新城疫病毒La Sota株HN基因的克隆与序列分析

旨在从分子生物学角度进一步研究新城疫病毒La Sota株HN基因,探究NDV La Sota HN基因的遗传变异情况。研究以试验室冻存的p NDV-HN为模版,根据Gen Bank中登录的NDV La Sota株序列设计引物扩增HN基因,将其克隆到pMD18-T载体后进行鉴定,对鉴定正确的重组质粒p MD-NDV-HN进行测序分析。应用生物信息学软件对新城疫病毒Lasota株HN基因进行系统进化树分析、氨基酸序列同源性分析、糖基化位点、蛋白结构跨膜区分析及磷酸化位点预测分析。核苷酸序列测定结果表明:HN基因的序列长度为1743bp,该基因的开放阅读框(Open Reading Frame,ORF)总长为1716 bp,编码571个氨基酸。生物信息学表明:试验获得的HN基因具有6个潜在糖基化位点及12个半胱氨酸残基,第5个潜在糖基化位点(508-510 aa)发生缺失及第123位半胱氨酸残基被色氨酸残基取代。La Sota株HN基因编码的蛋白质中有29个丝氨酸、10个酪氨酸和16个苏氨酸可能成为蛋白激酶磷酸化位点。将试验获得的La Sota株HN基因序列和推导的氨基酸序列与新城疫毒株的HN基因相应序列比较后发现,它们的核苷酸序列同源性和氨基酸序列同源性最高均可达到99%,表明HN基因在种属间具有较高的保守性。研究为新城疫病毒的分子病毒学研究奠定了基础。

Cloning and Sequence Analysis of HN Gene of La Sota Strain of Newcastle Disease Virus

This research aimed at further studying the HN gene of La Sota strain of Newcastle disease virus from the perspective of molecular biology and exploring the genetic variation of NDV La Sota HN gene. Taking laboratory frozen pNDV-HN as a template, Primer 5.0 analysis software was utilized to design a pair of primers according to the sequence of NDV La Sota strain published on the GenBank. HN gene was amplified and cloned to the pMD18-T vector. The recombinant plasmid pMD-NDV-HN was identified by restriction enzyme analysis, PCR identification and nucleotide sequence analysis. Boinformatics software was used to analyze La Sota strain of Newcastle disease virus HN gene with respect to phylogenetic tree analysis, amino acid sequence homology analysis, amino acid sequence homology analysis, glycosylation sites, transmembrane protein structure analysis and prediction of phosphorylation sites. The sequencing results revealed that HN gene was 1743 bp and the length of the open reading frame(ORF) was 1716 bp, encoding 571 amino acids. Bioinformatics showed that: HN gene had six potential glycosylation sites, no fifth site(508 - 510 aa), and 12 cysteine residues, the 123 cysteine residues were replaced by tryptophan residues. The protein La Sota strain HN gene encoded 29 serine, 10 tyrosine and 16 threonine, and those may become protein kinase phosphorylation sites. The highest nucleotide sequence homology and amino acid homology of NDV La Sota strain HN gene to NDV La Sota strain HN gene can reach 99%, indicating that HN gene conserved in higher species. The study will lay a foundation for the molecular virology research of NDV.