›› 2010, Vol. 37 ›› Issue (6): 171-173.

• 疾病防治 • 上一篇    下一篇

猪圆环病毒2型ORF3基因的原核表达

冉旭华，李树东，闻晓波，王密，杨焕民，倪宏波，侯喜林，朴范泽，邵磊，李晓娟，李冬野，周恩民   

  1. （黑龙江八一农垦大学动物科技学院 预防兽医学省重点实验室， 大庆 163319）
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-06-20 发布日期:2010-06-20

Cloning and Expression of ORF3 Gene of Porcine Circovirus Type 2 in E.coli

RAN Xu-hua,LI Shu-dong,WEN Xiao-bo,WANG Mi, YANG Huan-min,NI Hong-bo，HOU Xi-lin,PIAO Fan-ze,SHAO Lei,LI Xiao-juan,LI Dong-ye,ZHOU En-mim   

  1. (Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China)
  • Received:1900-01-01 Revised:1900-01-01 Online:2010-06-20 Published:2010-06-20

摘要： 用原核表达系统表达猪圆环病毒2型ORF3基因，分析ORF3蛋白的抗原性。根据GenBank上公布的猪圆环病毒2型(PCV-2)核苷酸序列进行分析，针对ORF3基因设计并合成1对特异性引物，PCR扩增该基因，并将此片段克隆入原核表达载体pET-32a(+)上，命名为pET32a-ORF3，转化大肠杆菌Rosetta-blue(DE3) pLacⅠ感受态细胞，1.0 mmol/L IPTG 37 ℃诱导表达。结果表明，PCR扩增得到315 bp的片段，重组蛋白大小约为29 ku，与预期大小相符。Western blotting分析结果表明，重组蛋白能与抗His-tag的单克隆抗体反应，不与PCV-2阳性血清发生反应。猪圆环病毒2型ORF3基因能在大肠杆菌中成功表达，但与阳性血清之间没有反应原性，为进一步研究PCV-2 ORF3蛋白的功能及特性奠定基础。

关键词: 猪圆环病毒2 型; ORF3基因; 原核表达

Abstract: To express ORF3 gene of porcine circovirus 2（PCV-2） in E.coli, analyze the antigenicity of ORF3 protein, according to the amino acid sequence of PCV-2 strain in GenBank, a pair of specific primer was designed to amplify the fragment encoding ORF3 gene. This fragment was cloned into the multiple cloning sites of pET-32a (+) vector. The recombinant plasmid was designated pET32a-ORF3 and was transformed into E.coli Rosetta-blue (DE3) pLacⅠ competent cells. Expression of recombinant protein was induced by addition of final concentration of 1.0 mmol/L IPTG under 37 ℃ condition. The length of PCR product of interest was 315 base pairs. The size of recombinant amino acid was 29 ku. The results of western blotting showed that it could be recognized by anti-His tagged mouse monoclonal antibody and could not be recognized by swine serum infected with PCV-2. The results showed that ORF3 of PCV-2 was expressed successfully in E.coli and the ORF3 protein didn’t exhibit reaction genicity. These results were benefit to research for function and characteristics of ORF3 of PCV-2.

Key words: porcine circovirus 2（PCV-2）; ORF3 gene; prokaryotic expression

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