摘要
通过PCR扩增猪圆环病毒2型HZ0201株的全长基因组,克隆到pBluescript SK+载体SacⅡ位点;同时以SacⅡ切出PCV2全长DNA,并进行自身环化,分别构建PCV2全长基因组重组质粒和首尾相接的环化DNA。利用脂质体分别将PCV2重组质粒和环化DNA转染PK-15和Vero细胞,免疫组织化学试验(IHC)结果显示二者均检出PCV2阳性抗原。其中PCV2环化DNA转染细胞的阳性率显著高于PCV2重组质粒。将转染细胞进行连续8次传代,以IHC对每代病毒检测表明,PCV2分子克隆在PK-15细胞中形成的病毒能稳定感染,并且滴度逐步增加;而在Vero细胞上复制能力不强,传到第四代即不能检出病毒。
The complete genome of porcine circovirus type 2 was amplified by polymerase chain reaction （PCR） and cloned into Sac Ⅱ site in pBluescript SK^＋ to construct PCV2 recombinant plasmid. The viral genomic sequence was then released from the plasmid by Sac Ⅱ digestion and circlized by self-ligation, The infectivity of the recombinant plasmid and circlized genome clones was analysed by lipofectamine transfection of the clones into PK-15 and vero cells, respectively. PCV2 antigen expressions could be detected on both cell lines by immunohistochemical analysis （IHC）, The antigen expression of circlized genome was more efficient than the recombinant plasmid. The transfected cells were passaged 8 times and antigen expression was monitored by IHC throughout the passaging period. The virus antigen could stably produced in PK-15 and the titer increased gradually. In comparison, only weak antigen expression was detected cells up to passage 3.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2007年第2期86-91,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(No.30370052)资助
关键词
猪圆环病毒2型
分子克隆
感染性分析
porcine cicrovirus type 2
molecular clone
infectivity analysis
