新城疫病毒SDWF2003株F基因的克隆与分子特征分析

Cloning and molecular characteristics analysis of F gene of Newcastle disease virus SDWF2003 isolate

将新城疫病毒(Newcastle disease virus,NDV)SDWF2003株经10日龄SPF鸡胚增殖后,提取病毒基因组RNA,并以此为模板,RT-PCR扩增获得与F基因预期大小一致的DNA片段,亚克隆到pMD18-T载体并酶切鉴定正确后进行测序。软件分析显示:基因全长1662bp,编码553个氨基酸,裂解位点为112R-R-Q-K-R-F117,是典型强毒株氨基酸序列结构;此分离毒株属于基因Ⅶ型,该型F蛋白第101位、第121位特征性氨基酸残基分别是K(赖氨酸)和V(缬氨酸),而本研究分离病毒F蛋白第101位、第121位分别是Q(谷氨酰胺)和V(缬氨酸);同源性比较,与LaSota、F48E9、Ch99核苷酸同源性分别为84.4%、87.1%、98.9%,氨基酸同源性分别为88.3%、91.7%、98.4%。

The F gene of the Newcastle disease virus （NDV） SDWF2003 isolate was amplified by RT-PCR from the virus genomic RNA, cloned into the pMD18-T vector, and sequenced. The full length gene comprised 1662 bp that encodes 553 amino acids with a deduced peptide of ^112R-R-Q-K-R-F^117 at the cleavage site, which is a featured motif of the virulent NDV strains. The F gene shared 84.4 %. 87.1%. 98.9 % nucleotide sequence homology with strain F48E9, La Sota, Ch99 respectively, and 88.3 %, 91.7 %, 98.4 % deduced amino acid identity with respective virus strains. A phylogenetic tree based on the published sequences of F genes was constructed and showed that this isolate belongs to the genotype Ⅶ. There were 2 amino acid alterations at position 101 and 121, where K （lysine） and V （valine） were substituted with Q （glutamine） and V （valine）, respectively.