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

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。

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

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.