猪圆环病毒2型CC株ORF3基因序列分析及真核表达

根据GenBank中猪圆环病毒2型(PCV2)的核酸序列,设计了1对引物,采用PCR方法从疑似断奶仔猪多系统衰竭综合征(PMWS)的死亡仔猪病料中扩增出了ORF3基因,将其克隆到高效真核表达载体pEGFP- N2中,筛选出含有ORF3基因的重组质粒,命名为pEGFP-N2-ORF3.对此重组质粒进行测序分析,结果表明,克隆的ORF3基因与其他PCV2的ORF3核苷酸序列相似性为96.2％-99.0％,氨基酸序列同源性为90.5 ％～98.1％.将重组质粒纯化后转染COS-7细胞,用PCR方法扩增出了特征性的基因片段,并采用特异性单抗进行间接免疫荧光,结果转染pEGFP- N2-ORF3重组蛋白在细胞核和细胞浆中均有表达,尤其在细胞核中表达量较高,且对细胞有一定的毒性.本研究为进一步探讨ORF3的结构和功能提供理论依据.

Sequence analysis of ORF3 gene of type 2 porcine circovirus and eukaryotic expressing of ORF3

A pair of primers were designed according to published ORF3 gene sequence of PCV2,and the ORF3 gene was amplified from the suspected PMWS samples by PCR.The PCR product was cloned into pEGFP-N2 vector,and the recombinant plasmid pEGFP-N2-ORF3 was obtained.The sequence of the cloned PCR product was analyzed and compared with other PCV2 isolates in GenBank.The results indicated that it shared 96.2% to 99.0% homology at their nucleotide level,and shared 90.5% to 98.1% identity at deduced amino acid level with other PCV2 strains.The purified recombinant plasmids were transfected into COS-7 cells,and the cloned gene fragment was amplified by PCR from transfected cells Using specific monoclonal antibody for indirect immunofluorescence(IFA),the recombinant protein was showed to be expressed in cellular nucleus and cytoplasms of COS-7 cells and showed some toxicity to COS-7 cells.This study provides a basis for further exploring the structure and function of ORF3.