鸡Toll样受体15单克隆抗体的制备及其初步应用

doi:10.11843/j.issn.0366-6964.2022.02.024

摘要/Abstract

摘要： 旨在制备鸡Toll样受体15（ChTLR15）的特异性单克隆抗体（mAb），并初步应用。利用PCR技术扩增ChTLR15第162-386位氨基酸，克隆于载体pET-30a中进行诱导表达，获得高纯度的重组ChTLR15（162-386 aa）蛋白。然后采用皮下多点注射方式将ChTLR15免疫6周龄的雌性BALB/c小鼠，常规获得针对ChTLR15蛋白的单克隆抗体。运用原核表达系统对ChTLR15基因进行截短表达，对单克隆抗体针对的ChTLR15抗原表位进行鉴定。利用6C7株单克隆抗体以激光共聚焦显微技术对鸡HD11细胞进行定位，同时，利用免疫组化技术确定ChTLR15在鸡部分组织中的分布情况。结果表明：成功构建重组质粒pET-30a-ChTLR15（162-386 aa），经IPTG诱导表达后，获得以包涵体形式存在的约26 ku的重组蛋白ChTLR15（162-386 aa）。方阵试验表明，最适血清稀释度为1：6 400，最适抗原包被浓度为0.35 μg·mL^-1。采用有限稀释法进行4轮筛选，最终获得4株针对ChTLR15蛋白的单克隆抗体，将其以2G4、6C7、6D10、7C4进行命名。2G4与6C7的亚类为IgG2a，6D10与7C4的亚类为IgG1。Western blot结果显示，4株单抗均能与ChTLR15发生特异性反应，而不与其他受检蛋白反应。运用原核表达系统对ChTLR15（162-386 aa）进行截短表达，对ChTLR15单克隆抗体的抗原表位进行鉴定。结果显示，单抗2G4与7C4识别的抗原表位为²³⁰QLGTVLEF²³⁷，6C7与6D10识别的抗原表位为²⁴⁵EMDLLS²⁵⁰。细胞定位结果显示，ChTLR15蛋白位于细胞表面。免疫组化结果显示，健康对照鸡肝、脾、肺、肾均有微弱阳性染色，禽致病性大肠杆菌（avian pathogenic Escherichia coli，APEC）感染鸡肝、脾、肺、肾阳性染色有所增强。本研究通过淋巴细胞杂交瘤技术成功获得4株抗ChTLR15蛋白的单克隆抗体，通过截短免疫原蛋白法对其识别的线性表位进行鉴定，可用于进一步研究ChTLR15的结构与功能，为后续信号通路、免疫机理、疫苗研发等相关领域的研究提供了有力工具。

关键词: ChTLR15, 单克隆抗体, 抗原表位, 免疫组化, 细胞定位

Abstract: The preparation and preliminary application of monoclonal antibodies (mAbs) specific to chicken Toll-like receptor 15 (ChTLR15) is described. The gene fragment encoded 162-386 amino acids of ChTLR15 were amplified by PCR and cloned into the vector pET-30a. IPTG was used to induce expression of the recombinant plasmid, the collected target protein was first purified by Ni-NTA affinity chromatography medium method, and the purified protein was renatured by dialysis using urea with concentration gradient. The renatured protein was purified again by gel filtration chromatography to obtain high-purity recombinant ChTLR15 (162-386 aa) protein. The 6-week-old female BALB/c mice were immunized with multi-loci subcutaneous injections, and the spleen cells of mice with higher serum antibody levels after immunization were fused with SP2/0 myeloma cells, and the limiting dilution method was used for multiple rounds of screening to obtain mAbs against ChTLR15 protein. The ChTLR15 gene was truncated and expressed by the prokaryotic expression system, and the ChTLR15 epitopes targeted by the mAbs were identified. The mAb 6C7 was used to locate the ChTLR15 in chicken HD11 cells with laser confocal microscopy. At the same time, the distribution of ChTLR15 in some chicken tissues was determined by immunohistochemistry. The recombinant plasmid pET-30a-ChTLR15 (162-386 aa) was successfully constructed, and the recombinant protein ChTLR15 (162-386 aa) at about 26 ku in the form of inclusion body was obtained after induced expression by IPTG. The optimal antigen coating concentration (0.35 μg·mL^-1) and the optimal serum dilution (1:6 400) were determined by indirect enzyme-linked immunosorbent assay (ELISA). A total of 4 mAb hybridoma cell lines that can stably secrete antibody against ChTLR15 were obtained, and they were named 2G4, 6C7, 6D10 and 7C4. The subclass of 2G4 and 6C7 was IgG2a, and the subclass of 6D10 and 7C4 was IgG1. Western blot results showed that the 4 mAbs can react specifically with ChTLR15, but not with other tested proteins. The prokaryotic expression system was used to express the truncated ChTLR15 (162-386 aa), and the epitopes of ChTLR15 mAbs were identified. The results showed that the epitope recognized by mAbs 2G4 and 7C4 was ²³⁰QLGTVLEF²³⁷, and the epitope recognized by 6C7 and 6D10 was ²⁴⁵EMDLLS²⁵⁰. Observation under a laser confocal microscopy suggested that ChTLR15 protein was located on the cell surface. Immunohistochemistry showed that the liver, spleen, lung, and kidney from the health birds had weak positive staining, and the positive staining of liver, spleen, lung, and kidney from the avian pathogenic Escherichia coli(APEC) challenged birds was enhanced. In this study, four mAbs against ChTLR15 protein were successfully developed and the epitopes recognized by them were identified, which is conducive to further research on the structural and functional characteristics of ChTLR15. It provides a robust tool for subsequent research in related fields such as signal pathways, immune mechanisms, and vaccine development.

Key words: ChTLR15, monoclonal antibody, antigenic epitope, immunohistochemistry, subcellular localization

中图分类号:

S852.4

李梦妮, 王航, 傅思瑶, 杨梓纯, 许炎辉, 樊毛迪, 高崧, 刘秀梵. 鸡Toll样受体15单克隆抗体的制备及其初步应用[J]. 畜牧兽医学报, 2022, 53(2): 576-587.

LI Mengni, WANG Hang, FU Siyao, YANG Zichun, XU Yanhui, FAN Maodi, GAO Song, LIU Xiufan. Preparation of Chicken Toll-like Receptor 15 Monoclonal Antibody and Its Preliminary Application[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 576-587.

参考文献 29

[1]	MANICASSAMY S, PULENDRAN B. Modulation of adaptive immunity with Toll-like receptors[J]. Semin Immunol, 2009, 21(4):185-193.
[2]	KAWAI T, AKIRA S. The roles of TLRs, RLRs and NLRs in pathogen recognition[J]. Int Immunol, 2009, 21(4):317-337.
[3]	HAJISHENGALLIS G, LAMBRIS J D. Microbial manipulation of receptor crosstalk in innate immunity[J]. Nat Rev Immunol, 2011, 11(3):187-200.
[4]	YUK J M, JO E K. Toll-like receptors and innate immunity[J]. J Bacteriol Virol, 2011, 41(4):225-235.
[5]	TAKEDA K, AKIRA S. Toll-like receptors in innate immunity[J]. Int Immunol, 2005, 17(1):1-14.
[6]	AKIRA S. Toll-like receptor signaling[J]. J Biol Chem, 2003, 278(40):38105-38108.
[7]	GRUEBER C E, WALLIS G P, JAMIESON I G. Episodic positive selection in the evolution of avian toll-like receptor innate immunity genes[J]. PLoS One, 2014, 9(3):e89632.
[8]	WERLING D, JANN O C, OFFORD V, et al. Variation matters:TLR structure and species-specific pathogen recognition[J]. Trends Immunol, 2009, 30(3):124-130.
[9]	KOBE B, KAJAVA A V. The leucine-rich repeat as a protein recognition motif[J]. Curr Opin Struct Biol, 2001, 11(6):725-732.
[10]	ZHAO Q P, GAO Q, ZHANG Y, et al. Identification of Toll-like receptor family members in Oncomelania hupensis and their role in defense against Schistosoma japonicum[J]. Acta Trop, 2018, 181:69-78.
[11]	HIGGS R, CORMICAN P, CAHALANE S, et al. Induction of a novel chicken Toll-like receptor following Salmonella enterica serovar typhimurium infection[J]. Infect Immun, 2006, 74(3):1692-1698.
[12]	BEUTLER B, REHLI M. Evolution of the TIR, Tolls and TLRs:Functional inferences from computational biology[J]. Curr Top Microbiol Immunol, 2002, 270:1-21.
[13]	韩焱, 徐明玉, 孙楠, 等. CpG寡核苷酸对鸡Toll样受体15表达量的影响[J]. 安徽农业科学, 2012, 40(14):8106, 8177.HAN Y, XU M Y, SUN N, et al. Effect of CpG-ODN on the expression of chicken TLR15[J]. J Anhui Agric Sci, 2012, 40(14):8106, 8177. (in Chinese)
[14]	俱雄, 陈春琳, 刘祥, 等. 重组大肠埃希菌外膜蛋白C的表达、纯化及多克隆抗体制备[J]. 陕西理工学院学报:自然科学版, 2015, 31(3):52-57.JU X, CHEN C L, LIU X, et al. Expression, purification and polyclonal antibody preparation of recombinant E.coli outer membrane protein OmpC[J]. Journal of Shaanxi University of Technology:Natural Science Edition, 2015, 31(3):52-57. (in Chinese)
[15]	赵东. 猪圆环病毒3型回顾性调查与cap蛋白单克隆抗体的研制[D]. 扬州:扬州大学, 2018.ZHAO D. Retrospective survey of procine circovirus type 3 in Jiangsu province and development of monoclonal antibodies against cap proteins of PCV3[D]. Yangzhou:Yangzhou University, 2018. (in Chinese)
[16]	刘秀梵. 单克隆抗体在农业上的应用[M]. 合肥:安徽科学技术出版社, 1994.LIU X F. Application of monoclonal antibodies in agriculture[J]. Hefei:Anhui Science and Technology Press, 1994. (in Chinese)
[17]	SKOWICKI M, LIPIŃSKI T. The development of methods for obtaining monoclonal antibody-producing cells[J]. Adv Hyg Exp Med, 2016, 70:367-379.
[18]	GAO Y L, HUANG X X, ZHU Y B, et al. A brief review of monoclonal antibody technology and its representative applications in immunoassays[J]. J Immunoassay Immunochem, 2018, 39(4):351-364.
[19]	LITTLE M, KIPRIYANOV S M, LE G F, et al. Of mice and men:Hybridoma and recombinant antibodies[J]. Immunol Today, 2000, 21(8):364-370.
[20]	闫慧丽, 席俊, 高学梅, 等. 抗Gly m Bd 28K单克隆抗体的制备及其免疫学特性[J]. 浙江农业学报, 2016, 28(5):748-752.YAN H L, XI J, GAO X M, et al. Preparation of monoclonal antibodies against Gly m Bd 28K and identification of their immunological properties[J]. Acta Agriculturae Zhejiangensis, 2016, 28(5):748-752. (in Chinese)
[21]	邢波建, 于淼, 吴迪, 等. 鸡Toll样受体15的生物信息学分析及组织表达[J]. 华北农学报, 2019, 34(1):232-238.XING B J, YU M, WU D, et al. Bioinformatics analysis and tissue expression of chicken Toll-like receptor 15[J]. Acta Agriculturae Boreali-Sinica, 2019, 34(1):232-238. (in Chinese)
[22]	陈大伟, 徐秀章, 夏文杰, 等. 一株鼠抗人CD36单克隆抗体的制备及应用[J]. 广州医药, 2020, 51(4):28-31, 42.CHEN D W, XU X Z, XIA W J, et al. Preparation and application of a mouse monoclonal antibody against human CD36[J]. Guangzhou Medical Journal, 2020, 51(4):28-31, 42. (in Chinese)
[23]	张志榜. 猪流行性腹泻病毒膜蛋白和核蛋白抗原表位的鉴定[D]. 北京:中国农业科学院, 2011.ZHANG Z B. Identification of antigenic epitopes on the membrane protein and nucleocapsid protein of porcine epidemic diarrhea virus[D]. Beijing:Chinese Academy of Agricultural Sciences, 2011. (in Chinese)
[24]	唐泽群, 罗海燕, 葛铭, 等. 抗chTLR3单克隆抗体制备及其初步应用[J]. 畜牧兽医学报, 2019, 50(6):1292-1300.TANG Z Q, LUO H Y, GE M, et al. Preparation of monoclonal antibody against chTLR3 and its preliminary application[J]. Acta Veterinaria Et Zootechnica Sinica, 2019, 50(6):1292-1300. (in Chinese)
[25]	GHOCHIKYAN A, PETRUSHINA I, DAVTYAN H, et al. Immunogenicity of epitope vaccines targeting different B cell antigenic determinants of human α-synuclein:Feasibility study[J]. Neurosci Lett, 2014, 560:86-91.
[26]	DE ZOETE M R, BOUWMAN L I, KEESTRA A M, et al. Cleavage and activation of a Toll-like receptor by microbial proteases[J]. Proc Natl Acad Sci USA, 2011, 108(12):4968-4973.
[27]	孙钰, 吴桂贤, 刘丽英, 等. 鼠伤寒沙门氏菌感染诱导鸡盲肠及脾脏TLR基因差异表达[J]. 中国农业大学学报, 2014, 19(6):161-167.SUN Y, WU G X, LIU L Y, et al. Differential expression of chicken TLR genes between spleen and cecum following the Salmonella typhimurium infection[J]. Journal of China Agricultural University, 2014, 19(6):161-167. (in Chinese)
[28]	ABASHT B, KAISER M G, LAMONT S J. Toll-like receptor gene expression in cecum and spleen of advanced intercross line chicks infected with Salmonella enterica serovar Enteritidis[J]. Vet Immunol Immunopathol, 2008, 123(3-4):314-323.
[29]	王燕, 周秀红, 祁克宗, 等. 雏鸡感染鸡白痢沙门菌后禽β-防御素6和鸡Toll样受体15在肠道转录与定位研究[J]. 畜牧兽医学报, 2014, 45(10):1711-1717.WANG Y, ZHOU X H, QI K Z, et al. Transcription and positioning of avian β-defensin 6 and chicken TLR15 within the intestinal tract of Salmonella pullorum infected chicks[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(10):1711-1717. (in Chinese)