猪流行性腹泻病毒CH/JL毒株S基因的克隆、序列分析及线性抗原表位区的鉴定  

Cloning and Sequence Analysis of the Spike Protein Gene of Porcine Epidemic Diarrhea Virus CH/JL Strain and Identification of Its Antigenic Region Containing Linear Epitopes

作　　者：孙东波[1] 冯力[1] 陈建飞[1] 时洪艳[1] 佟有恩[1] 刘胜旺[1] 陈洪岩[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪传染病研究室,哈尔滨150001

出　　处：《病毒学报》2007年第3期224-229,共6页Chinese Journal of Virology

摘　　要：以猪流行性腹泻病毒CH/JL毒株的RNA为模板,通过RT-PCR扩增获得的3个相互重叠的cDNA克隆覆盖了S基因,序列比对结果表明：PEDV CH/JL株S基因与CV777、Brl/87、JS、KPEDV和Chinju99毒株S基因核苷酸序列的同源性分别为96.97%、96.87%、96.41%、94.02%和93.93%,氨基酸序列的同源性分别为96.17%、95.88%、96.10%、92.36%和92.05%;分子进化树分析结果显示,PEDV CH/JL株S基因与JS毒株S基因亲缘关系最近,处于同一群。利用DNAstar Protean程序预测了PEDV CH/JL株S蛋白一个抗原表位区（83-276aa）,将其克隆到原核表达载体pGEX-6p-1后转化E.coliBL21（DE3）感受态细胞,在终浓度1.0mmol/L的IPTG诱导下获得了表达,Western blot结果显示,预测的抗原表位区GST融合蛋白能与猪流行性腹泻病毒多克隆抗血清反应,提示该抗原表位区含有线性抗原表位。Three overlapping cDNA clones of the spike gene of porcine epidemic diarrhea virus CH/JL strain was amplified by RT-PCR. Sequence analysis showed that the S gene of CH/JL strain was 96.97%, 96.87%, 96.41%, 94.02% and 93.93% identical at nucleotide sequence level and 96.17%, 95.88%, 96.10%, 92.36% and 92.05% identical at amino acid sequence level to that of strains CV777, Brl/87, JS, KPEDV and Chinju99, respectively. CH/JL strain and JS strain belonged to the same group in the phylogenetic tree based on the nucleotide sequences of the S genes of PEDV strains. An antigenic epitope region of CH/JL strain S protein was predicted by using DNAStar protean software, and was expressed in E. coli BL21（DE3）as a GST fusion protein. Western blotting showed that the fusion protein reacted positively with the polyclonal antibodies against PEDV, suggesting that the predicted antigenic region contained linear antigenic epitopes of PEDV spike protein.

关 键 词：猪流行性腹泻病毒 S基因 序列分析 线性抗原表位区 

分 类 号：S852.65[农业科学—基础兽医学]