湖北农业科学
湖北農業科學
호북농업과학
2009年
11期
2632-2635
,共4页
温国元%李坤%邵华斌%罗青平%张蓉蓉%王红琳%艾地云%罗玲%程国富%杨峻
溫國元%李坤%邵華斌%囉青平%張蓉蓉%王紅琳%艾地雲%囉玲%程國富%楊峻
온국원%리곤%소화빈%라청평%장용용%왕홍림%애지운%라령%정국부%양준
猪瘟病毒%E2蛋白%间接EUSA%原核表达
豬瘟病毒%E2蛋白%間接EUSA%原覈錶達
저온병독%E2단백%간접EUSA%원핵표체
classical swine fever virus%E2 protein%indirect ELISA%prokaryotic expression
以猪瘟病毒(Classical swine fever virus,CSFV)标准强毒石门株为模板,RT-PCR扩增CSFV E2基因N端的主要抗原区(△E2).PCR产物定向克隆至原核表达载体pET-32a中,构建了重组表达质粒pET-AE2,转化Eacterium coli BL21-Codonplus(DE3),IPTG诱导重组△E2蛋白表达,经SDS-PAGE和Western-blot鉴定蛋白的正确表达.KCl预染切胶法纯化△E2蛋白,以其为包被抗原,通过反应条件的优化,建立了CSFV抗体ELISA检测方法.用该方法对331份临床血清进行检测,并与IDEXX公司阻断ELISA试剂盒进行了相符性验证,符合率为74%.为研制猪瘟病毒抗体ELISA检测试剂盒奠定了基础.
以豬瘟病毒(Classical swine fever virus,CSFV)標準彊毒石門株為模闆,RT-PCR擴增CSFV E2基因N耑的主要抗原區(△E2).PCR產物定嚮剋隆至原覈錶達載體pET-32a中,構建瞭重組錶達質粒pET-AE2,轉化Eacterium coli BL21-Codonplus(DE3),IPTG誘導重組△E2蛋白錶達,經SDS-PAGE和Western-blot鑒定蛋白的正確錶達.KCl預染切膠法純化△E2蛋白,以其為包被抗原,通過反應條件的優化,建立瞭CSFV抗體ELISA檢測方法.用該方法對331份臨床血清進行檢測,併與IDEXX公司阻斷ELISA試劑盒進行瞭相符性驗證,符閤率為74%.為研製豬瘟病毒抗體ELISA檢測試劑盒奠定瞭基礎.
이저온병독(Classical swine fever virus,CSFV)표준강독석문주위모판,RT-PCR확증CSFV E2기인N단적주요항원구(△E2).PCR산물정향극륭지원핵표체재체pET-32a중,구건료중조표체질립pET-AE2,전화Eacterium coli BL21-Codonplus(DE3),IPTG유도중조△E2단백표체,경SDS-PAGE화Western-blot감정단백적정학표체.KCl예염절효법순화△E2단백,이기위포피항원,통과반응조건적우화,건립료CSFV항체ELISA검측방법.용해방법대331빈림상혈청진행검측,병여IDEXX공사조단ELISA시제합진행료상부성험증,부합솔위74%.위연제저온병독항체ELISA검측시제합전정료기출.
In order to express and purify the recombinant E2 protein of classical swine fever virus (CSFV), a truncated E2 gene fragment was amplified by RT-PCR with the CSFV virulent Shimen strain as template. After digestion with BamHl/ HindIII, the PCR product was cloned into pET-32a vector. The recombinant plasmid pET-△E2 was analyzed by resctriction enzyme digestion and sequencing, then was transformed into Eaeterium coli BL21-Condonplus (DE3). The recombinant △E2 protein was expressed by the induction of IPTG and identified by SDS-PAGE and Westem-blot analysis. An CSFV antibody indirect ELISA (△E2-ELISA) was developed and optimized with the purified recombinant △E2 protein as coating protein. Then, 331 swine sera samples were detected with the △E2-ELISA and IDEXX ELISA Kit, the coincidence rate between the △E2-ELISA and IDEXX ELISA Kit was determined as 74%. Further investigation was focused on the development of an indirect ELISA Kit for the detection of CSFV antibodies.
