一种克隆载体重组质粒pMD18-T Simple-PCV2的构建

Construction of a cloning vector recombinant plasmid pMD18-T Simple-PCV2

为了研究猪圆环病毒2型(PCV2)的全基因组变异特性,试验设计了1对特异性引物,并从疑似断乳仔猪多系统衰竭综合征(PMWS)的病料中经PCR直接扩增出PCV2全基因组,再与载体pMD18-T Simple连接后形成重组质粒pMD18-T Simple-PCV2(命名为P-S-PCV2),经酶切鉴定、PCR鉴定和序列测定分析的阳性重组质粒被证明含有长为1 767 bp的PCV2片段,同时通过Blast与GenBank中国内外的部分PCV2毒株进行同源性比较分析。结果表明:与国内外12个毒株间的同源性介于95.0%～100%之间,与国内的河北株(HB株)亲缘关系最近,同源性高达100%;与澳大利亚株(AUT2株)亲缘关系最远,同源性为95.0%。说明来源不同国家和地区的猪圆环病毒2型毒株存在地域性差异。

To study the complete genomic sequence of Porcine circovirus type 2 （ PCV2）, a pair of primers was designed, and the PCV2 complete genome was amplified directly by PCR from the pigs with doubtful post -weaning muhisystemic wasting syndrome （PMWS） and cloned into pMD18 -T Simple vector to construct the recombinant plasmid pMD18 -T Simple -PCV2 （named P -S -PCV2 ）. The positive recombinant plasnnid was digested with restriction enzyme and then identified by PCR and sequencing analysis, it contained a PCV2 segment of 1 767 bp. The sequence homology analysis was used to compare with part of the PCV2 strains at home and abroad listed in GenBank using Blast. The resuits showed that the strain shared homology of 95.0% ~ 100% compared with the other twelve strains listed in GenBank, the closest genetic relationship was with domestic strain of HB （ 100% homology）, the farthest genetic relationship was with Australia strain of AUT2 （ homology of 95.0% ）. It indicates that PCV2 isolates from different countries and areas have regional differences.