猪圆环病毒2型Cap原核表达与间接ELISA方法的建立

发布时间：2018-05-24 02:58

  本文选题：猪圆环病毒2型 + Cap基因　； 参考：《安徽农业大学》2015年硕士论文

【摘要】：猪圆环病毒(Porcine circovirus,PCV)是圆环病毒属的代表种,可分为PCV1和PCV2。PCV1没有致病性;PCV2引起的断奶仔猪多系统衰竭综合征(Postweaning multisystemic wasting syndrome,PMWS)是猪的免疫抑制病之一,给世界养猪业造成了严重的经济损失。目前,临床上还没有有效的方法控制和消灭PCV2,市场上流通的疫苗也不能起到很好的预防效果。因此,建立一种血清学检测方法,快速、及时掌握PCV2的流行情况,做好预防措施就显得十分关键。研究利用PCV2 ORF2的原核表达蛋白作为抗原,建立间接ELISA检测方法,并初步应用于PCV2血清抗体的临床检测。首先,根据实验室保存的PCV2鄂州株(Genebank未登录)基因序列设计特异性引物,分别在上、下游引物加入Sac I和Hind III酶切位点,扩增出PCV2鄂州株的ORF2基因,将该片段克隆至pET-28a原核表达载体,经PCR、酶切鉴定,表明成功构建了阳性重组表达质粒pET-ORF2。将其转化入大肠杆菌Rosetta中,经IPTG诱导表达,SDS-PAGE电泳分析初步确定重组蛋白为34.5KDa,主要以包涵体形式表达。最佳诱导条件为:37℃条件下,摇菌至OD值0.4-0.6左右时加入终浓度1mmol/L IPTG,诱导5h时目的蛋白表达量最高,可达0.46mg/mL。表达产物经His-tag镍柱纯化后进行Western-blot分析。结果表明表达的重组蛋白能够与PCV2标准阳性血清发生特异性反应,具有良好的反应原性。其次,用纯化的重组蛋白作为包被抗原,建立了PCV2抗体间接ELISA检测方法。其中,抗原包被浓度为4.6μg/mL,37℃孵育2h后4℃过夜;5%SMP/PBST,37℃封闭2h;待检血清的最佳稀释度为1∶40,37℃孵育1h;HRP-羊抗猪IgG稀释度为1∶3 000,37℃孵育1h;TMB显色15min。经统计学分析确定阴阳性临界值为0.421。并对优化后的间接ELISA方法的特异性、重复性进行测定并与商品化的试剂盒进行阳性符合率比较。结果显示,建立的间接ELISA方法与CSFV、PRRSV、FMDV、PRV、TGEV、PEDV的阳性血清无交叉反应,特异性好;变异系数最大为8.71%;与商品化试剂盒比较,阳性符合率达到96.7%。用建立的ELISA方法检测猪场送检血样223份,总阳性检出率为61.9%(138/223),其中保育猪阳性检出率为58.3%(63/108),育肥猪阳性检出率为65.2%(75/115)。结果表明建立的间接ELISA方法能用于临床PCV2抗体样本检测并为ELISA诊断试剂盒的开发奠定了基础。
[Abstract]:Porcine circovirus (PCV) is a representative species of the genus circovirus. It can be divided into two groups: PCV1 and PCV2.PCV1. The postweaning multisystemic wasting syndrome induced by PCV2 is one of the immunosuppressive diseases in pigs, which has caused serious economic losses to the world pig industry. At present, there is no effective method to control and eliminate PCV2 in clinic, and the marketable vaccine can not play a good preventive effect. Therefore, it is very important to establish a serological detection method to grasp the prevalence of PCV2 quickly and timely. To establish an indirect ELISA detection method using prokaryotic expression protein of PCV2 ORF2 as antigen, and to apply it to the clinical detection of PCV2 serum antibody. Firstly, specific primers were designed according to the sequence of PCV2 Ezhou strain (Genebank) kept in laboratory. The ORF2 gene of PCV2 Ezhou strain was amplified by adding Sac I and Hind III sites into the upstream and downstream primers, respectively. The fragment was cloned into the prokaryotic expression vector of pET-28a and identified by PCR and restriction endonuclease digestion. The positive recombinant expression plasmid pET-ORF2 was successfully constructed. It was transformed into Escherichia coli Rosetta and expressed by IPTG induced by SDS-PAGE. The recombinant protein was identified as 34.5KDa. it was mainly expressed as inclusion body. The optimal induction conditions were as follows: at 37 鈩，

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