60份,其中暘性52份,陰性8份;檢測臨床診斷手足口病患兒急性期血清標本88份.結果 PCR方法擴增的VP2基因長度約為762 bp;經酶切鑒定插入到錶達載體的基因片段與預期目的片段相一緻;SDS-PAGE結果顯示錶達產物相對分子質量約為71 500;經大腸桿菌高效錶達和純化穫得瞭重組蛋白EV71-VP2,通過Western blotting分析,該重組蛋白與手足口病患者血清反應產生特異雜交帶;建立ELISA檢測方法的靈敏度為87%,特異度為83%.在臨床診斷的88份手足口病患兒急性期血清標本中,抗EV71-IgM暘性48份,暘性率為55%.結論 本研究成功剋隆併構建瞭EV71 VP2基因高效原覈錶達繫統,初步結果顯示重組蛋白具有較好的抗原性,為EV71診斷試劑的研究奠定瞭基礎.
목적 극륭표체장도병독71형(EV71)외각단백VP2,감정중조단백면역활성,위EV71혈청학검측시제화역묘연구제공의거.방법 이용PCR기술종EV71/Henan/106/2009하남분리주기인조확증VP2기인,경매절련접도표체재체pMAL-c2X중,전화대장간균(E.coli TB1);용이병기류대반유당감(IPTG)유도목적기인표체,병대중조단백진행순화,SDS-PAGE화Western blotting방법대중조단백분석감정기면역활성;건립ELISA검측EV71 IgM항체방법,검측수족구병환인급성기중화항체양성혈청60빈,기중양성52빈,음성8빈;검측림상진단수족구병환인급성기혈청표본88빈.결과 PCR방법확증적VP2기인장도약위762 bp;경매절감정삽입도표체재체적기인편단여예기목적편단상일치;SDS-PAGE결과현시표체산물상대분자질량약위71 500;경대장간균고효표체화순화획득료중조단백EV71-VP2,통과Western blotting분석,해중조단백여수족구병환자혈청반응산생특이잡교대;건립ELISA검측방법적령민도위87%,특이도위83%.재림상진단적88빈수족구병환인급성기혈청표본중,항EV71-IgM양성48빈,양성솔위55%.결론 본연구성공극륭병구건료EV71 VP2기인고효원핵표체계통,초보결과현시중조단백구유교호적항원성,위EV71진단시제적연구전정료기출.
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.