【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。更多还原

【Abstract】 The nucleocapsid (N) gene of the porcine epidemic diarrhea virus (PEDV) strain LJB/03 which was previously isolated in Heilongjiang province was chined, sequenced and compared with published sequences of other PEDV strains.The nucleotide sequenco 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 LJB/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 kinaseⅡphosphorylatian sites,1 Tyrosine kinase phosphorylation site,2 cAMP-and cGMP-dependent protein kinase phosphorylation sites.To express N gene in E.coli,the amplified product of 1 326 bp was inserted into the expression vector pGEX-6p-1 and trans- formed into E.coli,then induced by IPTG at 37℃.The recombinant fusing protein was approximately 75 ku in molecular weight and reacted on antisera a- gainst PEDV by SDS-PAGE and Western-blotting analysis.更多还原