猪流行性腹泻病毒LJB/03地方株核蛋白基因序列分析及其原核表达  

Cloning and Expression of the N gene of Porcine Epidemic Diarrhea Virus LJB/03

作　　者：葛俊伟[1] 姜艳萍[1] 李宝贤[1] 裴敦国[1] 李一经[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《黑龙江畜牧兽医》2007年第3期12-14,共3页Heilongjiang Animal Science And veterinary Medicine

基　　金：黑龙江省十五科技攻关项目(GC01B501)

摘　　要：以猪腹泻粪便样品提取RNA,应用RT-PCR方法扩增获得了PEDV地方株LJB/03核衣壳蛋白(N)全基因序列,测序结果表明:N基因全长1326 bp,编码441个氨基酸,包含405个腺嘌呤(30．5%)、329个鸟嘌呤(24．8%)、294个胞嘧啶(22．2%)和298个胸腺嘧啶(22．5%)。与已发表的序列相比,LJB/03株N基因和其他分离株核苷酸的同源性在95%以上,与JS2004、chinju99、Brl/ 87、CV777同源性分别是97．4%、95．6%、96．6%、96．8%;氨基酸同源性在96．5%以上,与JS2004、chinju99、Br1/87、CV777的同源性分别是98．0%、96．90%、96．1%、96．4%。序列分析表明:N蛋白包含7个潜在的蛋白激酶C磷酸化位点、9个酪蛋白激酶Ⅱ磷酸化位点、1个酪氨酸激酶磷酸化位点和2个依赖cAMP和cGMP的蛋白激酶磷酸化位点。将N基因定向克隆到原核表达载体pGEX- 6p-1,转化宿主菌BL21中,经IPTG诱导后,N基因得以表达。表达产物经SDS-PAGE和Western Blot鉴定,确定表达的融合蛋白大小约为75ku,且具有良好的反应原性。The nucleoeapsid （ N ） gene of the porcine epidemic diarrhea virus （ PEDV ） strain LJB/03 which was previously isolated in Heilongjiang province was eloned, sequenced and compared with published sequenees of other PEDV strains. The nucleotide sequence encoding the entire N gene open reading frame （ORF） of LJB/03 was 1 326 base pairs and encoded a protein of 441 amino acids with predicted Mr of 49 ku. It consisted of 405 adenines （30.5%） , 294 cytosines （22. 1% ） , 329 guanines （ 24.8% ） and 298 thymines （22. 5% ） residues. Sequence comparison with other PEDV strains selected from Gen- Bank revealed that the IJB/03 N gene has a high sequence homology to those of other PEDV isolates, the amino acid sequence contained 7 potential protein kinase C phosphorylation sites, 9 Casein kinase Ⅱ phosphorylation sites, 1 Tyrosine kinase phosphorylation site, 2 cAMP - and cGMP- dependent protein kinase phosphorylation sites. To express N gene in E. coil, the amplified product of 1 326 bp was inserted into the expression vector pGEX -6p - 1 and transformed into E. coil, then induced by IPTG at 37 ℃. The recombinant fusing protein was approximately 75 ku in molecular weight and reacted on antisera against PEDV by SDS -PAGE and Western -blotting analysis

关 键 词：猪流行性腹泻病毒 N基因 克隆 表达 

分 类 号：S852.659.6[农业科学—基础兽医学]