鹅源副粘病毒NA-1株F蛋白基因的克隆及其载体的构建  

Cloning of Fusion Glycoprotein Gene of Avian Paramyxovirus NA-1 Strain and Contructed of Re-bacmid

作　　者：左玉柱[1] 乔木[2] 王学理[1] 向华[1] 黄海楠[1] 常爽[1] 丁壮[1] 

机构地区：[1]解放军军需大学军事兽医系,吉林长春130062 [2]沈阳市东陵区动物防疫站,辽宁沈阳110015

出　　处：《中国兽医学报》2004年第5期436-438,共3页Chinese Journal of Veterinary Science

基　　金：吉林省科委发展计划资助项目 (2 0 0 0 0 2 0 0 )

摘　　要：鹅源副粘病毒分离株 NA- 1经 1 1日龄鸡胚增殖后收集尿囊液 ,浓缩 ,提取病毒基因组总 RNA,采用 RT- PCR方法 ,一次性扩增出与预期大小相符的特异性条带。将扩增产物提纯回收后克隆入 p MD1 8- T载体 ,经转化、筛选及酶切鉴定后 ,对阳性克隆进行了序列测定和序列分析。测序后拼接的 F基因长 1 6 72 bp,包含完整的开放阅读框 ( 1 6 6 2bp) ,编码 5 5 3个氨基酸 ,F蛋白裂解位点的氨基酸顺序为 1 1 2 R- R- Q- K- R- F1 1 7,与 NDV的强毒株特征相符 ,同时也与鹅副粘病毒分离株致病性试验结果相符。根据 NDV基因分型标准 ,鹅源副粘病毒 NA- 1株归属基因 型 NDV。将 F基因克隆入转座载体 p Fast Bac ,得到重组转座载体 PFF,将 PFF转化大肠杆菌 DH1 0 Bac,提取质粒 ,得到阳性重组穿梭载体 re- Bacm id。The geese′s paramyxovirus isolate NA-1 was propagated in 11-day-old chicken embryos.The allantonic fluid of the virus were concentrated and the genomic RNA was extracted.The F gene of the virus has been successfully amplified by RT-PCR,and further cloned into pMD18-T vector,and a positive recombitant plasmid was screened by restriction enzyme analysis.The sequence analysis showed that the nucleotide sequence of this F gene was 1 672 bp and encoding a protein of 553 amino acids.The amino acid sequence of cleavage site region was ^(112)R-R-Q-K-R-F^(117),matching to virulent NDV strains,and at the same time it was consistent with the pathogenicity test isolates.The geese′s paramyxovirus isolate NA-1 belongs to genotype Ⅶ.A recombinant transposon vector(pFF)was contructed by inserting the F gene of strain siping into the transposon vector pFastBacⅠ.Then,pFF was transformed into E.coli DH10Bac,and The recombinant bacmid was contructed.The recombinant bacmid can be used to transfect into sf-9 cells to generate recombinant baculovirus expressing F protein of NA-1.

关 键 词：鹅副粘病毒病 F蛋白基因 RT-PCR 克隆 序列分析 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]