| 摘要: |
| 应用PCR扩增得到猪2型圆环病毒ORF2基因的3′端约600bp,该PCV2 ORF2基因已去除终止子,并分别在其上下游添加KpnⅠ和HindⅢ酶切位点.PCR产物用KpnⅠ和HindⅢ酶切后,与经同样处理过的pBAD/g ⅢB Vector连接,转化TOP10细胞后挑取阳性克隆,经酶切鉴定和测序验证后用L-arabinose诱导进行融合表达,表达的蛋白主要以包涵体存在。经过镍离子亲和层析柱纯化,SDS-PAGE显示1条约28 ku的目的条带,纯度达到85%以上。以该28 ku的多肽包被酶标板,建立了PCV2的间接ELISA检测方法. |
| 关键词: 猪圆环病毒 ORF2基因 大肠杆菌 表达 检测 |
| DOI： |
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| 基金项目:陕西省农业攻关项目（2003K02-G10） |
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| Development of indirect ELISA for detecting PCV2 antibody |
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| Abstract: |
| In order to express ORF2 gene of porcine circovirus type 2 (PCV-2),a pair of primers was designed according to the sequence of PCV2.The ORF2 gene was amplified by using the primers having HindⅢ and KpnⅠ site by PCR,and then the gene was subcloned into prokaryotic expressing vector pBAD/gⅢ.So the recombinant plasmid named pBAD/gⅢ-ORF2 was constructed.After that,pBAD/gⅢ-ORF2 was transformed into Escherichia coli TOP10 and induced by L-Arabinose.The results of SDS-PAGE and Western blot indicated that the ORF2 gene was expressed and the recombinant fusion protein was about 28 ku with purity over 85%.By using the fusion protein,a kind of ELISA method can be built for detecting PCV2 antibody. |
| Key words: porcine circovirus type 2 ORF2 gene expression in E.coli ELISA detecting |
