红嘴鸥TLR7蛋白多克隆抗体制备及在DF-1细胞中的表达特征分析

doi:10.16431/j.cnki.1671-7236.2023.07.026

摘要/Abstract

摘要： 【目的】制备红嘴鸥Toll样受体7（Toll-like receptor 7，TLR7）蛋白多克隆抗体，分析红嘴鸥TLR7蛋白在DF-1细胞中的表达特征，为深入了解红嘴鸥TLR7蛋白功能及作用机制提供材料。【方法】设计引物扩增红嘴鸥TLR7基因CDS区，并构建其原核表达载体与真核表达载体。将重组原核表达质粒pET32a-TLR7转化大肠杆菌Transetta （DE3）感受态细胞进行重组蛋白体外诱导表达，并对其诱导温度、诱导时间和IPTG浓度进行优化筛选。选用新西兰大白兔制备多克隆抗体，采用间接ELISA法检测血清抗体价效，利用Western blotting鉴定抗体特异性。将真核表达质粒pcDNA3.1-TLR7转染至DF-1细胞中过表达，利用间接免疫荧光技术检测红嘴鸥TLR7蛋白在转染DF-1细胞后不同时间点的表达特征。【结果】红嘴鸥TLR7基因CDS区长约1 182 bp，双酶切结果显示出2组大小不同的2条片段，分别为5 900、1 182 bp （原核表达载体）和5 428和1 182 bp （真核表达载体），测序后表明原核表达载体与真核表达载体构建成功。体外诱导温度为30 ℃、IPTG终浓度为1.0 mmol/L时培养5 h得到以包涵体形式大量表达的重组蛋白，蛋白大小为63 ku。间接ELISA法检测抗血清效价为1：256 000以上。Western blotting检测结果表明，制备的多克隆抗体具有较好的特异性。间接免疫荧光结果显示，转染DF-1细胞2 h后即可观察到绿色荧光，随着时间增加绿色荧光密度逐渐增加，且绿色荧光大部分聚集在细胞核及周围，少部分散布在细胞质中。【结论】原核表达的红嘴鸥TLR7蛋白具有良好的免疫原性，制备的多克隆抗体具有较高的反应性和特异性，TLR7蛋白可在DF-1细胞中成功表达。

关键词: 红嘴鸥; TLR7蛋白; 多克隆抗体; DF-1细胞; 表达

Abstract: 【Objective】 This study was aimed to prepare the polyclonal antibody of Toll-like receptor 7(TLR7) protein of Larus ridibundus, and analyze the expression characteristics of TLR7 protein in DF-1 cells, so as to provide materials for the in-depth understanding of the function and mechanism of TLR7 protein in Larus ridibundus.【Method】 Primers were designed to amplify the CDS region of TLR7 gene in Larus ridibundus, and its prokaryotic and eukaryotic expression vectors were constructed.The recombinant prokaryotic expression plasmid pET32a-TLR7 was converted to Escherichia coli Transetta (DE3) competent cells for in vitro induction of recombinant protein expression, and the conditions for inducing temperature, inducing time and IPTG concentration were optimally screened.New Zealand White rabbits were choosed to prepare polyclonal antibody, the serum antibody valence was detect by indirect ELISA method, and the antibody specificity was identified by Western blotting.The eukaryotic expression plasmid pcDNA3.1-TLR7 was overexpressed in DF-1 cells, and the expression characteristics of TLR7 protein in Larus ridibundus at different time points after transfection of DF-1 cells were detected by indirect immunofluorescence technology.【Result】 The CDS region of TLR7 gene in Larus ridibundus was 1 182 bp.The double digestion results showed two sets of fragments of different sizes of 5 900 and 1 182 bp (prokaryotic expression vectors), 5 428 and 1 182 bp (eukaryotic expression vector), respectively.Sequencing results showed that the prokaryotic and eukaryotic expression vectors were successfully constructed.When the induction temperature in vitro was 30 ℃ and the final concentration of IPTG was 1.0 mmol/L, a large number of recombinant proteins in the form of inclusion bodies were obtained by culture for 5 h, and the protein size was 63 ku.The antiserum valence of indirect ELISA method was more than 1:256 000.The Western blotting assay results showed that the prepared polyclonal antibody had good specificity.The indirect immunofluorescence results showed that green fluorescence could be observed 2 h after transfection of DF-1 cells, the green fluorescence density gradually increased with time, most of the green fluorescence gathered in and around the nucleus, and a small part was scattered in the cytoplasm.【Conclusion】 The prokaryotic expression of TLR7 protein in Larus ridibundus had good immunogenicity, the prepared polyclonal antibody had high reactivity and specificity, and TLR7 protein was successfully expressed in DF-1 cells.

Key words: Larus ridibundus; TLR7 protein; polyclonal antibody; DF-1 cells; expression

中图分类号:

S852.4⁺3

任胜杰, 申宏利, 项勋, 代飞燕, 朱茂银, 邬佳莉, 胡志辉, 段纲, 常华. 红嘴鸥TLR7蛋白多克隆抗体制备及在DF-1细胞中的表达特征分析[J]. 中国畜牧兽医, 2023, 50(7): 2865-2875.

REN Shengjie, SHEN Hongli, XIANG Xun, DAI Feiyan, ZHU Maoyin, WU Jiali, HU Zhihui, DUAN Gang, CHANG Hua. Polyclonal Antibody Preparation of TLR7 Protein and Its Expression Characteristics Analysis in DF-1 Cells of Larus ridibundus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2865-2875.

参考文献

[1] ZHOU R, LIU L, WANG Y.Viral proteins recognized by different TLRs[J]. Journal of Medical Virology, 2021, 93(11):6116-6123.
[2] SATTERTHWAITE A B.TLR7 signaling in lupus B cells:New insights into synergizing factors and downstream signals[J]. Current Rheumatology Reports, 2021, 23(11):80.
[3] ASAMI J, SHIMIZU T.Structural and functional understanding of the Toll-like receptors[J].Protein Science, 2021, 30(4):761-772.
[4] DUAN T, DU Y, XING C S, et al.Toll-like receptor signaling and its role in cell-mediated immunity[J].Frontiers in Immunology, 2022, 13:812774.
[5] MCGOWAN D C.Latest advances in small molecule TLR7/8 agonist drug research[J].Current Topics in Medicinal Chemistry, 2019, 19(24):2228-2238.
[6] 张莉莉, 陈玉秋, 侯雨彤, 等.鸭源新城疫强毒株对番鸭细胞因子及Toll样受体mRNA表达的影响[J].中国兽医科学, 2021, 51(1):90-97. ZHANG L L, CHEN Y Q, HOU Y T, et al.Duck mRNA expression of cytokines and Toll receptors induced by duck-origin virulent Newcastle disease virus[J].Chinese Veterinary Science, 2021, 51(1):90-97.(in Chinese)
[7] ZHOU Y C, CHEN X J, CAO Z J, et al.R848 is involved in the antibacterial immune response of golden pompano (Trachinotus ovatus) through TLR7/8-MyD88-NF-κB-signaling pathway[J].Frontiers in Immunology, 2021, 11:617522.
[8] 黄莉, 谢芝勋, 蓝元喜, 等.禽呼肠孤病毒感染对SPF鸡外周血淋巴细胞中天然免疫相关基因转录的影响[J].畜牧兽医学报, 2017, 48(1):116-123. HUANG L, XIE Z X, LAN Y X, et al.Effects of Avian reovirus infection on transscriptionof innate immune genes in peripheral blood lymphocytes of SPF chickens[J].Acta Veterinaria et Zootechnica Sinica, 2017, 48(1):116-123.(in Chinese)
[9] 闫冰.鹅TLR7和TLR21抗病毒免疫学特性及功能初探[D].雅安:四川农业大学, 2016. YAN B.Antiviral immune characters and function of goose TLR7 and TLR21[D].Ya'an:Sichuan Agricultural University, 2016.(in Chinese)
[10] WANG Y, WANG M J, ZHANG H, et al.Emergence, evolution, and biological characteristics of H10N4 and H10N8 Avian influenza viruses in migratory wild birds detected in eastern China in 2020[J].Microbiology Spectrum, 2022, 10(2):e00807-22.
[11] 常华, 段纲, 阮谦, 等.红嘴鸥TLR7基因克隆与生物信息学分析[J].中国畜牧兽医, 2022, 49(1):43-52. CHANG H, DUAN G, RUAN Q, et al.Cloning and bioinformatics analysis of TLR7 gene in Larus ridibundus[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(1):43-52.(in Chinese)
[12] KHANMOHAMMADI S, REZAEI N.Role of Toll-like receptors in the pathogenesis of COVID-19[J].Journal of Medical Virology, 2021, 93(5):2735-2739.
[13] 王文浩, 胡琳玉, 彭秋玲, 等.康乐黄鸡TLR7基因多态性及血清INF-γ水平相关性研究[J].黑龙江畜牧兽医, 2018, 17:89-91. WANG W H, HU Y L, PENG Q L, et al.Correlation between TLR7 gene polymorphism and serum INF-γ level in Kangle Yellow chicken[J]. Helongjiang Animal Science and veterinary Medicine, 2018, 17:89-91.(in Chinese)
[14] QI Y L, CHEN S, ZHAO Q R, et al.Molecular cloning, tissue distribution, and immune function of goose TLR7[J]. Immunology Letters, 2015, 163(2):135-142.
[15] XIONG D, SONG L, PAN Z M, et al.Identification and immune functional characterization of pigeon TLR7[J]. International Journal of Molecular Sciences, 2015, 16(4):8364-8381.
[16] SONG H, ZHANG M, GAO W, et al.Molecular cloning and functional analysis of peafowl (Pavo cristatus) Toll-like receptor 7[J].Poultry Science, 2018, 97(1):294-302.
[17] GU T T, LI G Q, WU X S, et al.Molecular cloning, tissue distribution and function analysis of duck TLR7[J].Animal Biotechnology, 2020, 33(2):234-241.
[18] IHA K, OMATSU T, WATANABE S, et al.Molecular cloning and expression analysis of bat Toll-like receptors 3, 7 and 9[J].The Journal of Veterinary Medical Science, 2010, 72(2):217-220.
[19] TRIPATHI N K, SHRIVASTAVA A.Recent developments in bioprocessing of recombinant proteins:Expression hosts and process development[J]. Frontiers in Bioengineering and Biotechnology, 2019, 7:420.
[20] 刘可姝, 吴义龙, 费荣梅, 等.扬子鳄TLR7蛋白多克隆抗体的制备及其在不同组织中的差异表达[J].中国兽医科学, 2015, 45(5):522-526. LIU K S, WU Y L, FEI R M, et al.Preparation of polyclonal antibody against TLR7 protein of Chinese alligator and its differential expression in different tissues[J].Chinese Veterinary Science, 2015, 45(5):522-526.(in Chinese)
[21] GAO Y, HUANG X, ZHU Y, et al.A brief review of monoclonal antibody technology and its representative applications in immunoassays[J]. Journal of Immunoassay and Immunochemistry, 2018, 39(4):351-364.
[22] NDZOUBOUKOU J B, ZHANG Y D, FAN X L.Recent developments in SARS-CoV-2 neutralizing antibody detection methods[J].Current Medical Science, 2021, 41(6):1052-1064.
[23] MCCANN K B, VAN ALSTINE J, MARTINEZ J, et al.Polyacrylic acid based plasma fractionation for the production of albumin and IgG:Compatibility with existing commercial downstream processes[J]. Biotechnology and Bioengineering, 2020, 117(4):1072-1081.
[24] KESIDIS A, DEPPING P, LODÉ A, et al.Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts[J]. Methods, 2020, 180:3-18.
[25] JIANG B, ZHANG W, LIU P, et al.The prokaryotic expression, polyclonal antibody preparation, and subcellular localization of the transmembrane protein NS2A of the Duck Tembusu virus[J]. Acta Virologica, 2020, 64(3):380-385.
[26] 弓莉, 覃宗华, 顾为望, 等.传染性腔上囊炎疫苗免疫鸡TLR7基因表达的动态变化[J].中国兽医科学, 2007, 37(6):486-490. GONG L, QIN Z H, GU W W, et al.Changes of expression levels of Toll-like receptor 7 gene in the spleen and bursa of chickens immunized with vaccine against IBD[J].Chinese Veterinary Science, 2007, 37(6):486-490.(in Chinese)
[27] 王秋玲.人工感染鸭源新城疫病毒对鸭免疫功能作用的分子机制[D].哈尔滨:东北农业大学, 2016. WANG Q L.The molecular mechanism of artificial infection of Newcastle disesae virus from duck on the immune function of ducks[D].Harbin:Northeast Agricultural University, 2016.(in Chinese)
[28] ANAM S, RAHMAN S U, ALI S, et al.Comparative growth kinetic study of Newcastle disease virus, Infectious bursal disease virus and Avian influenza virus in chicken embryo fibroblast and DF-1 cell lines[J]. Polish Journal of Veterinary Sciences, 2021, 24(2):287-292.