华中农业大学学报
華中農業大學學報
화중농업대학학보
JOURNAL OF HUAZHONG AGRICULTURAL UNIVERSITY
2009年
6期
710-714
,共5页
李坤%温国元%罗青平%商雨%艾地云%罗玲%王红琳%杨峻%邵华斌%程国富
李坤%溫國元%囉青平%商雨%艾地雲%囉玲%王紅琳%楊峻%邵華斌%程國富
리곤%온국원%라청평%상우%애지운%라령%왕홍림%양준%소화빈%정국부
猪圆环病毒2型%核衣壳基因%原核表达%间接ELISA
豬圓環病毒2型%覈衣殼基因%原覈錶達%間接ELISA
저원배병독2형%핵의각기인%원핵표체%간접ELISA
porcine circovius type 2%capsid gene%prokaryotic expression%indirect ELISA
建立了检测猪圆环病毒2型血清抗体的间接ELISA方法,并初步应用于临床血清样品的检测.用PCR方法从PCV2 HBZX株中获得截短的核衣壳(capsid,Cap)基因,将该基因克隆到原核表达载体pET-32a(+)中,构建重组表达质粒pET-Cap,将该重组质粒转化宿主菌E.coli BL21-CodonPlus(DE3),用IPTG诱导,SDS-PAGE和Western-blot鉴定蛋白的正确表达,表达蛋白cap分子质量为42 ku,可与6×HIS抗体反应;KCl预染切胶纯化目的蛋白,以其为抗原建立检测PCV2感染猪血清抗体的间接ELISA方法.ELISA检测5份血清样品结果变异系数均小于10%.用该方法分别检测湖北、河南等地的91份临床血清样品,阳性率为25%～100%.上述结果表明,所制备的目的蛋白具有较高的纯度和良好的免疫原性,所建立的间接ELISA检测抗体方法,具有良好的重复性,初步临床试用效果良好.
建立瞭檢測豬圓環病毒2型血清抗體的間接ELISA方法,併初步應用于臨床血清樣品的檢測.用PCR方法從PCV2 HBZX株中穫得截短的覈衣殼(capsid,Cap)基因,將該基因剋隆到原覈錶達載體pET-32a(+)中,構建重組錶達質粒pET-Cap,將該重組質粒轉化宿主菌E.coli BL21-CodonPlus(DE3),用IPTG誘導,SDS-PAGE和Western-blot鑒定蛋白的正確錶達,錶達蛋白cap分子質量為42 ku,可與6×HIS抗體反應;KCl預染切膠純化目的蛋白,以其為抗原建立檢測PCV2感染豬血清抗體的間接ELISA方法.ELISA檢測5份血清樣品結果變異繫數均小于10%.用該方法分彆檢測湖北、河南等地的91份臨床血清樣品,暘性率為25%～100%.上述結果錶明,所製備的目的蛋白具有較高的純度和良好的免疫原性,所建立的間接ELISA檢測抗體方法,具有良好的重複性,初步臨床試用效果良好.
건립료검측저원배병독2형혈청항체적간접ELISA방법,병초보응용우림상혈청양품적검측.용PCR방법종PCV2 HBZX주중획득절단적핵의각(capsid,Cap)기인,장해기인극륭도원핵표체재체pET-32a(+)중,구건중조표체질립pET-Cap,장해중조질립전화숙주균E.coli BL21-CodonPlus(DE3),용IPTG유도,SDS-PAGE화Western-blot감정단백적정학표체,표체단백cap분자질량위42 ku,가여6×HIS항체반응;KCl예염절효순화목적단백,이기위항원건립검측PCV2감염저혈청항체적간접ELISA방법.ELISA검측5빈혈청양품결과변이계수균소우10%.용해방법분별검측호북、하남등지적91빈림상혈청양품,양성솔위25%～100%.상술결과표명,소제비적목적단백구유교고적순도화량호적면역원성,소건립적간접ELISA검측항체방법,구유량호적중복성,초보림상시용효과량호.
An indirect ELISA method was developed based on a recombinant capsid protein of porcine circovirus type 2(PCV2).The DNA of the truncated capsid gene was amplified by PCR from the HBZX strain.The truncated cap gene of PCV2 strain HBZX was amplified by PCR and ligated into pET32a(+)vector.A clone containing the inserted DNA in the correct orientation was designated pET-Cap and transformed into Escherichia coli strain BL21-codonplus(DE3).The expressed pET-Cap gene product had a molecular mass of 42 ku identified by SDS-PAGE and Western-blot.The Cap protein was used as antigen to establish an indirect ELISA diagnostic method for PCV2 serum antibody detection.A total of 91 sera obtained from Henan and Hubei Province were tested by the Cap-based indirect ELISA method,the positive rate ranged between 25%～100%.The results obtained demonstrate that the Cap-based indirect ELISA method was able to detect PCV2 specific antibodies in swine sera and might be a useful tool for serodiagnosis.
