猪流行性腹泻病毒S2截短肽的原核表达及其线性B细胞表位区鉴定

Prokaryotic expression of truncated S2 subunit of porcine epidemic diarrhea virus and identification of its linear B cell epitopes containing regions

摘要:

【背景】由猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV)引起的猪流行性腹泻给养猪业造成了巨大的经济损失。PEDV S蛋白可以诱导宿主产生中和抗体。【目的】原核表达PEDV CV777疫苗株S2截短肽(aa：961?1 382)并制备其多克隆抗体；鉴定表达的S2截短肽上的线性B细胞表位区。【方法】将经密码子优化的PEDV S2截短肽编码DNA (s2t)克隆至载体pET-28a中并转化Escherichia coli BL21(DE3)，利用IPTG诱导S2截短肽表达。以经SDS-PAGE切胶纯化的重组S2截短肽免疫新西兰大白兔制备多克隆抗体。在E. coli BL21中GST融合表达覆盖S2截短肽序列全长、彼此重叠8个氨基酸残基的系列16肽。以制备的抗S2截短肽兔血清为一抗，通过western blot (WB)筛选系列16肽中的阳性反应性16肽，鉴定S2截短肽上的线性B细胞表位区。【结果】重组PEDV S2截短肽的相对分子质量约为50 kD；诱导4 h表达量最高，且主要形成包涵体。WB结果显示，纯化的S2截短肽能被猪抗PEDV血清识别；以纯化的S2截短肽免疫新西兰大白兔制备多抗血清，ELISA法检测抗体效价位于1:25 600?1:102 400之间。免疫组化和间接免疫荧光分析均表明，制备的多抗血清可以识别Vero细胞培养的PEDV DR13弱毒株。以制备的多抗血清通过WB从52个GST融合表达的16肽中鉴定到11个阳性反应性16肽。WB分析显示，得到的阳性反应性16肽都可以被猪抗PEDV血清识别。鉴定到的阳性16肽在S2截短肽上形成4个线性B细胞表位区(aa：969?984；1 065?1 096；1 225?1 280；1 361?1 382)。【结论】高效价抗PEDV S2截短肽多克隆抗体的制备和S2截短肽上线性B细胞抗原表位区的确定有助于了解S蛋白的结构与功能，为建立有效的PEDV检测方法奠定了基础。

Abstract:

[Background] Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) has caused significant economic losses in pig industry. PEDV S protein can induce the host to produce neutralizing antibodies. [Objective] To prokaryotically express the truncated S2 (aa: 961?1 382) of PEDV CV777 vaccine strain and prepare polyclonal antibodies with which to identify regions containing linear B cell epitopes on the truncated S2. [Methods] The codon-optimized DNA fragment encoding the truncated S2 of PEDV (s2t) was inserted into pET-28a. The resulting pET-28a-S2t was then transformed into Escherichia coli BL21(DE3) for expression under the induction of IPTG. Polyclonal antibody was prepared by immunizing New Zealand white rabbits with the truncated S2 purified by cutting out the aimed band from the SDS-PAGE. Serial 16-mers, overlapping 8 aa each other and covering the whole sequence of the truncated S2, were GST-fusion expressed in E. coli BL21. Positive 16-mers were identified by western blot (WB) using prepared polyclonal serum as the primary antibody. linear B cell epitopes containing regions were located. [Results] The truncated S2, about 50 kD, mainly existed in form of inclusion body, of which the expression level reached the maximum at 4 h post induction. The purified truncated S2 could be recognized by porcine anti-PEDV serum in WB analysis. The titer values of the polyclonal serum ranged from 1:25 600 to 1:102 400. Immunohistochemical and indirect immunofluorescence analyses indicated that the polyclonal antibodies showed specific reactivity with PEDV DR13 in Vero cells. Eleven positive 16-mers were identified from 52 GST-fused 16-mers by WB using the prepared serum. All the 11 16-mers could be recognized by porcine anti-PEDV serum in WB analysis. The identified positive 16-mers formed 4 linear B cell epitopes containing regions on the truncated S2 (aa: 969?984, 1 065?1 096, 1 225?1 280, and 1 361?1 382). [Conclusion] The preparation of high titer polyclonal antibodies against truncated S2 of PEDV and the identification of linear B cell epitopes containing sequences on it laid a foundation for understanding the structure and function of S protein, as well as developing effective PEDV diagnostic methods.