摘要
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Ecoli 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42ku，可与6×HIS抗体反应；KCI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。
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 pET- 32a（＋） vector. A clone containing the inserted DNA in the correct orientation was designated pET-Cap and transformed into Escherichia coli strain BL21-eodonplus（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 meth- od,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.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2009年第6期710-714,共5页
Journal of Huazhong Agricultural University
基金
湖北省科技攻关项目(2007AA402C55)资助
