应用在大肠杆菌中表达的猪2型圆环病毒ORF2蛋白建立一种ELISA诊断方法

The Establishment of an ELISA Assay with the ORF2 Protein of Porcine Circovirus type 2 Expressed in E.coli

根据GenBank中猪2型圆环病毒(PCV2)序列(AY035820),设计1对引物,并以包含PCV2全基因组的重组质粒pT-PCV为模板,扩增出含ORF2全基因的片段(709bp),回收PCR产物,将其克隆入pMD18-T载体,获得重组质粒pTORF2,并对其进行序列测定。重组质粒经Bal 和Sal 双酶切,回收ORF2基因,将其插入pGEX-KG的Sma 和Sal 位点间,构建原核表达质粒pGEXORF2,阳性重组质粒转化E.coliBL21(DE3),进行原核表达。用此表达产物包被酶标板,建立ELISA诊断方法,并对临床676份送检血清进行了检测,结果表明其总阳性率为62.7%(424/676),仔猪阳性率为38.9%(74/190),肥猪阳性率为63.2%(84/133),母猪阳性率为74.6%(249/334),公猪阳性率为89.5%(17/19)。

According to the whole genome sequence of PCV2 (GenBank No.AY035820), a pair of primers were designed to clone ORF2 gene by PCR from pT-PCV which contains the entire genome of PCV2. The PCR products was extracted, and was linked with pMD18-T vector (named as pTORF2). DNA sequence was determined. pTORF2 was digested with BalⅠ and SalⅠ to generate 640 bp fragment, laterly the acquired fragment were cloned into pGEX-KG and transformed into E.coli DH5α. The recombinant plasmids(pGEXORF2) were transformed into E.coli BL21. The expressed protein was used to establish an enzyme-linked immunosorbent assay (ELISA). 676 sera samples were examined with this ELISA. The total positive rate of these sera is 62.7%(424/676). The positive incidence in piglets, hogs, sows and boars is 38.9%(74/190), 63.2%(84/133), 74.6%(249/334) and 89.5%(17/19) respectively.