猪流行性腹泻病毒流行株S蛋白高变区B细胞线性表位肽的鉴定

【背景】对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)经典毒株与流行毒株S蛋白氨基酸序列比对显示,变异位点主要集中在S蛋白的N端。【目的】鉴定PEDV流行毒株S蛋白高变区(S10A,1-496 aa)的B细胞线性表位肽,特别是流行毒株特异性B细胞表位肽。【方法】以SDS-PAGE割胶纯化大肠杆菌表达的PEDV流行株SD2014 S蛋白高变区片段(S10ASD2014),免疫新西兰大白兔制备多克隆抗体;在E. coli中谷胱甘肽巯基转移酶(Glutathione S-transferase,GST)融合表达覆盖S10ASD2014序列全长且彼此重叠8个氨基酸残基的系列16肽。以制备的抗S10ASD2014多抗为一抗,通过Western blot筛选系列16肽中的阳性反应性16肽,鉴定S10ASD2014上的B细胞线性表位肽;利用抗PEDV经典毒株(CV777)S蛋白高变区片段(S10ACV777)多抗血清鉴定S10ASD2014上的保守性B细胞线性表位肽。【结果】重组表达的S10ASD2014相对分子质量约为72 kD;纯化的S10ASD2014表位肽能被PEDV阳性血清识别。制备的抗S10ASD2014多抗可以识别PEDV DR13弱毒株。利用抗S10ASD2014血清和抗S10ACV777血清从61个GST融合表达的16肽中鉴定到28个阳性反应16肽,其中13个为2种血清共同识别16肽。对24株PEDV不同亚群代表毒株的对应区域进行同源性分析表明2个阳性16肽为保守性表位肽。【结论】确定了PEDV流行株SD2014 S蛋白高变区的B细胞线性表位肽,B细胞线性表位肽,特别是流行毒株特异性表位肽的确定有助于了解S蛋白的结构与功能,为建立以表位为基础的PEDV感染检测方法打下良好基础。

Identification of B cell linear epitopes containing peptides on the hypervariable region of Porcine Epidemic Diarrhea Virus S protein

Background] Amino acid sequences alignment of the spike (S) proteins of porcine epidemic diarrhea virus (PEDV) classical and prevalent strains indicates that most of the variation maps to N-terminal region of the S protein. Objective] The objective of this study is to identify linear B cell epitopes (BCE) containing peptides, especially prevalent strain specific linear BCE containing peptides, on the hypervariable region (S10A, 1?496 aa) of the S protein of PEDV prevalent strain. Methods] The N-terminal hypervariable part of prevalent PEDV SD2014 S protein (S10ASD2014) was recombinantly expressed in Escherichia coli (E. coli). Polyclonal antibody was prepared by immunizing New Zealand white rabbits with S10ASD2014 purified by cutting out the aimed band from the SDS-PAGE. Serial Glutathione S-transferase (GST)-fusion expressed 16-mers with an overlap of 8 amino acids (aa) covering the entire S10ASD2014 sequence was produced in E. coli, and from which linear BCE containing 16-mers were identified by using western blot with prepared anti-S10ASD2014 serum as the primary antibody. The conserved linear BCE containing 16-mers on S10ASD2014 were identified with polyclonal serum against the counterpart of the S protein of PEDV CV777 vaccine strain (S10ACV777). Results] The relative molecular mass of the expressed S10ASD2014 was about 72 kD. The purified S10ASD2014 could be recognized by clinical porcine anti-PEDV serum. The polyclonal serum prepared by immunizing the rabbits with the purified S10ASD2014 could react with PEDV DR13 in Vero cells. From 61 16-mers, twenty-eight linear BCE containing 16-mers were identified by western blot using the prepared anti-S10ASD2014 and anti-S10ACV777 serum. Thirteen out of the 28 linear BCE containing 16-mers could be recognized by both of the 2 serum. Homology analysis of the corresponding region of 24 typical strains from different PEDV subgroups showed that 2 positive 16-mers were conserved. Conclusion] The identification of linear BCE containing peptides, especially the prevalent PEDV S protein-specific 16-mers laid a foundation for the understanding the S protein structure and function, as well as for developing epitope-based diagnostic methods.