鸡补体受体2基因克隆、蛋白表达纯化及其多克隆抗体的制备和鉴定

doi:10.16431/j.cnki.1671-7236.2020.01.024

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (1): 201-208.doi: 10.16431/j.cnki.1671-7236.2020.01.024

鸡补体受体2基因克隆、蛋白表达纯化及其多克隆抗体的制备和鉴定

孔子萌^1,2, 江波², 蔡云虹², 何后军¹, 李永清², 靳换²

1. 江西农业大学动物科学技术学院, 南昌 330045;
2. 北京市农林科学院, 畜禽疫病研究中心, 北京 100097

收稿日期:2019-06-24 出版日期:2020-01-20 发布日期:2020-01-17
通讯作者: 李永清, 靳换 E-mail:chunyudady@sina.com;jinhuan0717@126.com
作者简介:孔子萌(1996-),男,江西九江人,硕士生,研究方向:动物传染病,E-mail:743583554@qq.com
基金资助:
国家自然科学基金面上项目（31672588）；北京市博士后科研活动经费资助项目（2018-XX-053）；北京市农林科学院博士后基金（2018-ZZ-005）；北京市农林科学院科技创新能力建设专项（KJCX201914）

Cloning,Protein Expression and Purification of Chicken Complement Receptor 2 Gene and Preparation and Identification of Its Polyclonal Antibody

KONG Zimeng^1,2, JIANG Bo², CAI Yunhong², HE Houjun¹, LI Yongqing², JIN Huan²

1. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China;
2. Research Center for Infectious Diseases in Livestock and Poultry, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China

Received:2019-06-24 Online:2020-01-20 Published:2020-01-17

摘要/Abstract

摘要： 为初步鉴定鸡补体受体2（ChCR2）基因，本试验设计特异性引物，利用RT-PCR方法扩增ChCR2基因。利用同源臂连接的方法构建去掉跨膜区的ChCR2-△TM基因的原核表达载体pGEX-6P-1-ChCR2-△TM、pET-32a-ChCR2-△TM和真核表达载体pCMV-HA-ChCR2-△TM，将两个原核质粒转入大肠杆菌表达系统中表达GST-ChCR2-△TM和His-ChCR2-△TM蛋白。将纯化后的GST-ChCR2-△TM蛋白免疫BALB/c小鼠制备血清多克隆抗体，以His-ChCR2-△TM蛋白为包被原，ELISA检测血清多克隆抗体效价。利用间接免疫荧光试验和Western blotting对抗原抗体的特异结合性进行鉴定。结果显示，扩增到了一条大小为1 113 bp的ChCR2基因条带。成功构建了去掉跨膜区的原核表达载体pGEX-6P-1-ChCR2-△TM、pET-32a-ChCR2-△TM和真核表达载体pCMV-HA-ChCR2-△TM。诱导表达的GST-ChCR2-△TM蛋白在低温（16 ℃）诱导条件下可溶，而His-ChCR2-△TM蛋白以包涵体的形式存在。经ELISA测定，GST-ChCR2-△TM蛋白免疫的BALB/c小鼠血清效价为1∶512 000。间接免疫荧光试验和Western blotting结果显示，制备的抗GST-ChCR2-△TM蛋白血清多克隆抗体可与ChCR2蛋白发生特异性结合。本研究结果为ChCR2基因的进一步鉴定及鸡乃至禽类的相关免疫学研究提供了参考依据。

关键词: ChCR2基因; 蛋白表达和纯化; 多克隆抗体; ELISA; 间接免疫荧光; Western blotting

Abstract: In order to identify chicken complement receptor 2 (ChCR2) gene,we designed specific primers and amplified ChCR2 gene by RT-PCR.The prokaryotic expression vectors pGEX-6P-1-ChCR2-△TM and pET-32a-ChCR2-△TM and the eukaryotic expression vector pCMV-HA-ChCR2-△TM were constructed by homologous arm ligation.The two prokaryotic plasmids were transferred into E.coli expression system to express GST-ChCR2-△TM and His-ChCR2-△TM proteins.The polyclonal antibody was prepared by immunizing BALB/c mice with GST-ChCR2-△TM protein.The antibody titer was detected by ELISA.Indirect immunofluorescence test and Western blotting were used to identify the specific binding of antigen and antibody.The results showed that a ChCR2 gene with a size of 1 113 bp was amplified.The prokaryotic expression vectors pGEX-6P-1-ChCR2-ΔTM and pET-32a-ChCR2-ΔTM and the eukaryotic expression vector pCMV-HA-ChCR2-△TM with the transmembrane region removed were successfully constructed.The GST-ChCR2-△TM protein was soluble at low temperature (16 ℃),while His-ChCR2-△TM protein was in the form of inclusion body.The serum titer of BALB/c mice immunized with GST-ChCR2-△TM protein was 1:512 000 detected by ELISA.The results of indirect immunofluorescence assay and Western blotting showed that the polyclonal antibody against GST-ChCR2-△TM protein could specifically bind to ChCR2 protein.The results of this study provided a reference for further identification of ChCR2 gene and related immunological research of chicken and even poultry.

Key words: ChCR2 gene; expression and purification of protein; polyclonal antibody; ELISA; indirect immunofluorescence assay; Western blotting

中图分类号:

S831

孔子萌, 江波, 蔡云虹, 何后军, 李永清, 靳换. 鸡补体受体2基因克隆、蛋白表达纯化及其多克隆抗体的制备和鉴定[J]. 中国畜牧兽医, 2020, 47(1): 201-208.

KONG Zimeng, JIANG Bo, CAI Yunhong, HE Houjun, LI Yongqing, JIN Huan. Cloning,Protein Expression and Purification of Chicken Complement Receptor 2 Gene and Preparation and Identification of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 201-208.

参考文献

[1] TEDDER T F,ZHOU L J,ENGEL P.The CD19/CD21 signal-transduction complex of B-lymphocytes[J].Immunology Today,1994,15(9):437-442.
[2] ZHANG L,DING Z J,HEYMAN B.IgG3-antigen complexes are deposited on follicular dendritic cells in the presence of C1q and C3[J].Scientific Reports,2017,7(1):5400.
[3] JOG N R,YOUNG K A,MUNROE M E,et al.Association of Epstein-Barr virus serological reactivation with transitioning to systemic lupus erythematosus in at-risk individuals[J].Annals of the Rheumatic Diseases,2019,78(9):1235-1241.
[4] GASQUE P,CHAN P,MAUGER C,et al.Identification and characterization of complement C3 receptors on human astrocytes[J].Journal of Immunology,1996,156(6):2247-2255.
[5] YOUNG L S,SIXBEY J W,CLARK D,et al.Epstein-Barr virus receptors on human pharyngeal epithelia[J].Lancet,1986,1(8475):240-242.
[6] MOHAN R R,GORHAM R D,MORIKIS D.A theoretical view of the C3d:CR2 binding controversy[J].Molecular Immunology,2015,64(1):112-122.
[7] REID K B,DAY A J.Structure-function relationships of the complement components[J].Immunology Today,1989,10(7):177-180.
[8] KANE S J,SWANSON E,GORDON E O,et al.Relative impact of complement receptors CD21/35(Cr2/1) on scrapie pathogenesis in mice[J].mSphere,2017,2(6):e00493-17.
[9] AHEARN J M,FEARON D T.Structure and function of the complement receptors,CR1(CD35) and CR2(CD21)[J].Advances in Immunology,1989,46:183-219.
[10] HEESTERS B A,LINDQVIST M,VAGEFI P A,et al.Follicular dendritic cells retain infectious HIV in cycling endosomes[J].PLoS Pathogens,2015,11:e1005285.
[11] PAUL HANNAN J.The structure-function relationships of complement receptor type 2(CR2;CD21)[J].Current Protein and Peptide Science,2016,17(5):463-487.
[12] KANEKIYO M,BU W,JOYCE M G,et al.Rational design of an Epstein-Barr virus vaccine targeting the receptor-binding site[J].Cell,2015,162(5):1090-1100.
[13] PEKALSKI M L,GARCIA A R,FERREIRA R C,et al.Neonatal and adult recent thymic emigrants produce IL-8 and express complement receptors CR1 and CR2[J].Journal of Clinical Investigation Insight,2017,2(16):93739.
[14] BOHNSACK J F,COOPER N R.CR2 ligands modulate human B cell activation[J].Journal of Immunology,1988,141(8):2569-2576.
[15] VORUP-JENSEN T,JENSEN R K.Structural immunology of complement receptors 3 and 4[J].Frontiers in Immunology,2018,9:2716.
[16] TUVESON D A,AHEARN J M,MATSUMOTO A K,et al.Molecular interactions of complement receptors on B lymphocytes:A CR1/CR2 complex distinct from the CR2/CD19 complex[J].Journal of Experimental Medicine,1991,173(5):1083-1089.
[17] ZIJLSTRA L E,JUKEMA J W,MOOIJAART S P,et al.Association of complement receptor 1 gene polymorphisms with cognitive function[J].Physiological Genomics,2018,50(2):102-103.
[18] FRUSHICHEVA M P.Abstract A24:ZAP-70 and SYK regulation in the B cell receptor pathway in chronic lymphocytic leukemia[J].Cancer Research,2017,77(2 Supplement):A24.
[19] KELLER B,STUMPF I,STROHMEIER V,et al.High SYK expression drives constitutive activation of CD21 B cells[J].Journal of Immunology,2017,198(11):4285-4292.
[20] HOU G,ZHAO Q,ZHANG M Y,et al.LSD1 regulates Notch and PI3K/Akt/mTOR pathways through binding the promoter regions of Notch target genes in esophageal squamous cell carcinoma[J].Onco Targets and Therapy,2019,12:5215-5225.
[21] MONGINI P K,VILENSKY M A,HIGHET P F,et al.The affinity threshold for human B cell activation via the antigen receptor complex is reduced upon co-ligation of the antigen receptor with CD21(CR2)[J].Journal of Immunology,1997,159(8):3782-3791.
[22] CARTER R,FEARON D.CD19:Lowering the threshold for antigen receptor stimulation of B lymphocytes[J].Science,1992,256(5053):105-107.
[23] FEARON D T,CARTER R H.The CD19/CR2/TAPA-1 complex of B lymphocytes:Linking natural to acquired immunity[J].Annual Review of Immunology,1995,13(1):127-149.
[24] TAKAHASHI K.Mouse complement receptors type 1(CR1;CD35) and type 2(CR2;CD21):Expression on normal B cell subpopulations and decreased levels during the development of autoimmunity in MRL/Ipr mice[J].Journal of Immunology,1997,159(3):1557-1569.
[25] MOLINA H,KINOSHITA T,INOUE K,et al.A molecular and immunochemical characterization of mouse CR2.Evidence for a single gene model of mouse complement receptors 1 and 2[J].Journal of Immunology,1990,145(9):2974-2983.
[26] 万润,华敏,龙立书,等.H6N6亚型禽流感病毒N6基因的原核表达与多克隆抗体制备[J].中国畜牧兽医,2018,45(11):19-25. WAN R,HUA M,LONG L S,et al.Prokayotic expression and polyclonal antibody preparation of N6 gene of H6N6 subtype avian influenza virus[J].China Animal Husbandry & Veterinary Medicine,2018,45(11):19-25.(in Chinese)
[27] LINDBLOM R P,AEINEHBAND S,STROM M,et al.Complement receptor 2 is increased in cerebrospinal fluid of multiple sclerosis patients and regulates C3 function[J].Clinical Immunology,2016,166-167:89-95.
[28] GRATTONE M L,VILLIERS C L,VILLIERS M B,et al.Co-operation between human CR1(CD35) and CR2(CD21) in internalization of their ligands (C3b and IC3b) by murine transfected fibroblasts[J].Immunology,2010,98(1):152-157.
[29] THORARINSDOTTIR K,CAMPONESCHI A,JONSSON C,et al.CD21^－/low B cells associate with joint damage in rheumatoid arthritis patients[J].Scandinavian Journal of Immunology, 2019,90(2):e12792.
[30] 孙盈,闫敏鑫,黄秀芬,等.表达鸡补体因子C3d基因重组杆状病毒的构建及初步应用[J].中国兽医学报,2015,35(8):1254-1259. SUN Y,YAN M X,HUANG X F,et al.Preliminary application and construction of recombinant baculovirus expressing chicken complement component 3d gene[J].Chinese Journal of Veterinary Science,2015,35(8):1254-1259.(in Chinese)

鸡补体受体2基因克隆、蛋白表达纯化及其多克隆抗体的制备和鉴定

Cloning,Protein Expression and Purification of Chicken Complement Receptor 2 Gene and Preparation and Identification of Its Polyclonal Antibody

PDF

可视化

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

[1]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[2]	欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2487-2495.
[3]	张芳源, 蔺雅婷, 杨大为, 陈虎, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立[J]. 中国畜牧兽医, 2023, 50(5): 2012-2022.
[4]	王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.
[5]	胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.
[6]	王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.
[7]	牟一娇, 于瑞明, 张莉萍, 王永录. 基于猪流行性腹泻病毒变异毒株重组全长S2蛋白IgA抗体ELISA检测方法的建立及应用[J]. 中国畜牧兽医, 2023, 50(3): 1185-1194.
[8]	牛小杰, 徐明国, 肖莉, 刘文豪, 陈创夫, 王勇. 鸡白痢沙门氏菌IPAJ蛋白的表达纯化、多克隆抗体制备及ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(3): 1218-1228.
[9]	张凯, 张春平, 戈胜强, 孙春喜, 程杰, 伏春雨, 王刚, 牛星, 彭军. 非洲猪瘟病毒p22蛋白表达、多克隆抗体制备及间接ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(1): 270-279.
[10]	张梦杨, 何健, 刘阳坤, 姚伦广. 非洲猪瘟病毒p37蛋白生物信息学分析及其多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(1): 300-309.
[11]	朱丽霖, 王后坤, 陈雪阳, 刘晶, 梁雄燕, 方春, 杨玉莹. 鸡IFIT5对J亚群禽白血病病毒在DF-1细胞内复制的影响[J]. 中国畜牧兽医, 2023, 50(1): 310-316.
[12]	韩瑞, 杨行, 张文波, 陈佳玲, 周晓丽. 猪链球菌自溶素的原核表达及其抗体间接ELISA检测方法的建立[J]. 中国畜牧兽医, 2022, 49(9): 3508-3519.
[13]	席静, 易继海, 王月丽, 徐朕宇, 李培东, 张江伟, 陈创夫. 牛A群轮状病毒G10型XJ-2022株分离鉴定及基因型分析[J]. 中国畜牧兽医, 2022, 49(9): 3530-3538.
[14]	唐庆凤, 李玲, 黄加祥, 方文远, 农皓如, 唐艳, 陆呈委, 韦盘秋, 冯玲. 广西地区不同饲养模式及不同季节水牛奶霉菌毒素污染状况调查[J]. 中国畜牧兽医, 2022, 49(9): 3665-3675.
[15]	肖洋洋, 马忠臣, 李芮芮, 陈创夫, 郑炜, 王勇, 王鹏雁. 布鲁氏菌分泌蛋白BspI生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(8): 3112-3121.