猪圆环病毒2型ORF1和ORF2基因的克隆与表达  

Cloning and expression of ORF1 and ORF2 genes of porcine circovirus type 2

作　　者：王生育[1] 颜江华[1] 孔繁德[2] 姜海军[3] 张长弓[1] 黄印尧[2] 

机构地区：[1]厦门大学医学院抗癌研究中心,福建厦门361005 [2]厦门出入境检验检疫局,福建厦门361012 [3]福建农林大学动物科学学院,福建福州361006

出　　处：《中国预防兽医学报》2007年第12期934-937,共4页Chinese Journal of Preventive Veterinary Medicine

摘　　要：根据GenBank猪圆环病毒2型基因序列,设计合成两对特异性引物,对厦门株-1进行PCR扩增,得到ORF1和ORF2基因。将PCR产物酶切后,插入到pET-22b载体中,构建原核表达载体pET-22b/ORF1、pET-22b/ORF2,转化大肠杆菌BL21(DE_3),经IPTG诱导表达,收集茵液进行SDS-PAGE分析。确定重组蛋白主要以包涵体的形式表达,包涵体洗涤溶解后,采用Ni^(2+)离子金属螯合亲和层析柱纯化蛋白,逐步透析法进行复性。Western blot和ELISA分析结果表明Rep蛋白和Cap蛋白均能与猪圆环病毒2型阳性血清发生特异性反应。ORFI and ORF2 genes of porcine circovirus type 2 （PCV-2） were obtained by PCR with specific primers designed according to PCV-2 sequence in GenBank and cloned into pET-22b, respectively. The resultant constructs were transformed into E.coli BL21 （DE3） and expressed by IPTG induction. The expression products, which existed mainly as inclusion bodies, was purified by Ni^2＋-affinity chromatographic columnwas and refolded following dialysis. Western blot and ELISA analysis showed that the purified products could react with PCV-2 specific serum.

关 键 词：猪圆环病毒 REP蛋白 CAP蛋白 表达 

分 类 号：Q786[生物学—分子生物学] S852.659.2[农业科学—基础兽医学]