大肠杆菌FliC蛋白D0/D1结构域与Pal蛋白融合蛋白的生物信息学分析及原核表达

doi:10.16431/j.cnki.1671-7236.2025.10.034

摘要/Abstract

摘要： 【目的】构建并表达大肠杆菌FliC蛋白D0/D1结构域与Pal蛋白的融合蛋白，为开发新型禽致病性大肠杆菌(avian pathogenic Escherichia coli，APEC)疫苗提供研究基础。【方法】分析GenBank数据库中大肠杆菌O1血清型菌株的FliC及Pal蛋白氨基酸序列特征，选择FliC蛋白的D0/D1结构域(N-端5－140位氨基酸和C-端499－583位氨基酸)及Pal蛋白主要结构(21－173位氨基酸)构建“三明治型”融合蛋白。对融合蛋白进行理化性质、二级结构、三级结构、抗原性、致敏性及线性和不连续表位预测。通过分子对接和免疫模拟分别评估融合蛋白与免疫受体的结合能力及免疫效果。合成融合蛋白碱基序列并使用pET-28a(+)进行载体构建，采用大肠杆菌BL21(DE3)感受态细胞进行融合蛋白表达，利用镍-琼脂糖凝胶6FF纯化融合蛋白并进行复性，通过Western blotting验证融合蛋白的反应原性。【结果】融合蛋白分子质量为42.72 ku，是亲水蛋白；α-螺旋、延伸链、β-转角和无规则卷曲分别占48.79%、14.25%、6.76%和30.19%，其中α-螺旋主要处于FliC蛋白D0/D1结构域；三级结构拉氏图显示，优势区域中含有的残基数占96.9%。该融合蛋白具有良好的抗原性，无致敏性，含有8个线性表位和5个不连续表位。该融合蛋白与Toll样受体2、4、5均能进行分子对接，且免疫模拟结果显示，该蛋白能引起良好的体液免疫和细胞免疫。经PCR鉴定和测序确定嵌合基因FliCN-Pal-FliCC阳性单克隆并转化大肠杆菌BL21(DE3)感受态细胞，成功表达纯化出融合蛋白FliCN-Pal-FliCC。融合蛋白能与大肠杆菌O1血清型阳性小鼠的血清发生反应。【结论】本研究成功构建并表达了以APEC O1血清型的FliC和Pal蛋白为基础的“三明治”型融合蛋白FliCN-Pal-FliCC，为研发APEC疫苗候选抗原提供研究基础。

关键词: 大肠杆菌; FliC蛋白; 结构域; Pal蛋白; 融合蛋白; 生物信息学分析

Abstract: 【Objective】 The purpose of this study was to construct and express the fusion protein of Escherichia coli (E.coli) FliC protein D0/D1 domain and Pal protein,provide a basis for developing a novel avian enteropathogenic E.coli (APEC) vaccine.【Method】 The amino acid sequence characteristics of FliC and Pal proteins of E.coli O1 serotype strains in GenBank database were analyzed.The D0/D1 domain of FliC protein (N-terminal 5-140 amino acids and C-terminal 499-583 amino acids) and the main structure of Pal protein (21-173 amino acids) were selected to construct the "sandwich-type" fusion protein.The physicochemical properties,secondary structure,tertiary structure,antigenicity,sensitization,linear and discontinuous epitopes of the fusion protein were predicted.The binding ability and immune effect of the fusion protein with immune receptors were evaluated through molecular docking and immune simulation.The base sequence of the fusion protein was synthesized and the vector was constructed using pET-28a(+).The fusion protein was expressed using E.coli BL21(DE3) competent cells.The fusion protein was purified using nickel-agarose gel 6FF and renatured.The reactivity of the fusion protein was verified by Western blotting.【Result】 The molecular mass of the fusion protein was precisely determined to be 42.72 ku,and it was identified as a hydrophilic protein.The composition of the protein’s secondary structure was precisely quantified,with the alpha helix,extended strand,beta turn and random coil accounting for 48.79%,14.25%,6.76% and 30.19%,respectively.Notably,the alpha helix was mainly located within the D0/D1 domain of the FliC protein.The Ramachandran plot of the tertiary structure revealed that the advantage region encompassed an impressive 96.9% of the residues.The fusion protein had outstanding antigenicity,no sensitization,and contained 8 linear epitopes and 5 discontinuous epitopes.Additionally,the fusion protein demonstrated the ability to molecularly dock with Toll-like receptors 2,4 and 5,and the immune simulation results exhibited promising and significant outcomes.The chimeric gene FliCN-Pal-FliCC positive monoclonal was identified by PCR and sequencing and transformed into E.coli BL21 (DE3) competent cells.The fusion protein FliCN-Pal-FliCC was successfully expressed and purified.The fusion protein could react with the serum of E.coli O1 serotype-positive mice.【Conclusion】 In this study,"sandwich" fusion protein FliCN-Pal-FliCC based on FliC and Pal proteins of APEC O1 serotype was successfully constructed and expressed,which provided a research basis for the research and development of candidate antigens of APEC vaccine.

Key words: Escherichia coli; FliC protein; domain; Pal protein; fusion protein; bioinformatics analysis

中图分类号:

S852.61

陈洪, 车业贵, 陈长春, 丁小丽, 邱树磊, 陆辉, 王永娟, 温洪伟, 周子祥. 大肠杆菌FliC蛋白D0/D1结构域与Pal蛋白融合蛋白的生物信息学分析及原核表达[J]. 中国畜牧兽医, 2025, 52(10): 4894-4903.

CHEN Hong, CHE Yegui, CHEN Changchun, DING Xiaoli, QIU Shulei, LU Hui, WANG Yongjuan, WEN Hongwei, ZHOU Zixiang. Bioinformatics Analysis of Fusion Protein of Escherichia coli FliC Protein D0/D1 Domain and Pal Protein and Prokaryotic Expression[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4894-4903.

参考文献

[1] NAWAZ S,WANG Z,ZHANG Y,et al.Avian pathogenic Escherichia coli (APEC):Current insights and future challenges[J].Poultry Science,2024,103(12):104359.
[2] GUABIRABA R,SCHOULER C.Avian colibacillosis:Still many black holes[J].Fems Microbiology Letters,2015,362(15):fnv118.
[3] KATHAYAT D,LOKESH D,RANJIT S,et al.Avian pathogenic Escherichia coli (APEC):An overview of virulence and pathogenesis factors,zoonotic potential,and control strategies[J].Pathogens,2021,10(4):467.
[4] 马兴树.禽大肠杆菌病疫苗研究进展[J].中国畜牧兽医,2015,42(1):234-244. MA X S.Research progress on vaccines of avian colibacillosis[J].China Animal Husbandry & Veterinary Medicine,2015,42(1):234-244.(in Chinese)
[5] 李世念,刘婉宁,陈亚萍,等.基于免疫信息学方法设计针对猪急性腹泻综合征冠状病毒S、M及E蛋白的多表位疫苗[J].中国畜牧兽医,2022,49(3):1057-1066. LI S N,LIU W N,CHEN Y P,et al.Design of multi epitope vaccine against Swine acute diarrhea syndrome coronavirus S,M and E proteins based on immunoinformatics methods[J]. China Animal Husbandry & Veterinary Medicine,2022,49(3):1057-1066.(in Chinese)
[6] IL K M,LEE C,PARK J,et al.Crystal structure of Bacillus cereus flagellin and structure-guided fusion-protein designs[J].Scientific Reports,2018,8(1):5814.
[7] SONG W S,JEON Y J,NAMGUNG B,et al.A conserved TLR5 binding and activation hot spot on flagellin[J].Scientific Reports,2017,7:40878.
[8] SAMATEY F A,IMADA K,NAGASHIMA S,et al.Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling[J].Nature,2001,410(6826):331-337.
[9] ACHARYA D,SULLIVAN M J,DUELL B L,et al.Rapid bladder interleukin-10 synthesis in response to uropathogenic Escherichia coli is part of a defense strategy triggered by the major bacterial flagellar filament FliC and contingent on TLR5[J].mSphere,2019,4(6):e00545-19.
[10] PUTH S,VERMA V,HONG S H,et al.An all-in-one adjuvanted therapeutic cancer vaccine targeting dendritic cell cytosol induces long-lived tumor suppression through NLRC4 inflammasome activation[J].Biomaterials,2022,286:121542.
[11] BARKHORDARI M,BAGHERI M,IRIAN S,et al.Comparison of flagellin and an oil-emulsion adjuvant in inactivated Newcastle disease vaccine in stimulation of immunogenic parameters[J].Comparative Immunology Microbiology and Infectious Diseases,2021,75:101622.
[12] 王亚贞.FliC蛋白和PEI作为H9N2禽流感灭活疫苗免疫佐剂的研究[D].广州：华南农业大学,2020. WANG Y Z.The effects of FliC protein and PEI as adjuvants for H9N2 inactivated avian influenza vaccine[D].Guangzhou:South China Agricultural University,2020.(in Chinese)
[13] 任桂萍,张腾,吴超,等.GM-CSF和FliC作为新型复合生物佐剂对猪圆环病毒疫苗的免疫增强效果[J].东北农业大学学报,2018,49(10):70-78. REN G P,ZHANG T,WU C et,al.Immune enhancement effect of GM-CSF and FliC as a new compound biological adjuvant on Porcine circovirus vaccine[J].Journal of Northeast Agricultural University,2018,49(10):70-78.(in Chinese)
[14] ALIPRANTIS A O,YANG R B,MARK M R,et al.Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2[J].Science,1999,285(5428):736-739.
[15] LIEN E,SELLATI T J,YOSHIMURA A,et al.Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products[J].Journal of Biological Chemistry,1999,274(47):33419-33425.
[16] HELLMAN J,ROBERTS J J,TEHAN M M,et al.Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in Gram-negative sepsis and causes inflammation and death in mice[J].Journal of Biological Chemistry,2002,277(16):14274-14280.
[17] LIANG M D,BAGCHI A,WARREN H S,et al.Bacterial peptidoglycan-associated lipoprotein:A naturally occurring Toll-like receptor 2 agonist that is shed into serum and has synergy with lipopolysaccharide[J].Journal of Infectious Diseases,2005,191(6):939-948.
[18] WILKINS M R,GASTEIGER E,BAIROCH A,et al.Protein identification and analysis tools in the ExPASy server[J].Methods in Molecular Biology,1999,112:531-552.
[19] GEOURJON C,DELEAGE G.SOPMA:Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Computer Applications in the Biosciences,1995,11(6):681-684.
[20] DU Z,SU H,WANG W,et al.The trRosetta server for fast and accurate protein structure prediction[J].Nature Protocols,2021,16(12):5634-5651.
[21] LASKOWSKI R A,JABLONSKA J,PRAVDA L,et al.PDBsum:Structural summaries of PDB entries[J].Protein Science,2018,27(1):129-134.
[22] DOYTCHINOVA I A,FLOWER D R.VaxiJen:A server for prediction of protective antigens,tumour antigens and subunit vaccines[J].BMC Bioinformatics,2007,8:4.
[23] DIMITROV I,BANGOV I,FLOWER D R,et al.AllerTOP v.2—A server for in silico prediction of allergens[J].Journal of Molecular Modeling,2014,20(6):2278.
[24] PONOMARENKO J,BUI H H,LI W,et al.ElliPro:A new structure-based tool for the prediction of antibody epitopes[J].BMC Bioinformatics,2008,9:514.
[25] SINGH A,COPELAND M M,KUNDROTAS P J,et al.GRAMM Web server for protein docking[J].Methods in Molecular Biology,2024,2714:101-112.
[26] RAPIN N,LUND O,BERNASCHI M,et al.Computational immunology meets bioinformatics:The use of prediction tools for molecular binding in the simulation of the immune system[J].PLoS One,2010,5(4):e9862.
[27] CHEN C H,CHEN C C,WANG W B,et al.Intranasal immunization with Zika virus envelope domain Ⅲ-flagellin fusion protein elicits systemic and mucosal immune responses and protection against subcutaneous and intravaginal virus challenges[J].Pharmaceutics,2022,14(5):1014.
[28] 蒋路遥,祝谢民,吴佳炎,等.禽致病性大肠杆菌lpxL和lpxM缺失减毒活疫苗的安全性和保护效果评价[J].中国家禽,2025,47(1):1-9. JIANG L Y,ZHU X M,WU J Y,et al.Evaluation of efficacy and safety of the live attenuated vaccine based on avianpathogenic Echerichia coli with deficient lpxL and lpxM[J].China Poultry,2025,47(1):1-9.(in Chinese)
[29] 李胜男,张雨薇,古丽米热·对山巴依,等.鞭毛蛋白FliC对牛源金黄色葡萄球菌EsxB的免疫增强作用[J].新疆农业大学学报,2020,43(2):101-107. LI S N,ZHANG Y W,DUISHANBAYI G,et al.Immune enhancement effects of flagellin protein FliC on EsxB protein of bovine Staphylococcus aureus[J].Journal of Xinjiang Agricultural University,2020,43(2):101-107.(in Chinese)
[30] YUKI K,MITSUI Y,SHIBAMURA-FUJIOGI M,et al.Anesthetics isoflurane and sevoflurane attenuate flagellin-mediated inflammation in the lung[J].Biochemical and Biophysical Research Communications,2021,557:254-260.
[31] LI S,WEN M,WEN G,et al.Structure and biological activity in vitro of flagellin and its mutants from Escherichia coli Nissle 1917[J].Archives of Microbiology,2024,206(5):221.
[32] 吴文文,庞胜美,丁雪燕,等.大肠杆菌鞭毛蛋白不同结构域与F4菌毛主要结构亚单位FaeG嵌合基因的构建及其表达蛋白的功能研究[J].中国预防兽医学报,2020,42(7):656-661. WU W W,PANG S M,DING X Y,et al.Construction and functional study of chimeric genes of different domains of E.coli flagel in with FaeG,the main structural subunit of F4 fimbriae[J].Chinese Journal of Preventive Veterinary Medicine,2020,42(7):656-661.(in Chinese)
[33] KHIM K,BANG Y J,PUTH S,et al.Deimmunization of flagellin for repeated administration as a vaccine adjuvant[J].NPJ Vaccines,2021,6(1):116.
[34] GODLEWSKA R,WISNIEWSKA K,PIETRAS Z,et al.Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria:Function,structure,role in pathogenesis and potential application in immunoprophylaxis[J].FEMS Microbiology Letters,2009,298(1):1-11.
[35] MCMAHON M,MURPHY T F,KYD J,et al.Role of an immunodominant T cell epitope of the P6 protein of nontypeable Haemophilus influenzae in murine protective immunity[J].Vaccine,2005,23(27):3590-3596.
[36] CORDWELL S J,LEN A C,TOUMA R G,et al.Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies[J].Proteomics,2008,8(1):122-139.
[37] KIM S J,SIN J I,KIM M J.CD8⁺ T cells directed against a peptide epitope derived from peptidoglycan-associated lipoprotein of Legionella pneumophila confer disease protection[J].Frontiers in Immunology,2020,11:604413.
[38] LYNNE A M,KARIYAWASAM S,WANNEMUEHLER Y,et al.Recombinant Iss as a potential vaccine for avian colibacillosis[J].Avian Diseases,2012,56(1):192-199.
[39] GYIMAH J E,PANIGRAHY B,WILLIAMS J D.Immunogenicity of an Escherichia coli multivalent pilus vaccine in chickens[J].Avian Diseases,1986,30(4):687-689.
[40] HU R,LI J,ZHAO Y,et al.Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC)[J].Microbial Cell Factories,2020,19(1):119.
[41] GERRITZEN M,SALVERDA M,MARTENS D E,et al.Spontaneously released Neisseria meningitidis outer membrane vesicles as vaccine platform:Production and purification[J].Vaccine,2019,37(47):6978-6986.