论文

麻花鸡副黏病毒ZH-1株F基因真核表达载体的构建

  • 李红丽 ,
  • 詹丽娥 ,
  • 乔忠 ,
  • 王彩先 ,
  • 陆冰洋
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  • 山西省农科院畜牧兽医研究所, 山西太原 030032
李红丽(1972-), 女, 山两翼城人, 助理研究员, 学士, 主要从事动物传染病研究工作.

收稿日期: 2009-01-28

  网络出版日期: 2014-10-14

基金资助

山西省自然科学基金资助项目(2008011072)

Construction of the F Gene Eukaryotic Expression Vector of the Paramyxovirus Strain ZH-1 From Brambling

  • LI Hong li ,
  • ZHAN Li e ,
  • QIAo Zhong ,
  • WANG Cai xian ,
  • LU Bing yang
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  • Institute of Animal Husbandry and Veterinavy,Shanxi Academy of Agricultural Sciences,Taiyuan 030032,China

Received date: 2009-01-28

  Online published: 2014-10-14

摘要

将麻花鸡副黏病毒ZH-1株毒种接种于11日龄SPF鸡胚,收集48~96h死亡的鸡胚尿囊液.参考已发表的鸡源副黏病毒F基因序列,设计并合成了一对特异性引物,用以扩增麻花鸡副黏病毒ZH-1株F基因,预期扩增的F基因片段包含完整的开放阅读框.通过RT-PCR扩增出麻花鸡副黏病毒ZH-1株F基因片段,琼脂糖凝胶电泳回收、纯化,得到F基因片段,经EcoR I和Sal I消化,将F基因克隆进入PCI-neo载体,转化大肠杆菌DHSa,挑选菌落,经限制性核酸内切酶Sal I和EcoR I双酶切及PCR鉴定,结果证明重组真核表达载体PCI-neo-F构建成功,为下一步在哺乳动物细胞vero细胞中表达打下良好的基础.扩增的F基因测序后,与其他禽副黏病毒1型F基因进行系统发育树的分析比较,为阐明麻花鸡副黏病毒ZH-1株的遗传背景奠定了基础.

本文引用格式

李红丽 , 詹丽娥 , 乔忠 , 王彩先 , 陆冰洋 . 麻花鸡副黏病毒ZH-1株F基因真核表达载体的构建[J]. 华北农学报, 2009 , 24(3) : 54 -58 . DOI: 10.7668/hbnxb.2009.03.012

Abstract

The allantoic fluid was collected from the dead chicken embryo within 48-96 hours after the 11-day-old SPF brambling chicken embryos by type ZH-1. To amplify the sequence of the fusion gene( F- gene) and to determine whether the amplified sequence contains the integrated sequence of open reading frame(ORF),one pair of specific primer was designed and synthesized basedon the published sequence of the F gene and then was used to amplify the F-gene in brambling by RT-PCR. The amplified fragment was taken back and purified, and then was cleaved by both EcoR I and Sal I enzymes. Later,the F gene was transformed into DH5αby PCI-neo vector. The detected results of positive isolates by both PCRanalysis and enzyme cleaving response with EcoRI and Sal I,which showed that the expression vector PCI- neo-F has been constructed successfully,and thispaved the way to study the Vero cell of mammals. The information about the amplified sequence and the compared knowledge in nucleotide composition, amino acid sequence and systemically de- velopment tree between the amplified F gene sequence and the other ZH-1 F gene,were useful for the further elucidate the genetic background of paramyxovirus type ZH-1.

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