棘突蛋白氨基端嵌入标签(记)蛋白的重组猪流行性腹泻病毒的拯救和鉴定

Rescue and identification of a recombinant porcine epidemic diarrhea virus carrying a tagged peptide at the N terminus of S protein

摘要:

【背景】猪流行性腹泻(porcine epidemic diarrhea,PED)是严重影响养猪业健康发展的动物传染病，其致病原为猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)。PEDV结构蛋白之一——棘突蛋白(spike protein,S protein)负责病毒的侵染、入胞等过程，该蛋白结构和功能研究是PEDV分子生物学研究的热点。S蛋白分为两个功能域，N端的S1亚单位和C端的S2亚单位，以往研究表明PEDV DR13^att毒株S1^N(19−233 aa)结构域缺失，不影响DR13^att毒株的存活和增殖。【目的】基于靶向RNA重组技术，在PEDV (DR13^att) S1^N结构域分别引入HA标签基因、抗坏血酸过氧化物酶(APEX2)基因，构建重组PEDV，考察S1^N结构域能否被标签(记)蛋白或多肽替换。【方法】利用靶向RNA重组技术的PEDV反向遗传学操作平台，将转移载体p-PEDV-DR13^att的S1^N结构域(1−234 aa)分别替换成HA基因、APEX2基因，进行重组转移载体的构建，再分别将线性化的载体体外转录的RNA转染至mPEDV感染的LR7细胞，然后在Vero细胞上拯救重组PEDV。通过观察细胞病变效应(cytopathic effect,CPE)、RT-PCR检测、测序、间接免疫荧光分析(immunofluorescence assay,IFA)、Western blotting检测对重组病毒进行验证，最后测定重组病毒滴度并绘制重组病毒生长曲线，探究重组病毒与亲本病毒的生长特性。【结果】构建的重组病毒经过拯救和传代，发现引入HA标签蛋白的重组猪流行性腹泻病毒rPEDV-DR13-S1^N-HA (rPEDV-DSH)在P1代出现细胞病变，引入APEX2蛋白的重组猪流行性腹泻病毒rPEDV-DR13-S1^N-APEX2(rPEDV-DSA)盲传至P4代，未出现细胞病变。对P4代的rPEDV-DSH、rPEDV-DSA进行RT-PCR检测、测序、间接免疫荧光分析、蛋白质印迹法验证，证实引入HA标签的重组病毒rPEDV-DSH拯救成功，而携带APEX2的重组病毒未能拯救成功。重组病毒rPEDV-DSH生长曲线表明rPEDV-DSH与亲本毒株DR13^att有相似的生长趋势，但病毒增殖水平显著低于亲本毒株。【结论】HA标签替换S1^N(1−234 aa)结构域的重组PEDV rPEDV-DSH成功拯救，为进一步研究S蛋白与宿主细胞的互作机制建立了基础。

关键词:猪流行性腹泻病毒;HA标签;靶向RNA重组技术;S1^N结构域

Abstract:

[Background] Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) is a severe porcine infectious disease causing serious losses to the pig industry. The spike (S) protein, one of the structural proteins of PEDV, is responsible for viral infection and entry in host cells. The structure and function of S protein are a research hotspot in the molecular biology of PEDV. The S protein of PEDV has two functional domains: the N-terminal S1 domain and the C-terminal S2 domain. Deletion of the S1^N domain (position: 19−233 aa) did not impair the survival or propagation of the PEDV strain DR13^att in vitro. [Objective] To know whether a tagged protein or peptide can substitute the S1^N domain, we introduced the HA tag and soybean ascorbate peroxidase (APEX2) gene respectively into the S1^Ndomain of DR13^att to establish recombinant viruses by targeted RNA recombination. [Methods] The S1^N domain (position: 1−234 aa) of the transfer vector p-PEDV-DR13^att was respectively substituted with HA gene and APEX2 gene and the transfer vectors were linearized. The RNA of linearized transfer vectors were electrotransferred to the LR7 cells infected with mPEDV, and the recombinant PEDV was rescued on Vero cells. The recombinant virus was validated by observation of cytopathic effect (CPE), RT-PCR, sequencing, indirect immunofluorescence assay (IFA), and Western blotting. Finally, the titer of each recombinant virus was measured and the growth curve of each recombinant virus was plotted to reveal the growth characteristics of the recombinant viruses and their parent virus. [Results] The constructed recombinant viruses were rescued and passaged. The recombinant virus rPEDV-DR13-S1^N-HA (rPEDV-DSH) carrying the HA tag instead of the S1^N domain showed CPE in P1. The recombinant virus rPEDV-DR13-S1^N-APEX2 (rPEDV-DSA) carrying APEX2 had not presented CPE until blind passage to P4. The RT-PCR, sequencing, IFA, and Western blotting of rPEDV-DSH and rPEDV-DSA in P4 confirmed that rPEDV-DSH with the HA tag was successfully rescued, while rPEDV-DSA was not rescued. The growth curves indicated that rPEDV-DSH had a similar growth trend but decreased proliferation level compared with the parent strain DR13^att. [Conclusion] The recombinant virus rPEDV-DSH carrying a HA tag in the S1^N domain (position: 1−234 aa) was successfully rescued, which laid a foundation for further research on the interaction mechanism between the S protein and host cells.

Key words:porcine epidemic diarrhea virus;HA tag;targeted RNA recombination;S1^N domain