麻花鸡副黏病毒ZH-1株F基因真核表达载体的构建

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

将麻花鸡副黏病毒ZH^-1株毒种接种于11日龄SPF鸡胚,收集48-96 h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EcoRⅠ和SalⅠ消化,将F基因克隆进入PCI-neo载体,转化大肠杆菌DH5α,挑选菌落,经限制性核酸内切酶SalⅠ和EcoRⅠ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。

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 based on 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 DH5a by PCI-neo vector. The detected results of positive isolates by both PCR analysis and enzyme cleaving response with Eco R Ⅰand Sal Ⅰ, which showed that the expression vector PCI- neo-F has been constructed successfully, and this paved 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 development 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.