The Expression of Senecavirus VP2 Protein Promoted by Molecular Chaperones TF in Escherichia coli
YANG Rui1,2,3, RU Yi2, HAO Rong-Zeng2, LI Ya-Jun2, YANG Yang2, REN Rui-Fang2, LU Bing-Zhou2, MAO Yu-Han2, ZHANG Yue2, ZHENG Hai-Xue2, ZHANG Yong1, ZHAO Xing-Xu1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences/College of Veterinary Medicine, Lanzhou University/State Key Laboratory for Animal Disease Control and Prevention, Lanzhou 730046, China; 3 China Agricultural Veterinarian Biology Science and Technology Co., Ltd., Lanzhou 730046, China
Abstract：Senecavirus A (SVA), an emerging RNA virus in recent years, which seriously endangers the development of global pig industry. Its viral structural protein 2 (VP2) plays an important role in inducing the body's immune response. To prepare a soluble structural protein VP2 of SVA and systematically analyze its immunogenicity, firstly, the SVA-VP2 protein recombinant expression plasmid pET28a-SVA-VP2 was constructed based on the gene sequence of the SVA/CH-FJ-2017 strain, and then transferred the pET28a-SVA-VP2 recombinant plasmid and the molecular chaperones pTf16 plasmid into Escherichia coli BL21 (DE3) competent cells. The soluble recombinant SVA-VP2 protein was induced to express by using the characteristics of molecular chaperones trigger factor (TF) to promote the soluble expression of protein in the prokaryotic expression system. BALB/c mice (Mus musculus) was immunized by the purified target protein which was identified by SDS-PAGE and Western blot. Serum antibody levels was detected by indirect ELISA, and neutralizing antibody titers was measured by virus neutralization experiments, and the CD4+ and CD8+ T lymphocyte subpopulations in the spleen and the proliferation and cytokine production of spleen lymphocytes were detected by flow cytometry. The results showed that the soluble recombinant VP2 protein was successfully prepared by using molecular chaperones TF16. The recombinant VP2 protein could react specifically with SVA positive serum. The serum antibody titer level of immunized mice could reach 1:32 000, and the neutralizing antibody level could reach 1:128. The percentage of CD4+ and CD8+ T lymphocytes in the spleen of immunized mice was very extremely significantly higher than that of the control group (P<0.001), and the expression levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-10 were very extremely significantly higher than those in the control group (P<0.001). In conclusion, this study successfully prepared soluble recombinant VP2 protein by co-expression of molecular chaperones TF, and the recombinant VP2 protein had good immunogenicity both in vivo and in vitro, which can trigger strong humoral immune response and cellular immune response of the body. This study provided experimental materials and theoretical basis for the development of SVA subunit vaccine and diagnostic agent.
[1] 崔川, 刘佳欢, 韩伟建, 等. 2020. A型塞内卡病毒 VP2 蛋白原核表达及免疫原性分析[J]. 中国动物传染病学报, 30(06): 91-98. (Cui C, Liu J H, Han W J, et al.2020. Prokaryotic expression and immunogenicity of Senecavirus A VP2 protein[J]. Chinese Journal of Animal Infectious Diseases, 30(06): 91-98.) [2] 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 chaperones on soluble protein expression of recombinant A-FMDV VP1 in E. coli[J]. Journal of Henan Agricultural University, 52(3): 350-355.) [3] 郭丽莹, 冯霞, 潘颂佳, 等. 2022. 塞内卡病毒A结构蛋白T细胞抗原表位的筛选与鉴定[J]. 中国兽医科学, 52(01): 77-84. (Guo L Y, Feng X, Pan S J, et al.2022. The screening and identification of T cell epitopes in the structural protein of Senecavirus A[J]. Chinese Veterinary Science, 52(01): 77-84.) [4] 李磊. 2014. 分子伴侣Trigger Factor新功能位点的鉴定[D]. 硕士学位论文, 吉林大学, 导师: 杨立泉. pp. 2. (Li L.2014. Identification of novel chaperone sites in chaperone trigger factor[D]. Thesis for M.S., Jilin University, Supervisor: Yang L Q, pp. 2.) [5] 孟媛, 曾嘉庆, 于成东, 等. 2021. 塞尼卡病毒VP1蛋白亚单位候选疫苗小鼠免疫评价[J]. 中国兽医学报, 41(11): 2085-2090. (Meng Y, Zeng J Q, Yu C D, et al.2017. Immune evaluation of candidate Seneca Valey virus VP1 protein subunit vaccine in mice[J]. Chinese Veterinary Science, 47(9): 1118-1123.) [6] 莫亚霞, 宋品, 穆素雨, 等. 2019. A型塞内卡病毒衣壳蛋白的原核表达及其五聚体的组装[J]. 中国兽医科学, 49(08): 969-976. (Mo Y X, Song p, Mu S Y, et al.2019. Prokaryotic expression of capsid protein of Seneca virus type A and assembly of its pentamer[J]. Chinese Veterinary Science, 49(08): 969-976.) [7] 冉云伟, 张改平, 刘运超, 等. 2017. 分子伴侣促进截短型人乳头瘤病毒58亚型L1蛋白在大肠杆菌中的可溶性表达[J]. 生物技术通讯, 28(03): 295-300. (RanY W, Zhang G P, Liu Y C, et al.2017.Recombinant expression of truncated human papillomavirus 58 L1 protein with molecular chaperones in E. coli[J]. Letters in Biotechnology, 28(03): 295-300.) [8] 陶倩, 曹飞, 彭珂楠, 等. 2022. 猪塞内卡病毒病原学研究进展[J]. 病毒学报, 38(02): 505-512. (Tao Q, Cao F, Peng K N, et al.2022. Research progress on etiology of porcine Seneca virus[J]. Chinese Journal of Virology, 38(02): 505-512.) [9] 陶世宇, 茹毅, 陈娇, 等. 2023. A型塞内卡病毒VP2蛋白多表位抗原免疫原性分析[J]. 农业生物技术学报, 31(6): 1284-1295. (Tao S Y, Ru Y, Chen J, et al.2023. Immunogenicity analysis of multi-epitopes antigen of Senecavirus A VP2 protein[J]. Journal of Agricultural Biotechnology, 31(6): 1284-1295.) [10] 王迁迁, 于永乐, 李月华, 等. 2022. A型塞尼卡病毒检测方法及疫苗研究进展[J]. 中国畜牧兽医, 49(6): 2336-2346. (Wang Q Q, Yu Y L, Li Y H, et al.2022. Advance in detection methods and vacines of Senecavirus A[J]. China Animal Husbandry & Veterinary Medicine, 49(6): 2336-2346.) [11] 魏婷, 杨帆, 张伟, 等. 2020. A型塞内卡病毒P12A-3C基因真核表达质粒的构建及其免疫效力的研究[J]. 中国兽医科学, 50(07): 861-868. (Wei T, Yang F, Zhang W, et al.2020. Construction of eukaryotic expression plasmid for P12A-3C gene of Senecavirus A and immunity experiments in mouse[J]. Chinese Veterinary Science, 50(07): 861-868.) [12] 伍春平, 茹毅, 田宏, 等. 2021. A型塞内卡病毒的病毒样颗粒的制备及其免疫原性分析[J]. 生物工程学报, 37(9): 3211-3220. (Wu C P, Ru Y, Tian H, et al.2021. Generation and immunogenicity evaluation of Senecavirus A virus-like particles[J]. Chinese Journal of Biotechnology, 37(9): 3211-3220.) [13] 伍春平. 2021. A型塞内卡病毒结构蛋白B细胞抗原表位的鉴定[D]. 硕士学位论文, 中国农业科学院, 导师: 郑海学, pp. 25-41. (Wu C P.2021. Identification of B cell epitopes on the structural protein of Senecavirus A[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences, Supervisor: Zheng H X, pp. 25-41.) [14] 姚飞. 2020. 塞内卡病毒A VP1、VP2蛋白B细胞表位的筛选与鉴定[D]. 硕士学位论文, 中国农业科学院,导师: 潘丽. pp. 27-37. (Yao F.2020. Screening and identification of B cell epitopes of VP1 and VP2 proteins of Senecavirus A[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences, Supervisor: Pan L, pp. 27-37.) [15] 张涛, 王会宝, 孙燕燕, 等. 2022. A型塞内卡病毒VP1蛋白单克隆抗体制备及胶体金试纸条的试制[J]. 农业生物技术学报, 30(11): 2255-2266. (Zhang T, Wang H B, Sun Y Y, et al.2022. Preparation of monoclonal antibody based on VP1 protein against Senecavirus A (SVA) and trial rroduction of colloidal gold test strips for SVA detection[J]. Journal of Agricultural Biotechnology, 30(11): 2255-2266.) [16] 张永宁, 张舟, 诸明欣, 等. 2019. A 型塞内卡病毒 VP2蛋白原核表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 46(1): 256-263. (Zhang Y N,Zhang Z,Zhu M X, et al.2019. Prokaryotic expresion of the VP2 protein of Senecavirus A and preparation of its polyclonal antibodies[J]. China Animal Husbandry & Veterinary Medicine, 46(1): 256-263.) [17] Balchin D, Hayer-Hartl M, Hartl F U.2020. Recent advances in understanding catalysis of protein folding by molecular chaperones[J]. FEBS Letters, 594(17): 2770-2781. [18] Canning P, Canon A, Bates J L, et al.2016. Neonatal mortality, vesicular lesions and lameness associated with Senecavirus A in a U.S. sow farm[J]. Transboundary and Emerging Diseases, 63(4): 373-378. [19] Dvorak C M, Akkutay-Yoldar Z, Stone S R, et al.2017. An indirect enzymelinked immunosorbent assay for the identification of antibodies to Senecavirus A in swine[J]. BMC Veterinary Research, 13(1): 50. [20] 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. [21] Gimenez-Lirola L G, Rademacher C, Linhares D, et al.2016. Serological and molecular detection of Senecavirus A associated with an outbreak of swine idiopathic vesicular disease and neonatal mortality[J]. Journal of Clinical Microbiology, 54(8): 2082-2089. [22] Hales L M, Knowles N J, Reddy P S, et al.2008. Complete genome sequence analysis of Seneca valley virus-001, a novel oncolytic picornavirus[J]. Journal of General Virology, 89: 1265-1275. [23] Leme R A, Zotti E, Alcantara B K, et al.2015. Senecavirus A: An emerging vesicular infection in Brazilian pig herds[J]. Transboundary and Emerging Diseases, 62(6): 603-611. [24] Li C, Wu X Y, Wang X L, et al.2022. Complete genome and pathogenesis of a novel recombinant Senecavirus A isolate in P.R. China[J]. Journal of General Virology,103(11): 1788. [25] Li N, Qiao Q L, Guo H F, et al.2021a. Evaluation of immunogenicity and protective eficacy of a novel Senecavirus A strain-based inactivated vacine in mice[J]. Research in Veterinary Science, 142: 133-140. [26] Li Y W, Zhang Y Y, Liao Y X, et al.2021b. Preliminary evaluation of protective eficacy of inactivated Senecavirus A on pigs[J]. Life (Basel), 11(2): 157. [27] Liu F,Wang Q,Huang Y,et al.2020. A 5-year review of Senecavirus A in China since its emergence in 2015[J]. Frontiers in Veterinary Science, 7: 56792. [28] Malekian R, Sima S, Jahanian-Najafabadi A, et al.2019. Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones[J]. Protein Expression and Purification ,160: 66-72. [29] Maggioli M F, Lawson S, Lima M D, et al.2018. Adaptive immune responses following Senecavirus A infection in pigs[J]. Journal of Virology, 92(3): e01717-17. [30] Mu S Y, Sun S Q, Dong H, et al.2020. Potent protective immune responses to Senecavirus induced by virus-like particle vaccine in pigs[J]. Vaccines (Basel), 8(3): 532. [31] Nitika P, Shashank.2022. Role of molecular and chemical chaperon in assisting refolding of BMP2 in E. coli[J]. International Journal of Biological Macromolecules, 220: 204-210. [32] Palmer I, Wingfield P T.2012. Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli[J]. Current Protocols in Protein Science, 6: 631-632. [33] Pasma T, Davidson S, Shaw S L.2008. Idiopathic vesicular disease in swine in Manitoba[J]. Canadian Veterinary Journal-revue Veterinaire Canadienne, 49(1): 84-85. [34] Piao D C, Shin D W, Kim I S, et al.2016. Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli[J]. BMC Biotechnology, 16(1): 39. [35] Segales J, Barcellos D, Alfieri A, et al.2017. Senecavirus A: An emerging pathogen causing vesicular disease and mortality in pigs?[J]. Veterinary Pathology, 54: 11-21. [36] Sharma B, Fernandes M H V, Lima M D, et al.2019. A novel live attenuated vaccine candidate protects against heterologous Seneca virus A challenge[J]. Frontiers in Immunology, 10: 2660. [37] Singh K, Corner S, Clark S G, et al.2012. Seneca Valley virus and vesicular lesions in a pig with idiopathic vesicular disease[J]. Journal of Veterinary Science & Technology, 3(6): 1-3. [38] Venkataraman S, Reddy P S, Loo J, et al.2008. Structure of Seneca valley virus-001: An oncolytic picornavirus representing a new genus[J]. Structures, 16(10): 1555-1561. [39] Wen W, Chen X H, Lv Q, et al.2022. Identification of a conserved neutralizing epitope in Seneca Valley virus VP2 protein: New insight for epitope vaccine designment[J]. Virology Journal, 19(1): 65. [40] William S, Song T.2008. Recombinant protein complex expression in E. coli[J]. Current Protocols in Protein Science, 5: Unit 5.21. [41] Wu Q, Zhao X, Bai Y, et al.2017. The first identification and complete genome of Senecavirus A affecting pig with idiopathic vesicular disease in China[J]. Transboundary and Emerging Diseases, 64(5): 1633-1640. [42] Yang F, Zhu Z X, Cao W J, et al.2018. Immunogenicity and protective efficacy of an inactivated cell culture-derived Seneca Valley virus vaccine in pigs[J]. Vaccine, 36(6): 841-846. [43] Zhu Z, Yang F, Chen P, et al.2017. Emergence of novel Seneca Valley virus strains in China[J]. Transboundary and Emerging Diseases, 64(4): 1024-1029.
