基于减法蛋白组学和反向疫苗学方法设计禽致病性大肠杆菌O1和O78血清型多表位疫苗

doi:10.16431/j.cnki.1671-7236.2024.12.029

摘要/Abstract

摘要： 【目的】设计针对禽致病性大肠杆菌(avian pathogenic Escherichia coli，APEC)O1和O78血清型的多表位疫苗(multi-epitope vaccine，MEV)，为APEC新型疫苗的研制奠定基础。【方法】本研究结合减法蛋白组学和反向疫苗学进行研究。通过CD-HIT、BLASTP等工具去除APEC O1、O78蛋白序列中的冗余和不相似蛋白。将APEC O1、O78中相似蛋白与鸡参考蛋白质组进行BLASTP比对，去除同源蛋白，保留非同源蛋白。采用DEG、VFDB等数据库筛选具有毒力的必需蛋白。利用在线工具PSORTb和VaxiJen v 2.0筛选候选蛋白。使用在线工具NetCTL 1.2和NetMHCⅡpan 4.0预测T淋巴细胞主要组织相容性复合体Ⅰ和Ⅱ类分子结合表位，用在线工具IEDB预测B淋巴细胞表位。经在线工具VaxiJen v 2.0评估抗原性后，将合格的表位通过柔性linker串联成多表位疫苗。对设计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。通过分子对接和免疫模拟分别评估多表位疫苗与免疫受体的结合能力及免疫效果，优化密码子便于克隆表达。【结果】经筛选后，选择12个CLT表位、12个HTL表位和12个B淋巴细胞表位构建多表位疫苗MEV-O1O78。多表位疫苗MEV-O1O78分子质量为69.81 ku，为稳定亲水蛋白，具有良好的抗原性，存在7个潜在的N-糖基化位点。二级结构中，α-螺旋、延长链和无规则卷曲分别占7.93%、10.81%和81.27%。三级结构拉氏图显示，优势区域中含有的残基数占95.6%。免疫模拟结果显示，多表位疫苗MEV-O1O78能引起良好的体液免疫，并提高部分细胞因子的表达，经密码子优化确保设计的多表位疫苗MEV-O1O78在大肠杆菌K12表达系统中高效、稳定地表达。【结论】本研究成功设计了含36个优势表位的APEC O1和O78多表位疫苗MEV-O1O78，可为研发禽致病性大肠杆菌病的多表位疫苗提供理论依据和数据支持。

关键词: 禽致病性大肠杆菌(APEC); 血清型; 多表位疫苗

Abstract: 【Objective】 This study was aimed to design a multi-epitope vaccine (MEV) against avian pathogenic Escherichia coli (APEC) O1 and O78 serotypes,laying a foundation for the development of new vaccines for APEC.【Method】 This study combined subtraction proteomics and reverse vaccinology.Redundant and non-similar proteins in the protein sequences of APEC O1 and O78 were removed using CD-HIT and BLASTP tools.Similar proteins in APEC O1 and O78 were removed by BLASTP and compared with reference proteomes of chicken,retaining non-homologous proteins.Essential toxigenic proteins were screened using DEG,VFDB,etc.databases.Candidate proteins were selected using PSORTb and VaxiJen v 2.0.T-cell major histocompatibility complex (MHC) class Ⅰ and Ⅱ molecule-binding epitopes were predicted using NetCTL 1.2 and NetMHCⅡpan 4.0,and B-cell epitopes were predicted using IEDB.Multi-epitope vaccine were evaluated for antigenicity by VaxiJen v 2.0,and the qualified epitopes were linked together by flexible linkers to form a multi-epitope vaccine.The antigenicity,physical and chemical properties,N-glycosylation sites,secondary structure and tertiary structure of the constructed multi-epitope vaccine were predicted.The binding ability of the multi-epitope vaccine to immune receptors was evaluated by molecular docking,and the immune effect was evaluated by immune simulation.Finally,the codons were optimized for cloning and expression.【Result】 After screening,12 MHC Ⅰ,12 MHC Ⅱ and 12 B lymphocyte epitope-dominant epitopes were selected to construct the multi-epitope vaccine MEV-O1O78.The molecular mass of the multi-epitope vaccine MEV-O1O78 was 69.81 ku,a stable hydrophilic protein with good antigenicity,containing 7 potential N-glycosylation sites.In the secondary structure,alpha-helix,extended chain and random coil accounted for 7.93%,10.81% and 81.27%,respectively.The Ramachandran plot of the tertiary structure showed that the epitope-rich region contained 95.6% of the residues.The immune simulation results showed that multi-epitope MEV-O1O78 could induce good humoral immunity and enhance the expression of some cytokines.The codon optimization ensured that the designed multi-epitope MEV-O1O78 was efficiently and stably expressed in the Escherichia coli K12 expression system.【Conclusion】 In this study,the APEC O1 and O78 multi-epitope vaccine MEV-O1O78 containing 36 dominant epitopes was successfully designed,which provided theoretical basis and data support for the development of multi-epitope vaccine for avian pathogenic colibacillosis.

Key words: avian pathogenic Escherichia coli (APEC); serotype; multi-epitope vaccine

中图分类号:

S852.4

陈洪, 吴双, 车业贵, 邱树磊, 周子祥, 王奕, 王永娟, 袁橙. 基于减法蛋白组学和反向疫苗学方法设计禽致病性大肠杆菌O1和O78血清型多表位疫苗[J]. 中国畜牧兽医, 2024, 51(12): 5425-5438.

CHEN Hong, WU Shuang, CHE Yegui, QIU Shulei, ZHOU Zixiang, WANG Yi, WANG Yongjuan, YUAN Cheng. Design of Multi-epitope Vaccines for Avian Pathogenic Escherichia coli O1 and O78 Serotypes Based on Subtractive Proteomics and Reverse Vaccinology[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5425-5438.

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