摘要
【目的】设计针对猪急性腹泻综合征冠状病毒(Swine acute diarrhea syndrome coronavirus,SADS-CoV)S、M及E蛋白的多表位疫苗。【方法】本研究选用IEDB预测SADS-CoV S、M及E蛋白T淋巴细胞主要组织相容性复合体Ⅰ(MHCⅠ)类分子结合表位;用NetMHCIIpan 4.0 Serve预测T淋巴细胞MHCⅡ类分子结合表位;用Immunomedicine Group、IEDB预测B淋巴细胞表位。将各个服务器预测结果筛选出重叠表位区域作为优势表位,运用IEDB、AllerTOP v 2.0 Servers筛选出高保守性、非致敏性的优势表位区域,通过柔性linker串联成多表位疫苗。对构建的多表位疫苗进行抗原性、理化性质、N-糖基化位点、二级结构和三级结构预测。使用分子对接评估多表位疫苗与免疫受体的结合能力,最后进行基因克隆。【结果】经筛选后的高保守性、非致敏性优势表位构建的多表位疫苗相对分子质量为35.30 ku,为稳定亲水蛋白,具有良好的抗原性,存在1个N-糖基化位点,在二级结构中α-螺旋、β-折叠、无规则卷曲和β-转角分别占22.11%、20.35%、50.88%和6.67%。三级结构Ramachandran作图显示优势区域中含有残基数占到90.00%,进行细化后优势区域的残基数增加到91.92%,三级结构突出表位作图也证明多表位疫苗具有良好的免疫原性,分子对接表明多表位疫苗与TLR3具有高亲和力。经密码子优化、反向翻译和基因克隆确保设计的多表位疫苗在大肠杆菌K12表达系统中高效、稳定的表达。【结论】构建的多表位疫苗抗原可有效表达并可能诱导强烈的T细胞和B细胞免疫应答。本研究为SADS-CoV多表位疫苗的设计提供了一种新的方法,为SADS-CoV多表位疫苗的研发提供了理论依据及数据支持。
【Objective】The study was aimed to design a multi-epitope vaccine against Swine acute diarrhea syndrome coronavirus(SADS-CoV)S,M and E proteins.【Method】IEDB server was used to predict the MHC classⅠmolecular binding epitopes of SADS-CoV S protein,M protein and E proteins of T lymphocytes.T lymphocyte MHC classⅡmolecule binding epitopes were predicted by NetMHCIIpan 4.0 Serve.Immunomedicine Group and IEDB server were used to predicted B lymphocyte epitopes.The overlapping epitope regions were selected as the dominant epitopes from the prediction results of each server,and the highly conservative and non-sensitizing dominant epitope regions were selected by using IEDB and AllerTOP v 2.0 servers,which were connected in series into a multi epitope vaccine through a flexible linker.The antigenicity,physicochemical properties,N-glycosylation sites,secondary and tertiary structures of the constructed multi-epitope vaccine were evaluated.The binding capacity of multi-epitope vaccines to immune receptors was evaluated by molecular docking and silico cloning was carried out.【Result】The results showed that the multi-epitope vaccine constructed with highly conservative and non-sensitizing dominant epitopes had a relative molecular weight of 35.30 ku.It was a stable hydrophilic protein with good antigenicity.There was one N-glycosylation site,and the alpha helix,beta sheet,random coil and beta turn were 22.11%,20.35%,50.88%and 6.67%in the secondary structure,respectively.Ramachandran mapping of tertiary structure found that the residue base in the dominant region accounted for 90.00%.After refinement,the residue base in the dominant region increased to 91.92%.Mapping of prominent epitopes of tertiary structure also proved that the multi-epitope vaccine had good immunogenicity,and molecular docking showed that the multi epitope vaccine had high affinity with TLR3.Finally,codon optimization,reverse translation and silico cloning ensure the efficient and stable expression of the designed multi epitope vaccine in E.coli K12 expression system.【Conclusion】The constructed multi-epitope vaccine might effectively express and induce strong T-cell and B-cell immune responses.This study provided a new method for the design of SADS-CoV multi-epitope vaccine,and provided theoretical basis and data support for the research and development of SADS-CoV multi-epitope vaccine.
作者
李世念
刘婉宁
陈亚萍
王金涛
LI Shinian;LIU Wanning;CHEN Yaping;WANG Jintao(College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319,China)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第3期1057-1066,共10页
China Animal Husbandry & Veterinary Medicine
基金
黑龙江八一农垦大学引进人才科研启动计划(XYB201913)。
