犬瘟热病毒核衣壳蛋白N基因的真核表达及鉴定

简中友,,贾 赟,王全凯,徐立秋,吴 斌,肇慧君,李振荣

中国农学通报. 2008, 24(4): 8-12

中国农学通报 ›› 2008, Vol. 24 ›› Issue (4) : 8-12. DOI: 10.11924/j.issn.1000-6850.20085719
畜牧 动物医学 蚕 蜂

犬瘟热病毒核衣壳蛋白N基因的真核表达及鉴定

  • 简中友,,贾 赟,王全凯,徐立秋,吴 斌,肇慧君,李振荣
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Eukarytic Expression and Identification of Nucleocapsid Protein N Gene of Canine Distemper Virus

  • Jian Zhongyou, , Jia Yun, Wang Quankai, Xu Liqiu, Wu Bin, Zhao Huijun, Li Zhenrong
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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CDV N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。

Abstract

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.

关键词

犬瘟热病毒；N基因；潮霉素；CHO-K1细胞

Key words

canine distemper virus;N gene;hygromycine;CHO-K1 cell

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简中友,,贾 赟,王全凯,徐立秋,吴 斌,肇慧君,李振荣. 犬瘟热病毒核衣壳蛋白N基因的真核表达及鉴定. 中国农学通报. 2008, 24(4): 8-12 https://doi.org/10.11924/j.issn.1000-6850.20085719
Jian Zhongyou, , Jia Yun, Wang Quankai, Xu Liqiu, Wu Bin, Zhao Huijun, Li Zhenrong. Eukarytic Expression and Identification of Nucleocapsid Protein N Gene of Canine Distemper Virus. Chinese Agricultural Science Bulletin. 2008, 24(4): 8-12 https://doi.org/10.11924/j.issn.1000-6850.20085719

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