摘要
根据GenBank发表的犬瘟热病毒N基因全序列,设计合成了1对特异扩增CDVN基因的引物。以山东泰安分离的CDV-FOX-TA株细胞毒中提取病毒RNA来制模板,利用RT-PCR扩增出了1.6kb的N基因,将其克隆到pIREShyg载体上,构建了pIRES-N真核表达载体。然后通过磷酸钙共沉淀法转染CHO-K1细胞,通过潮霉素筛选得到阳性克隆,间接免疫荧光实验(IFA)鉴定N基因在CHO细胞中的表达,并用RT-PCR方法从转录水平证实N基因在CHO-K1细胞中的表达,最终建立了CHO/CDV-N细胞株,为犬瘟热病毒的血清学检测和基因疫苗的研制奠定了基础。
According to the published sequence of N gene of CDV, a pair of primers was designed and synthesized, which can amplify CDV N gene. N gene’s cDNA fragment of about 1.6kb was obtained through RT-PCR from FOX TA strain of CDV, and was cloned into pIREShyg Vector Eukarytic Expression Vector named as pIRES-N was constructed. pIRES-N was transfected into CHO-K1 cells with coprecipitation, and positive cells were screened with hygromycine selection. The N gene protein was expressed in the CHO cells proved by IFA. The N gene was transcripted as proved with RT-PCR. CHO/CDV-N cell strain was constructed, which provided evidence for research of CDV serumal detection and gene vaccine.
出处
《中国农学通报》
CSCD
2008年第4期8-12,共5页
Chinese Agricultural Science Bulletin
基金
国家质检总局课题资助(项目号:2005IK047)
