鸡新城疫病毒F基因的克隆及序列分析  

Cloning and Sequence Analysis of F Gene of Newcastle Disease

作　　者：安琦[1,2] 申燕[1,2] 原韬[1,2] 刘颖[1,2] 金丽颖[1,2] 高冬妮[1,2] 葛菁萍[1,2] 平文祥[1,2] 

机构地区：[1]黑龙江大学生命科学学院/微生物省高校重点实验室,哈尔滨150080 [2]农业微生物技术教育工程研究中心,哈尔滨150500

出　　处：《中国农学通报》2014年第26期11-16,共6页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金"表达IBDV主要保护性抗原的重要杆状病毒构建及其作为疫苗的免疫效果研究"(31270143);国家自然科学基金"菌群演替与温水沤麻系统关键酶产生菌代谢组学特征的耦合机制"(31270534);黑龙江省高等学校科技创新团队"农业微生物发酵技术"(2012td009);黑龙江大学高层次人才(创新团队)支持计划"功能微生物选育及其产物应用"(Hdtd2010-17)

摘　　要：研究旨在从分子生物学角度研究新城疫病原F基因,探究NDVF基因的遗传变异情况及其蛋白结构。提取新城疫病毒基因组RNA,根据GenBank中已知的NDVLa Sota株序列设计引物扩增F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pMD-NDV-F进行测序分析。应用生物信息学软件对新城疫病毒F基因进行系统进化树分析、氨基酸序列同源性分析、蛋白结构跨膜区分析及F蛋白三级结构的预测。核苷酸序列测定结果表明:F基因为1792 bp,共编码553个氨基酸;生物信息学分析结果表明:F基因在种属间具有较高的保守性,试验获得的F基因与NDV La Sota、Clone30、V4、B1等弱毒株的一致性较高,具有典型的弱毒株特性,F基因的氨基酸序列与新城疫病毒La Sota株F基因的序列同源性最高可达100%;F蛋白的三级结构预测结果显示:F蛋白部分片段与新城疫病毒F蛋白融合片段的相似性为96%。研究为新城疫病毒的分子病毒学研究奠定了基础。This research aims at studying the F pathogen gene of Newcastle disease from the angle of molecular biology, exploring the genetic variation and protein structure of（Newcastle Disease Virus, NDV）F gene. The RNA genome of NDV La Sota strain was extracted, Primer5.0 analysis software was utilized to design primers according to the sequence of NDV La Sota strain published on the GenBank. F gene was amplified and cloned to the pMD18-T vector. The recombinant plasmid pMD-NDV-F was identified by restriction enzyme analysis, PCR identification and nucleotide sequence analysis. Boinformatics software was used to analyze Fgene on phylogenetic tree analysis, amino acid sequence homology analysis, transmembrane region analysisand the tertiary structure prediction of F protein. The sequencing results revealed that F gene was 1792 bp and encoded 553 amino acids. The analysis of bioinformatics indicated that F gene was conserved among speciesand was homologous with NDV La Sota, Clone30, V4, B1 and other weak strains with typical attenuated strain characteristics. The highest amino acid homology of F gene to NDV La Sota strain F gene can reach 100%. The tertiary structure prediction results manifested that the similarity between partial fragment of F protein and NDV F protein fusion fragment was 96%. The study may lay a foundation for the molecular virology research on NDV.

关 键 词：新城疫病毒 F基因 RT-PCR 序列分析 

分 类 号：Q786[生物学—分子生物学]