兔白细胞介素-10基因的克隆、原核表达及其多克隆抗体的制备

doi:10.11843/j.issn.0366-6964.2013.05.017

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (5): 788-795.doi: 10.11843/j.issn.0366-6964.2013.05.017

兔白细胞介素-10基因的克隆、原核表达及其多克隆抗体的制备

万小颖^1,2，秦立志^1,2，施丽娟^1,2，王文洲^1,2，李挺^1,2，李碧春^1,2，吴信生^{1, 2*}

（1. 扬州大学动物科学与技术学院，扬州 225009； 2. 江苏省动物遗传繁育与分子设计重点实验室，扬州 225009）

收稿日期:2012-11-02 出版日期:2013-05-23 发布日期:2013-05-23
通讯作者: 吴信生，教授，博士，E-mail: xswu@yzu.edu.cn
作者简介:万小颖（1989-），女，江苏海安人，硕士生，主要从事动物遗传育种与繁殖的研究，E-mail：xywan1024@163.com
基金资助:
现代农业产业技术体系建设专项资金资助（CARS-44-1）；江苏高校优势学科建设工程资助项目（苏政办发【2011】137号）；江苏省研究生科研创新计划（CXLX11_1027）

Cloning, Prokaryotic Expression and Polyclonal Antibody Preparation of Rabbit IL-10 Gene

WAN Xiao-ying^{1, 2}, QIN Li-zhi^{1, 2}, SHI Li-juan^{1, 2}, WANG Wen-zhou^{1, 2}, LI Ting^{1, 2},LI Bi-chun^{1, 2}, WU Xin-sheng^{1, 2*}

（1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China;2. Jiangsu Key Laboratory of Animal Genetics & Breeding and Molecular Design, Yangzhou 225009, China）

Received:2012-11-02 Online:2013-05-23 Published:2013-05-23

摘要/Abstract

摘要：

白细胞介素-10（IL-10）是一种多功能的细胞因子，参与免疫调节和抗炎反应。本研究旨在克隆并表达IL-10基因，以制备兔IL-10多克隆抗体。运用RT-PCR技术从新西兰白兔脾脏总RNA中扩增IL-10基因，将该基因亚克隆入原核表达载体pET-32a并转入大肠杆菌BL21（DE3）中诱导表达获得融合蛋白，融合蛋白经纯化后免疫豚鼠制备多克隆抗体，最后分别用间接ELISA和Western blot检测抗体的效价和特异性。结果表明：克隆得到了兔IL-10基因，大小为537 bp，经核苷酸测序与GenBank已登录的基因序列（NM_001082045）相似性为99%；重组载体pET-IL-10构建成功；SDS-PAGE电泳结果显示融合蛋白成功表达；制备的多克隆抗体效价高达1∶56 000，且具有很高的特异性。成功实现了兔IL-10基因的原核表达，并制备了豚鼠抗兔IL-10多克隆抗体，为深入研究兔IL-10基因的功能奠定了基础。

Abstract:

Interleukin-10 (IL-10) has been recently identified as a multifunctional cytokine because of its close link with immune regulation and anti-inflammatory responses. The aim of this study was to clone and express rabbit IL-10 gene in order to prepare the polyclonal antibody against recombinant IL-10. The IL-10 gene of New Zealand white rabbit was amplified by RT-PCR from spleen total RNA, then was sub-cloned into prokaryotic expression vector pET-32a and expressed in E. coli BL21 (DE3) host cells. Fusion protein was purified and immunized into guinea pigs so as to prepare polyclonal antibody. Finally, the sensitivity and specificity of the polyclonal antibody were detected through indirect ELISA and Western blot, respectively. As a result, rabbit IL-10 gene was cloned, and the obtained 537 bp fragment had 99% identities to the previously identified rabbit IL-10 gene (NM_001082045) at nucleotide levels; recombinant vector pET-IL-10 was successfully constructed; SDS-PAGE electrophoresis showed that the fusion protein was well expressed; the anti-IL-10 polyclonal antibody showed high sensitivity (1:56 000) and specificity. The above results indicate that IL-10 gene has been successfully cloned and expressed, and the guinea pig anti-rabbit IL-10 polyclonal antibody has been prepared by immunization with the purified recombinant IL-10 protein, which lays the foundation for further studies on rabbit IL-10 gene functions.

中图分类号:

Q78
S829.1

万小颖，秦立志，施丽娟，王文洲，李挺，李碧春，吴信生. 兔白细胞介素-10基因的克隆、原核表达及其多克隆抗体的制备[J]. 畜牧兽医学报, 2013, 44(5): 788-795.

WAN Xiao-ying, QIN Li-zhi, SHI Li-juan, WANG Wen-zhou, LI Ting,LI Bi-chun, WU Xin-sheng. Cloning, Prokaryotic Expression and Polyclonal Antibody Preparation of Rabbit IL-10 Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(5): 788-795.

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.0366-6964.2013.05.017

https://www.xmsyxb.com/CN/Y2013/V44/I5/788

参考文献

FIORENTINO D F, BOND M W, MOSMANN T R. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones［J］. J Exp Med, 1989, 170(6): 2081-2095.

王浩, 汪文娟. hsCRP, TNF-α和IL-10在小儿败血症早期诊断中的价值［J］. 中国卫生检验杂志, 2011, 21(11): 2710-2711.

URBONAS V, EIDUKAITE A, TAMULIENE I. Increased interleukin-10 levels correlate with bacteremia and sepsis in febrile neutropenia pediatric oncology patients［J］. Cytokine, 2012, 57(3): 313-315.

TESSE R, DEL VECCHIO G C, DE MATTIA D, et al. Association of interleukin-(IL)10 haplotypes and serum IL-10 levels in the progression of childhood immune thrombocytopenic purpura［J］. Gene, 2012, 505(1): 53-56.

GIRARD-MADOUX M J H, KEL J M, REIZIS B, et al. IL-10 controls dendritic cell-induced T-cell reactivation in the skin to limit contact hypersensitivity［J］. J Allergy Clin Immun, 2012, 129(1): 143-150.

PESTKA S, KRAUSE C D, SARKAR D, et al. Interleukin-10 and related cytokines and receptors［J］. Annu Rev Immunol, 2004, 22: 929-979.

ALBA-SOTO C D, SOLANA M E, PONCINI C V, et al. Dendritic cells devoid of IL-10 induce protective immunity against the protozoan parasite Trypanosoma cruzi［J］. Vaccine, 2010, 28(46): 7407-7413.

ZHANG Y G, ZHANG J, TIAN C, et al. The -1082G/A polymorphism in IL-10 gene is associated with risk of Alzheimer's disease: A meta-analysis［J］. J Neurol Sci, 2011, 303(1-2): 133-138.

LIO D, SCOLA L, FORTE G I, et al. TNFα, IFNγ and IL-10 gene polymorphisms in a sample of Sicilian patients with coeliac disease［J］. Digest Liver Dis, 2005, 37(10): 756-760.

BLANCO E, MONUX G, MAS A, et al. Role of IL-10 promoter polymorphisms in the development of severe aorto-iliac occlusive disease［J］. Hum Immunol, 2008, 69(10): 651-654.

OPDAL S H. IL-10 gene polymorphisms in infectious disease and SIDS［J］. FEMS Immunol Med Mic, 2004, 42(1): 48-52.

王少扬, 孙顺来, 马卫闽, 等. IL-10启动子基因多态性与乙型肝炎肝硬化相关性探讨［J］. 免疫学杂志, 2010, 26(8): 694-697.

张柳平, 段艳, 潘耀谦. IL-2在自然感染兔脑炎原虫獭兔体内免疫作用的研究［J］. 动物医学进展, 2004, 25(1): 115-116.

姚艳敏, 徐彤彤, 武琦, 等. 兔急性肺血栓栓塞症血清TNF-α，IL-1β和IL-4的变化［J］. 中国实验方剂学杂志, 2012, 18(8): 222-224.

孟庆玲, 乔军, 陈创夫, 等. 兔IL-15蛋白在毕赤酵母中的表达及其免疫学活性［J］. 石河子大学学报, 2008, 26(6): 700-703.

孟庆玲, 乔军, 才学鹏, 等. 兔IFN-γ基因的克隆、遗传进化分析及其表达［J］. 甘肃农业大学学报, 2008, 4: 7-11.

王芳, 胡波, 任雪枫, 等. 新西兰白兔γ-干扰素在昆虫细胞中的表达及其活性测定［J］. 华北农学报, 2010, 3: 9-13.

THEODOSSIADIS P G, LIARAKOS V S, SFIKAKIS P P, et al. Intravitreal administration of the anti-TNF monoclonal antibody Infliximab in the rabbit ［J］. Graefes Arch Clin Exp Ophthalmol, 2009, 247: 273-281.

聂永梅, 陈槐卿, 成敏, 等. IL-8在兔动脉粥样硬化模型中斑块处的蛋白和基因表达［J］. 生物医学工程学杂志, 2005, 22(2): 258-264.

于征淼, 陈晶, 刘志辉, 等. 清瘟败毒饮对兔全身炎症反应综合症IL-1、6、10、18、TNF-α、IFN-γ的影响［J］. 中药药理与临床, 2009, 25(1): 3-6.

HADDAD J J, SAADE N E, SAFIEHGARABEDIAN B. Interleukin-10 and the regulation of mitogen-activated protein kinases: are these signalling modules targets for the anti-inflammatory action of this cytokine? ［J］. Cell Signal, 2003, 15(3): 255-267.

LI M C, HE S H. IL-10 and its related cytokines for treatment of inflammatory bowel disease［J］. World J Gastroentero, 2004, 10(5): 620-625.

孙琳琳, 孙亿, 臧丽, 等. 猪生长分化因子11的原核表达及抗体制备［J］. 畜牧兽医学报, 2009, 40(11): 1577-1581.

翟军军, 窦永喜, 张海瑞, 等. 小反刍兽疫病毒P蛋白的表达、纯化及抗体的制备及初步应用［J］. 畜牧兽医学报, 2010, 41(8): 955-1000.

李辉, 吴旭东, 龚燕华, 等. PCGF1蛋白的表达、纯化及其单克隆抗体的制备与鉴定［J］. 细胞与分子免疫学杂志, 2011, 27(1): 71-73.

单丽伟, 唐如春, 刘三阳, 等. 小麦种子过氧化物酶WP1基因的原核表达、纯化及多克隆抗体制备［J］. 生物工程学报, 2011, 27(1): 26-30.

WAN S B, WANG W, LUO M, et al. cDNA cloning, prokaryotic expression, polyclonal antibody preparation of the auxin-binding protien 1 gene［J］. Plant Mol Biol Rep, 2010, 28: 373-380.

[1]	戴学宇, 张乾义, 徐璐, 赵启祖, 王琴, 夏应菊. CRISPR/Cas9基因编辑技术在重要猪病毒病防控中的研究与应用[J]. 畜牧兽医学报, 2020, 51(5): 943-951.
[2]	吴锦艳, 田宏, 蒙学莲, 惠小婷, 王耀杰, 关玉华, 尚佑军, 刘永生, 张志东. 慢病毒介导稳定表达增强小反刍兽疫病毒复制的山羊Vero/SLAM细胞系的建立[J]. 畜牧兽医学报, 2019, 50(1): 218-226.
[3]	胡慧艳, 贾青, 侯胜奎, 刘津, 张婧, 张伟峰, 锡建中. 猪StAR基因启动子区克隆及其转录活性研究[J]. 畜牧兽医学报, 2018, 49(7): 1524-1532.
[4]	胡慧艳, 贾青, 侯胜奎, 刘津, 赵思思, 张伟峰, 张军, 张建亭, 边慧敏. 猪DKK1基因启动子区的克隆及其活性分析[J]. 畜牧兽医学报, 2017, 48(6): 1150-1157.
[5]	许倩茹, 张二芹, 郭军庆, 杨继飞, 刘运超, 王爱萍, 王丽, 万博, 汪磊, 蒋大伟, 邓瑞广, 张改平. 新城疫病毒F蛋白在水稻中的表达及检测[J]. 畜牧兽医学报, 2017, 48(6): 1167-1172.
[6]	胡燕红，余玲莹，黄晓红. 酵母表达的长角血蜱谷氨酰胺转移酶及其活性分析[J]. 畜牧兽医学报, 2017, 48(2): 331-339.
[7]	何祖勇，刘志国，孙燕霞，丛佩清，陈瑶生. 共表达3种纤维素降解酶的转基因小鼠模型的建立与分析[J]. 畜牧兽医学报, 2016, 47(6): 1162-1169.
[8]	胡湘云，高小龙，付向晶，刘丹丹，范文涛，王燕平，杜恩岐，王兴龙，党如意，杨增岐. 双峰驼天然重链抗体可变区酵母双杂交文库的构建与鉴定[J]. 畜牧兽医学报, 2015, 46(6): 1071-1076.
[9]	符梅，陈亚冰，李键，熊显荣,兰道亮. NOBOX基因在麦洼牦牛卵母细胞、早期胚胎及胎儿卵巢的表达[J]. 畜牧兽医学报, 2014, 45(11): 1746-1751.
[10]	刘志国. CRISPR/Cas9系统介导基因组编辑的研究进展[J]. 畜牧兽医学报, 2014, 45(10): 1567-1583.
[11]	陈利，赵薇，占思远，李丹，李利，仲涛，王林杰，张红平. 山羊不同组织及不同发育时期骨骼肌内参基因的表达稳定性分析[J]. 畜牧兽医学报, 2014, 45(8): 1228-1236.
[12]	张春香，赵辉，张国林，郭丽娜，任有蛇. 绵羊ApoER2 基因克隆及其干扰质粒对睾丸共培养细胞硒蛋白表达的影响[J]. 畜牧兽医学报, 2014, 45(8): 1237-1245.
[13]	王敬姣，张明月，谭贝贝，张萃，李冬杰，戴蕴平，李宁，李世杰. 转基因克隆牛外源基因整合位点的检测[J]. 畜牧兽医学报, 2014, 45(4): 553-558.
[14]	冀元凯，田雪，范瑞文，于秀菊，赵园园，马淑慧，董常生. MATP在不同毛色羊驼皮肤中的表达、定位及与其他基因的相关性分析[J]. 畜牧兽医学报, 2014, 45(3): 502-508.
[15]	王向鹏，魏蕊芳，肖书奇，周恩民. 慢病毒载体介导稳定表达CD163 的PAM细胞系的建立及对PRRSV感染的研究[J]. 畜牧兽医学报, 2013, 44(11): 1797-1804.

兔白细胞介素-10基因的克隆、原核表达及其多克隆抗体的制备

Cloning, Prokaryotic Expression and Polyclonal Antibody Preparation of Rabbit IL-10 Gene

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价