抗chTLR3单克隆抗体制备及其初步应用

doi:10.11843/j.issn.0366-6964.2019.06.020

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (6): 1292-1300.doi: 10.11843/j.issn.0366-6964.2019.06.020

抗chTLR3单克隆抗体制备及其初步应用

唐泽群^1,2, 罗海燕^1,2, 葛铭^1,2, 郭荣^1,2, 许丹蕾^1,2, 张瑞莉^1,2*

1. 东北农业大学动物医学学院, 哈尔滨 150030;
2. 黑龙江省实验动物与比较医学重点实验室, 哈尔滨 150030

收稿日期:2018-12-20 出版日期:2019-06-23 发布日期:2019-06-23
通讯作者: 张瑞莉,主要从事兽医病理学研究,E-mail:zhangruil@neau.edu.cn
作者简介:唐泽群(1993-),男,黑龙江大庆人,硕士生,主要从事兽医病理学研究;罗海燕(1989-),男,湖南新邵人,硕士,主要从事兽医病理学研究。
基金资助:
国家自然科学基金（31272533）

Preparation of Monoclonal Antibody against chTLR3 and Its Preliminary Application

TANG Zequn^1,2, LUO Haiyan^1,2, GE Ming^1,2, GUO Rong^1,2, XU Danlei^1,2, ZHANG Ruili^1,2*

1. College of Veterinary Medicine, Northeast Agriculture University, Harbin 150030, China;
2. Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, Harbin 150030, China

Received:2018-12-20 Online:2019-06-23 Published:2019-06-23

摘要/Abstract

摘要： Toll样受体3（TLR3）是激发机体天然免疫的模式识别受体，为了制备能与天然结构的鸡TLR3（chTLR3）蛋白结合的单克隆抗体，将前期构建的可以在细胞内表达的真核重组质粒pVAX1-Igk-chTLR3免疫BALB/c小鼠，采用杂交瘤技术制备抗chTLR3单克隆抗体；并应用该抗体采用Western blot、流式细胞术及间接免疫荧光技术检测镉中毒雏鸡外周血淋巴细胞与脾内chTLR3的表达变化。结果发现，获得1株阳性杂交瘤细胞，该细胞株单个细胞染色体数量为（94±2）条，细胞上清效价为1∶2⁵，抗体亚类鉴定为IgG3，细胞上清能够与原核表达的chTLR3胞外区蛋白及鸡外周血淋巴细胞中的chTLR3特异性结合。应用此抗体采用Western blot、流式细胞术及间接免疫荧光技术均能够在对照组和染镉组雏鸡外周血淋巴细胞及脾内检测到chTLR3的蛋白表达。结果表明，成功制备了具有天然活性的chTLR3单克隆抗体，该抗体可用于Western blot、间接免疫荧光技术及流式细胞术研究chTLR3在鸡组织细胞的分布定位、表达变化，为示踪chTLR3蛋白分布、表达及研究其在鸡发病机制中的作用提供了重要的物质基础。

Abstract: Toll-like receptor 3 (TLR3) is a pattern recognition receptor that stimulates innate immunity. In order to prepare a monoclonal antibody that can bind to chicken TLR3 (chTLR3) protein with natural structure, BALB/c mice were immunized with recombinant plasmid pVAX1-Igk-chTLR3, which could be expressed in cells, and monoclonal antibodies against chTLR3 were prepared by hybridoma technique. The expression of chTLR3 expression of chTLR3 in peripheral blood lymphocytes and spleen of cadmium poisoned chickens were detected by Western blot, flow cytometry and indirect immunofluorescence. The results showed that a positive hybridoma cell line was obtained. The number of chromosomes of the cell line was 94±2, the titer of the cell supernatant was 1:2⁵, and the antibody subclass was identified as IgG3. The cell supernatant not only specifically bound to the prokaryotic expression protein of chTLR3, but also bound to chTLR3 protein in chicken peripheral blood lymphocytes. Western blot, flow cytometry and indirect immunofluorescence were used to detect the expression of chTLR3 in peripheral blood lymphocyte and spleen of chickens in control group and cadmium-exposed group. The results indicated that the monoclonal antibody against chTLR3 with natural activity was successfully prepared. The antibody can be used to study the distribution, localization and expression of chTLR3 in chicken tissues and cells by Western blot, indirect immunofluorescence and flow cytometry. It provides an important material basis for tracing the distribution and expression of chTLR3 protein and its role in the pathogenesis of chicken diseases.

中图分类号:

Q813

唐泽群, 罗海燕, 葛铭, 郭荣, 许丹蕾, 张瑞莉. 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 Zequn, LUO Haiyan, GE Ming, GUO Rong, XU Danlei, ZHANG Ruili. Preparation of Monoclonal Antibody against chTLR3 and Its Preliminary Application[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1292-1300.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2019.06.020

https://www.xmsyxb.com/CN/Y2019/V50/I6/1292

参考文献

[1] AKIRA S, TAKEDA K. Toll-like receptor signalling[J]. Nat Rev Immunol, 2004, 4(7):499-511.
[2] AKIRA S, UEMATSU S, TAKEUCHI O. Pathogen recognition and innate immunity[J]. Cell, 2006, 124(4):783-801.
[3] ALEXOPOULOU L, HOLT A C, MEDZHITOV R, et al. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3[J]. Nature, 2001, 413(6857):732-738.
[4] JIN M S, LEE J O. Structures of TLR-ligand complexes[J]. Curr Opin Immunol, 2008, 20(4):414-419.
[5] RAUF A, KHATRI M, MURGIA M V, et al. Differential modulation of cytokine, chemokine and Toll like receptor expression in chickens infected with classical and variant infectious bursal disease virus[J]. Vet Res, 2011, 42(1):85-96.
[6] 王成成, 王杲强,葛铭,等. 感染传染性法氏囊病病毒的DF-1细胞中chTLR3表达的动态变化[J]. 中国兽医科学, 2014, 44(12):1304-1308.
WANG C C, WANG G Q, GE M, et al. Dynamic changes of chTLR3 expression in DF-1 cells infected with infectious bursal disease virus[J]. Chinese Veterinary Science, 2014, 44(12):1304-1308. (in Chinese)
[7] JANEWAY C A Jr, MEDZHITOV R. Innate immune recognition[J]. Annu Rev Immunol, 2002, 20:197-216.
[8] FITZGERALD K A, MCWHIRTER S M, FAIA K L, et al. IKKε and TBK1 are essential components of the IRF3 signaling pathway[J]. Nat Immunol, 2003, 4(5):491-496.
[9] LIU W J, GE M, HU X Q, et al. The effects of Agaricus blazei murill polysaccharides on cadmium-induced apoptosis and the TLR4 signaling pathway of peripheral blood lymphocytes in chicken[J]. Biol Trace Elem Res, 2017, 180(1):153-163.
[10] 郭虹,陈观今,郑焕钦,等. 弓形虫ROP1基因真核表达重组质粒免疫小鼠后的免疫应答[J]. 中国寄生虫学与寄生虫病杂志, 1999, 17(6):334-337.
GUO H, CHEN G J, ZHENG H Q, et al. Immune responses in mice vaccinated with recombinant plasmid pcDNA3 containing ROP1 gene from Toxoplasma GondⅡ[J]. Chinese Journal of Parasitology and Parasitic Diseases, 1999, 17(6):334-337. (in Chinese)
[11] 岳锋,杨健,周娟娟,等. 抗猪PD-L1单克隆抗体的制备与其生物学特性鉴定[J]. 基因组学与应用生物学, 2018, 37(6):2368-2373.
YUE F, YANG J, ZHOU J J, et al. Preparation of anti-porcine PD-L1 monoclonal antibody and identification of its biological characteristics[J]. Genomics and Applied Biology, 2018, 37(6):2368-2373. (in Chinese)
[12] HU X Q, ZHANG R L, XIE Y Y, et al. The protective effects of polysaccharides from Agaricus blazei murill against Cadmium-induced oxidant stress and inflammatory damage in chicken livers[J]. Biol Trace Elem Res, 2017, 178(1):117-126.
[13] 陈建忠, 朱海红,陈智,等. 小鼠TLR3真核表达质粒的构建及其活性初步鉴定[J]. 中华微生物学和免疫学杂志, 2004, 24(6):447-450.
CHEN J Z, ZHU H H, CHEN Z, et al. Construction of eukaryotic expression plasmid of murine TLR3 and identification of its biological activity[J]. Chinese Journal of Microbiology and Immunology, 2004, 24(6):447-450. (in Chinese)
[14] VAINE M, WANG S X, HACKETT A, et al. Antibody responses elicited through homologous or heterologous prime-boost DNA and protein vaccinations differ in functional activity and avidity[J]. Vaccine, 2010, 28(17):2999-3007.
[15] 刘淑颖, 陈雨欣,王世霞,等. DNA免疫技术在高质量单克隆抗体诱导开发中的应用进展[J]. 药学进展, 2017, 41(9):662-669.
LIU S Y, CHEN Y X, WANG S X, et al. Progress in application of DNA immunization technique in R&D of high-quality monoclonal antibodies[J]. Progress in Pharmaceutical Sciences, 2017, 41(9):662-669. (in Chinese)
[16] ALEXOPOULOU L, HOLT A C, MEDZHITOV R, et al. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3[J]. Nature, 2001, 413(6857):732-738.
[17] 郑泓,韩双艳,林影,等. 质粒DNA单次脾内注射制备单克隆抗体[J]. 生物工程学报, 2007, 23(4):710-714.
ZHENG H, HAN S Y, LIN Y, et al. Preparation of monoclonal antibody based on single intrasplenic immunization of plasmid DNA[J]. Chinese Journal of Biotechnology, 2007, 23(4):710-714. (in Chinese)
[18] 梁文花, 贾云慧,许程志,等. 两株H1N1亚型猪流感病毒HA蛋白中和性单克隆抗体的制备及抗病毒活性研究[J]. 畜牧兽医学报, 2018, 49(7):1460-1466.
LIANG W H, JIA Y H, XU C Z, et al. Preparation and antiviral activity of two neutralizing monoclonal antibodies against HA protein of H1N1 swine influenza virus[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(7):1460-1466. (in Chinese)
[19] 蔡瑞丽, 王帅,缪静雅,等. 抗CMG2单克隆抗体的制备及其初步应用[J]. 免疫学杂志, 2018, 34(7):630-635.
CAI R L, WANG S, MIAO J Y, et al. Preparation of mouse anti-CMG2 monoclonal antibody and its preliminary application[J]. Immunological Journal, 2018, 34(7):630-635. (in Chinese)

抗chTLR3单克隆抗体制备及其初步应用

Preparation of Monoclonal Antibody against chTLR3 and Its Preliminary Application

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 10

编辑推荐

Metrics

本文评价

[1]	陈奡蕾, 黄德如, 安娅菲, 李守军. 动物肠类器官培养技术[J]. 畜牧兽医学报, 2023, 54(7): 2743-2750.
[2]	史明艳, 高雪, 杨学义, 窦忠英, 许尚忠. 构建具有毛囊结构的组织工程皮肤及移植研究[J]. 畜牧兽医学报, 2019, 50(10): 2097-2104.
[3]	史明艳, 高雪, 易力, 周新雨, 亢君芳, 李志超. 过表达IL-6对山羊毛囊干细胞增殖和迁移的影响[J]. 畜牧兽医学报, 2019, 50(6): 1154-1161.
[4]	霍书英, 李玉荣, 武现军. 肾上腺素能受体β2和胆碱能受体M1在小鼠睾丸的表达定位及功能研究[J]. 畜牧兽医学报, 2017, 48(6): 1028-1034.
[5]	徐礼杰，李钟淑，方南洙. 吡格列酮对MZT期小鼠胚胎发育阻滞的影响[J]. 畜牧兽医学报, 2016, 47(11): 2240-2247.
[6]	田青,王洪荣,王梦芝. 氢化可的松对奶牛乳腺上皮细胞酪蛋白合成的影响[J]. 畜牧兽医学报, 2014, 45(10): 1663-1670.
[7]	吕婧玉，许晓婷，陈晓佩，翟明胜，冯建昆，于文浩，王新庄. 5-aza-dC体外诱导兔骨髓间充质干细胞分化为胰岛细胞的研究[J]. 畜牧兽医学报, 2014, 45(9): 1538-1543.
[8]	马瑞丽，张彦明，崔红杰，郭抗抗. 新生仔猪口腔黏膜上皮细胞的体外培养方法[J]. 畜牧兽医学报, 2013, 44(9): 1445-1453.
[9]	李小佳，张君涛，徐秋勤，王鸽，曹书郡，王新庄，张志平. 饲养层密度对兔类ES细胞分离培养的影响[J]. 畜牧兽医学报, 2013, 44(7): 1051-1056.
[10]	廖庆红，丁培阳，赵丹丹，王爱华，齐雪峰. 山羊子宫内膜细胞与性腺激素对uNK细胞分泌活性的调节作用[J]. 畜牧兽医学报, 2013, 44(6): 866-870.