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

Abstract

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.

CLC Number:

Q78
S829.1

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
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2013.05.017

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

References

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]	DAI Xueyu, ZHANG Qianyi, XU Lu, ZHAO Qizu, WANG Qin, XIA Yingju. Research Progress and Application of CRISPR/Cas9 Gene Editing Technology in Prevention and Control of Important Swine Virus Diseases [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 943-951.
[2]	WU Jinyan, TIAN Hong, MENG Xuelian, HUI Xiaoting, WANG Yaojie, GUAN Yuhua, SHANG Youjun, LIU Yongsheng, ZHANG Zhidong. Establishment of Vero/SLAM Cell Lines with Stable Expression SLAM to Enhanced PPRV Replication Using the Lentiviral Expression System [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 218-226.
[3]	HU Hui-yan, JIA Qing, HOU Sheng-kui, LIU Jin, ZHANG Jing, ZHANG Wei-feng, XI Jian-zhong. Cloning and Transcriptional Activity of Swine StAR Gene Promoter [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1524-1532.
[4]	HU Hui-yan, JIA Qing, HOU Sheng-kui, LIU Jin, ZHAO Si-si, ZHANG Wei-feng, ZHANG Jun, ZHANG Jian-ting, BIAN Hui-min. Cloning and Activity Analysis of the Promoter Region of Swine DKK1 Gene [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1150-1157.
[5]	XU Qian-ru, ZHANG Er-qin, GUO Jun-qing, YANG Ji-fei, LIU Yun-chao, WANG Ai-ping, WANG Li, WAN Bo, WANG Lei, JIANG Da-wei, DENG Rui-guang, ZHANG Gai-ping. Expression and Detection of NDV-F Protein in Rice Endosperm [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1167-1172.
[6]	HU Yan-hong, YU Ling-ying, HUANG Xiao-hong. Analysis on a Transglutaminase from Haemaphysalis longicornis Expressed in Pichia pastoris [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 331-339.
[7]	HE Zu-yong，LIU Zhi-guo，SUN Yan-xia，CONG Pei-qing，CHEN Yao-sheng. Generation of a Transgenic Mouse Model Co-expressing Three Fibrolytic Enzyme Genes [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1162-1169.
[8]	HU Xiang-yun,GAO Xiao-long，FU Xiang-jing，LIU Dan-dan，FAN Wen-tao，WANG Yan-ping，DU En-qi，WANG Xing-long，DANG Ru-yi，YANG Zeng-qi. Construction and Characterization of a Naive Camelus Bactrianus Variable Domain of Heavy Chain of Heavy-Chain Antibody（VHH）Yeast Two-Hybrid Library [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(6): 1071-1076.
[9]	FU Mei，CHEN Ya-bing，LI Jian，XIONG Xian-rong，LAN Dao-liang. NOBOX Gene Expression in Maiwa Yak Oocytes，Early Embryogenesis and Fetal Ovaries [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(11): 1746-1751.
[10]	LIU Zhi-guo. Research Progress on CRISPR/Cas9 Mediated Genome Editing [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(10): 1567-1583.
[11]	CHEN Li，ZHAO Wei，ZHAN Si-yuan，LI Dan，LI Li，ZHONG Tao，WANG Lin-jie，ZHANG Hong-ping. The Expression Stability Analysis of Reference Genes in the Different Tissues and Skeletal Muscle of Different Development Periods in Goat [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1228-1236.
[12]	ZHANG Chun-xiang，ZHAO Hui，ZHANG Guo-lin，GUO Li-na，REN You-she. The Clone of ApoER2 Gene and the Inhibitory Effect of Interference Plasmid ApoER2-siRNA on Selenoprotein Expression in Co-culture Sertoli Cells of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1237-1245.
[13]	WANG Jing-jiao，ZHANG Ming-yue，TAN Bei-bei，ZHANG Cui，LI Dong-jie，DAI Yun-ping，LI Ning,LI Shi-jie. Integration Site Detection of Exogenous Gene in Transgenic Cloned Cattle [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(4): 553-558.
[14]	JI Yuan-kai,TIAN Xue，FAN Rui-wen，YU Xiu-ju,ZHAO Yuan-yuan，MA Shu-hui，DONG Chang-sheng. Study on the Expression and Localization of MATP in Different Colors of Alpaca Skin and the Correlation Analysis of Other Genes [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(3): 502-508.
[15]	WANG Xiang-peng, WEI Rui-fang, XIAO Shu-qi, ZHOU En-min. Generation of a Porcine Alveolar Macrophage Cell Line Stably Expressing CD163 by Lentiviral Vector for the Production of Porcine Reproductive and RespiratorySyndrome Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(11): 1797-1804.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments