Soluble Expression of Foot-and-mouth Disease Multi-epitope Recombinant Protein in Escherichia coli and Evaluation of Its Immunization Effect
JIANG Cheng-Hui1,2,3, RU Yi2, HAO Rong-Zeng2, LI Ya-Jun2,3, ZHAO Long-He2, YANG Yang2, LU Bing-Zhou2, ZHENG Hai-Xue2, ZENG Qiao-Ying1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences/College of Animal Medicine and Biosafety, Lanzhou University/National Key Laboratory of Animal Disease Prevention and Control, Lanzhou 730046, China; 3 China Agricultural Vet. Bio. Science and Technology Co., Ltd., Lanzhou 730046, China
Abstract：Foot-and-mouth disease (FMD), caused by the Foot-and-mouth disease virus (FMDV), is a highly contagious disease among cloven-hoofed animals, posing a serious threat to the development of the livestock industry. In order to develop a novel epitope vaccine for FMD, this study, based on the gene sequence of the prevalent swine (Sus scrofa) O-type FMDV strain (O/MYA98/BY/2010), constructed a recombinant expression plasmid pColdⅡ-7B2T containing 7 B-cell epitopes and 2 T-cell epitopes. The recombinant plasmid was co-expressed with the molecular chaperone TF16 in Escherichia coli, followed by a series of analytical procedures including affinity purification, SDS-PAGE, and Western blot analysis. The recombinant protein was then used to immunise BALB/c mice (Mus musculus), after which its humoral and cellular immune responses were evaluated. The results demonstrated that the 7B2T recombinant protein, prepared using the molecular chaperone TF16, was expressed in a soluble form with a molecular weight of approximately 26 kD and could induce a specific immune response with FMDV-positive serum. Furthermore, the 7B2T protein was found to induce significant specific IgG production in the host, with an average neutralising antibody titer of 1∶96. In addition, the 7B2T protein had been observed to induce robust cellular immune responses in murine models, promoting the differentiation of CD4+ and CD8+ T lymphocyte subsets within the spleen. Lymphocyte proliferation assays and enzyme-linked immunospot assays (ELISPOTS) revealed that the splenic lymphocyte proliferation levels and interferon-γ (IFN-γ) spot counts in the 7B2T protein-treated group were extremely significantly higher than those in the inactivated vaccine (IV) group and PBS group (P<0.01), while the IL-4 spot counts were comparable to those of the IV group. In summary, the present study demonstrated that the soluble multi-epitope 7B2T recombinant protein, prepared using the molecular chaperone TF16, was capable of inducing both humoral and cellular immune responses in mice. This study provides experimental ideas and data references for the preparation of FMD multi-epitope recombinant proteins and the development of multi-epitope vaccines.
[1] 崔颖磊, 刘运超, 冯华, 等. 2018. 分子伴侣对A型FMDV结构蛋白VP1在大肠杆菌中可溶性表达的促进作用[J]. 河南农业大学学报, 52(3): 350-355. (Cui Y L, Liu Y C, Feng H, et al.2018. Facilitaion of molecular chaperoneson soluble protein expression of recombinant A-FMDV VP1 in E.coli[J]. Journal of Henan Agricultural University, 52(3): 350-355.) [2] 付丽新, 唐冬梅, 周彪, 等. 2018. 基于O型口蹄疫病毒VP21-33肽段增强VP1141-160肽段免疫原性的新型纳米颗粒疫苗研究[J].中国人兽共患病学报, 34(8): 8. (Fu L X, Tang D M, Zhou B, et al. 2018. Research on a novel nanoparticle vaccine enhancing the immunogenicity of VP1141-160 peptide based on VP21-33 peptide of Foot-and-mouth disease type O virus[J]. Chinese Journal of Zoonoses, 34(8): 8.) [3] 刘家勐, 宋丹, 罗朝唯, 等. 2021. 猪O 型口蹄疫病毒抗原表位/VP1蛋白的融合表达及免疫原性研究[J]. 华南农业大学学报, 42(3): 5-9. (Liu J M, Song D, Luo C W, et al.2021. Fusion expression and immunogenicity of porcine Foot-and-mouth disease type O virus epitope /VP1 protein[J]. Journal of South China Agricultural University, 42(3): 5-9.) [4] 马超锋, 刘宏斌, 郑汝平, 等. 2009. 大肠杆菌疫苗研究进展[J]. 河南畜牧兽医, 30(23): 13-14. (Ma Z F, Liu H B, Zheng R P, et al.2009. Progress in Escherichia coli vaccine research[J]. Henan Animal Husbandry and Veterinary Science, 30(023): 13-14.) [5] 邵军军, 常惠芸. 2008. 口蹄疫多表位疫苗的研究进展[J]. 中国兽医科学, 38(11): 1009-1012. (Shao J J, Chang H Y.2008. Advances in multiple-epitopes vaccines against foot-and-mouth disease[J]. Chinese Veterinary Science, 38(11): 1009-1012.) [6] 王国强. 2018. O型口蹄疫病病毒多表位免疫原和表位嵌合病毒样颗粒研制及其免疫原性评价[D]. 博士学位论文, 河南农业大学, 导师: 王艳玲, pp. 25-53. (Wang G Q.2018. Preparation and immunogenicity evaluation of multi-epitope-immunogen and epitope-chimeric virus-like particles of Foot-and-mouth disease type O virus in Escherichia coli[D]. Thesis for Ph.D., Henan Agricultural University, Supervisor: Wang Y L, pp. 25-53.) [7] 杨锐, 茹毅, 郝荣增, 等. 2023. 分子伴侣TF促进塞内卡病毒VP2蛋白在大肠杆菌表达[J]. 农业生物技术学报, 31(9): 1944~1955. (Yang R, Ru Y, Hao R Z, et al.2023. Molecular chaperone TF promotes the expression of Seneca virus VP2 protein in Escherichia coli[J]. Journal of Agricultural Biotechnology, 31(9): 1944-1955.) [8] 张宇萌, 童梅, 陆小冬, 等. 2016. 提高大肠杆菌可溶性重组蛋白表达产率的研究进展[J].中国生物工程杂志, 36(5): 7. (Zhang Y M, Tong M, Lu X D, et al. et al. 2016. Research progress on improving the expression and production of soluble recombinant protein in Escherichia coli[J]. Chinese Journal of Bioengineering, 36(5): 7.) [9] Barnett P V, Ouldridge E J, Rowlands D J, et al.1989. Neutralizing epitopes of type o Foot-and-mouth disease virus. I. identification and characterization of three functionally independent, conformational sites[J]. Journal of General Virology, 70(6): 1483-1491. [10] Blanco E, Garcia-Briones M, Sanz-Parra A, et al.2001. Identification of T-cell epitopes in nonstructural proteins of Foot-and-mouth disease virus[J]. Journal of Virology, 75(7): 3164-3174. [11] Cao Y, Lu Z, Li D, et al.2014. Evaluation of cross-protection against three topotypes of sero type O Foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I:C)[J]. Veterinary Microbiology, 168(2-4): 294-301. [12] Chen Y, Zhu J, Wang S, et al.2024. Modular nano-antigen display platform for pigs induces potent immune responses[J]. ACS Nano, 18(42): 29152-29177. [13] Crowther J R, Farias S, Carpenter W C, et al.1993. Identification of a fifth neutralizable site on type O Foot-and-mouth disease virus following characterization of single and quintuple monoclonal antibody escape mutants[J]. Journal of General Virology, 74(8): 1547-1553. [14] Gerner W, Denyer M S, Takamatsu H H, et al.2006. Identification of novel Foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine[J]. Virus Research, 121(2): 223-228. [15] Dorner B G, Dorner M B, Zhou X, et al.2009. Selective expression of the chemokine receptor XCR1 on cross-presenting dendritic cells determines cooperation with CD8+T cells[J]. Immunity, 31(5): 823-833. [16] Fang M, Li J, Wang H, et al.2012. Correlation between efficacy and structure of recombinant epitope vaccines against bovine type O Foot-and-mouth disease virus[J]. Biotechnology Letters, 34(5): 839-847. [17] Ferbitz L, Maier T, Patzelt H, et al.2004. Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins[J]. Nature, 431: 590-596. [18] Jara M, Frias-De-Diego A, Dellicour S, et al.2019. Global phylogeographical patterns in the spread of Foot-and-mouth disease virus[J]. Cold Spring Harbor Laboratory, 168(3): 294-301. [19] Jian Z Y, Liu M Q, Zhu C Z, et al.2004. Recombinant bivalent vaccine against Foot-and-mouth disease virus serotype O/A infection in guinea pig[J]. Acta Biochimica Et Biophysica Sinica, 36(09): 589-596. [20] Jo H E, Ko M K, Choi J H, et al.2019. New foot-and-mouth disease vaccine, O JC-R, induce complete protection to pigs against Sea topotype viruses occurred in south Korea[J]. Journal of Veterinary Science, 20(4): 2014-2015. [21] Knight-Jones T J D, Rushton J.2013. The economic impacts of foot and mouth disease-What are they, how big are they and where do they occur?[J]. Preventive Veterinary Medicine, 112(3): 161-173. [22] Lee H, Piao D, Lee J, et al.2017. Artificially designed recombinant protein composed of multiple epitopes of Foot-and-mouth disease virus as a vaccine candidate[J]. Microbial Cell Factories, 16(1): 16-33. [23] Lei Y, Shao J, Zhao F, et al.2019. Artificially designed Hepatitis B virus core particles composed of multiple epitopes of type A and O Foot-and-mouth disease virus as a bivalent vaccine candidate[J]. Journal of Medical Virology, 91(12): 2142. [24] Li H, Liu P, Dong H, et al.2024. Foot-and-mouth disease virus antigenic landscape and reduced immunogenicity elucidated in atomic detail[J]. Nature Communications, 15(1): 8774. [25] Li K, He Y, Wang L, et al.2021. Two cross-protective antigen sites on Foot-and-mouth disease virus serotype O structurally revealed by broadly neutralizing antibodies from cattle[J]. Journal of Virology, 95(21): e0088121. [26] Lu B, Ru Y, Hao R, et al.2024. A ferritin-based nanoparticle displaying a neutralizing epitope for Foot-and-mouth disease virus (FMDV) confers partial protection in guinea pigs[J]. BMC Veterinary Research, 20(1): 301. [27] Mclaughlin K, Seago J, Robinson L, et al.2010. Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro[J]. Veterinary Research, 41(3): 36-48. [28] Poonsuk K, Luis G L, Zimmerman J J.2018. A review of Foot-and-mouth disease virus (FMDV) testing in livestock with an emphasis on the use of alternative diagnostic specimens[J]. Animal Health Research Reviews, 19(2): 100-112. [29] Ru J, Chen Y, Tao S, et al.2024. Exploring hollow mesoporous silica nanoparticles as a nanocarrier in the delivery of Foot-and-mouth disease virus-like particle vaccines[J]. ACS Applied Bio Materials, 7(2): 9. [30] Sanyal A, Gurumurthy C B, Venkataramanan R, et al.2003. Antigenic characterization of Foot-and-mouth disease virus serotype Asia1 field isolates using polyclonal and monoclonal antibodies[J]. Veterinary Microbiology, 93(1): 1-11. [31] Shao J, Liu W, Gao S, et al.2024. A recombinant multi-epitope trivalent vaccine for Foot-and-mouth disease virus serotype O in pigs[J]. Virology, 596(16): 110103. [32] Song J X, Wang M X, Zhou L.2023. A candidate nanoparticle vaccine comprised of multiple epitopes of the African swine fever virus elicits a robust immune response[J]. Journal of Nanobiotechnology, 21(1): 424-442. [33] Yamamoto T, Ayae N, Takuto N, et al.2025. Cross-reactive fc-mediated antibody responses to influenza ha stem region in human sera following seasonal vaccination[J]. Vaccines, 13(14): 345-364. [34] Zamorano P, Wigdorovitz A, Chaher M T, et al.1994. Recognition of B and T cell epitopes by cattle immunized with a synthetic peptide containing the major immunogenic site of VP1 FMDV 01 Campos[J]. Virology, 201(2): 383-397.
