布鲁氏菌分泌蛋白BspD多克隆抗体制备及其生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.02.030

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (2): 676-684.doi: 10.16431/j.cnki.1671-7236.2021.02.030

布鲁氏菌分泌蛋白BspD多克隆抗体制备及其生物信息学分析

李芮芮^1,2,3, 马忠臣^1,2,3, 张弘扬¹, 王震^1,2,3, 努尔赛力克·努素甫⁴, 王勇^1,2,3, 陈创夫^1,2,3

1. 石河子大学动物科技学院, 石河子 832000;
2. 西部地区高发人兽共患传染性疾病防治协同创新中心, 石河子 832000;
3. 新疆生产建设兵团动物疾病防控重点实验室, 石河子 832000;
4. 伊犁哈萨克自治州新源县农业农村局, 伊犁 835800

收稿日期:2020-06-19 出版日期:2021-02-20 发布日期:2021-02-23
通讯作者: 王勇, 陈创夫 E-mail:wangyong@zhzu.edu.cn;chunagfu-chen@163.com
作者简介:李芮芮(1995-),女,河南三门峡人,硕士生,研究方向:兽医学,E-mail:3233395945@qq.com
基金资助:
国家重点研发计划"畜禽重要胞内菌基因调控及其与宿主互作的分子机制"（2017YFD0500302、2017YFD0500304）

Polyclonal Antibody Preparation and Bioinformatics Analysis of Brucella Secreted Protein BspD

LI Ruirui^1,2,3, MA Zhongchen^1,2,3, ZHANG Hongyang¹, WANG Zhen^1,2,3, NUSUFU·Nuersailike⁴, WANG Yong^1,2,3, CHEN Chuangfu^1,2,3

1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
2. Collaborative Innovation Center for Prevention and Control of Zoonotic Infectious Diseases in Western China, Shihezi 832000, China;
3. Key Laboratory of Animal Disease Prevention and Control, Xinjiang Production and Construction Corps, Shihezi 832000, China;
4. Agricultural and Rural Bureau of Xinyuan County, Ili Kazakh Autonomous Prefecture, Ili 835800, China

Received:2020-06-19 Online:2021-02-20 Published:2021-02-23

摘要/Abstract

摘要： 本研究旨在获得布鲁氏菌BspD蛋白，制备其多克隆抗体，并分析其潜在生物学功能。根据流产布鲁氏菌（Brucella abortus）2308的BspD基因序列（GenBank登录号：NC_007618.1）获得BspD基因序列，对布鲁氏菌BspD氨基酸序列进行生物信息学分析，然后将目的基因嵌入pMD19-T载体，使用限制性内切酶切下目的基因重组入pET-28a（+）载体，构建pET28a-BspD重组载体，经过双酶切、测序验证后，IPTG诱导表达菌株，SDS-PAGE法鉴定BspD的表达，His标签蛋白纯化柱纯化BspD蛋白，BCA试剂盒检测蛋白浓度。用纯化BspD蛋白免疫新西兰大白兔，Western blotting鉴定多克隆抗体的特异性，间接ELISA法检测抗体的效价及反应原性。结果表明，成功表达出37 ku BspD蛋白，BCA方法测得纯化BspD浓度为2 000 μg/mL；Western blotting结果显示制备的抗体具有良好的特异性，间接ELISA检测出BspD多克隆抗体的效价为1：12 800，成功制备出BspD多克隆抗体，但其反应原性较低。生物学信息分析得出BspD蛋白具有亲水性，存在跨膜区，无信号肽，有17个磷酸化位点和11个抗原决定簇，BspD蛋白二级结构以α-螺旋为主，达到86.80%，还存在少量延伸链、无规则卷曲、β-折叠等；在线软件Phyre 2构建BspD三级结构证实其多为α-螺旋。以上结果可为进一步研究布鲁氏菌分泌蛋白BspD的功能及分子机制提供参考。

关键词: 布鲁氏菌; BspD分泌蛋白; 生物信息学分析; 多克隆抗体

Abstract: The purpose of this study was to obtain Brucella BspD protein,analyze its potential biological functions,and prepare its polyclonal antibody.After the synthesis of BspD gene sequences and the bioinformatics analysis of the amino acid sequence of Brucella BspD,according to the BspD gene sequence of Brucella abortus 2308 (GenBank accession No:NC_007618.1),the target gene was inserted into pMD19-T vector,and the target gene was digested by restriction endonuclease and recombined into pET-28a(+) vector to construct the pET28a-BspD recombinant vector.After double enzyme digestion and sequencing verification,IPTG was used to induce expression strain,SDS-PAGE was used to identify the expression of BspD,His label protein purification column was used to purify BspD protein,BCA kit was used to detecte the protein concentration,purified BspD protein was used to immunize New Zealand White rabbits,Western blotting was used to identify the specificity of polyclonal antibodies.Indirect ELISA was used to detect the titer and reactivity of antibody.The 37 ku BspD protein had been expressed successfully,and the purified BspD concentration was 2 000 μg/mL by BCA method.The Western blotting results showed that the prepared antibody had good specificity,and the titer of BspD polyclonal antibody detected by indirect ELISA was 1:12 800.The polyclonal antibody against BspD had been successfully prepared,but its reactivity was low.According to the analysis of biological information,BspD protein was hydrophilic,existed transmembrane region,had no signal peptide,possessed 17 phosphorylation sites and 11 antigenic epitopes.The secondary structure of BspD protein was mainly α-helix,reaching 86.80%,and there were a few extended chains,random coil,β-folding and other structures.In addition,the tertiary structure of BspD was constructed by Phyre 2,an online software,also confirmed that it was α-helix.The results provided references for further study on the function and molecular mechanism of BspD secreted by Brucella.

Key words: Brucella; BspD secreted protein; bioinformatics analysis; polyclonal antibody

中图分类号:

S852.6⁺14

李芮芮, 马忠臣, 张弘扬, 王震, 努尔赛力克·努素甫, 王勇, 陈创夫. 布鲁氏菌分泌蛋白BspD多克隆抗体制备及其生物信息学分析[J]. 中国畜牧兽医, 2021, 48(2): 676-684.

LI Ruirui, MA Zhongchen, ZHANG Hongyang, WANG Zhen, NUSUFU·Nuersailike, WANG Yong, CHEN Chuangfu. Polyclonal Antibody Preparation and Bioinformatics Analysis of Brucella Secreted Protein BspD[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 676-684.

参考文献

[1] GOODWIN Z I,PASCUAL D W.Brucellosis vaccines for livestock[J].Veterinary Immunology & Immuno-pathology,2016,181:51-58.
[2] FRANCO M P,MULDER M,GILMAN R H,et al.Human brucellosis[J].The Lancet Infectious Diseases,2007,7(12):775-786.
[3] 纪太旺,张辉,郭飞,等.布鲁氏菌043强毒株BR0524基因缺失突变株的构建与毒力的初步评价[J].石河子大学学报自然科学版,2014,32(5):529-535. JI T W,ZHANG H,GUO F,et al.Construction and preliminary evaluation of virulence of BR0524 gene deficient Brucella melitensis 043 strain[J].Journal of Shihezi University (Natural Science Edition),2014,32(5):529-535.(in Chinese)
[4] 王慧,张亚丽,王远志,等.布鲁氏菌16M omp31基因缺失株的构建与鉴定[J].石河子大学学报自然科学版,2013,31(1):30-34. WANG H,ZHANG Y L,WANG Y Z,et al.Construction and identification of omp31-deleted mutant of Brucella standard strain 16M[J].Journal of Shihezi University (Natural Science Edition),2013,31(1):30-34.(in Chinese)
[5] ANDREINI C,CAVALLARO G,ROSATO A,et al.Regulation of Brucella,virulence by the two-component system BvrR/BvrS[J].Veterinary Microbio-logy,2002,90(1):329-339.
[6] 张沾,陈创夫,张辉,等.布鲁氏菌Ⅳ型分泌系统分泌子VceC功能的初步研究[J].中国预防兽医学报,2011,33(9):685-688. ZHANG Z,CHEN C F,ZHANG H,et al.Preliminary study on the function of Brucella type Ⅳ secretion system (T4SS) effector VceC[J].Chinese Journal of Preventive Veterinary Medicine,2011,33(9):685-688.(in Chinese)
[7] MYENI S,CHILD R,NG T W,et al.Brucella modulates secretory trafficking via multiple type Ⅳ secretion effector proteins[J].PLoS Pathogens,2013,9(8):e1003556.
[8] LACERDA T L,SALCEDO S P,GORVEL J P.Brucella T4SS:The VIP pass inside host cells[J].Current Opinion in Microbiology,2013,16(1):45-51.
[9] LLOSA M,ROV C,DEHIO C.Bacterial type IV secretion systems in human disease[J].Molecular Microbiology,2009,73:141-15.
[10] KE Y H,WANG Y F,LI W F,et al.Type Ⅳ secretion system of Brucella spp.and its effectors[J].Frontiers in Cellular and Infection Microbiology,2015,5:72.
[11] MAARTEN F,SUN Y H,ANDREAS B,et al.Identification of VceA and VceC,two members of the VjbR regulon that are translocated into macrophages by the Brucella type Ⅳ secretion system[J].Molecular Microbiology,2008,70,1378-1396.
[12] DE JONG M F,STARR T,WINTER M G,et al.Sensing of bacterial type Ⅳ secretion via the unfolded protein response[J].mBio,2013,4(1):e00418-00412.
[13] ALAIDAROUS M,VE T,CASEY L W,et al.Mechanism of bacterial interference with TLR4 signaling by Brucella Toll/interleukin-1 receptor domain-containing protein TcpB[J].Journal of Biological Chemistry,2014,289(2):654-668.
[14] JAKKA P,NAMANI S,MURUGAN S,et al.The Brucella effector protein TcpB induces degradation of inflammatory caspases and thereby subverts non-canonical inflammasome activation in macrophages[J].Journal of Biological Chemistry,2017,292(50):20613-20627.
[15] DE BARSY M,JAMET A,FILOPON D,et al.Identification of a Brucella spp.secreted effector specifically interacting with human small GTPase Rab2[J].Cellular Microbiology,2011,13(7):1044-1058.
[16] MARCHESINI M I,HERRMANN C K,SALCEDO S P,et al.In search of Brucella abortus type Ⅳ secretion substrates:Screening and identification of four proteins translocated into host cells through VirB system[J].Cellular Microbiology,2011,13(8):1261-1274.
[17] 李瑾,肖婷,孙慧,等.猪囊尾蚴排泄分泌抗原Ts8B3基因的原核表达、纯化以及免疫反应性分析[J].中国病原生物学杂志,2018,13(5):480-483. LI J,XIAO T,SUN H,et al.Prokaryotic expression,purification,and immunological characterization of the ES antigen gene Ts8B3 of cysticercus cellulosae[J].Chinese Journal Etiology,2018,13(5):480-483.(in Chinese)
[18] 成璐,张冬星,吴娟,等.布鲁菌L7/L12蛋白的原核表达及多克隆抗体的制备[J].动物医学进展,2018,39(12):10-14. CHENG L,ZHANG D X,WU J,et al.Prokaryotic expression polyclonal antibody preparation of L7/L12 protein of Brucella suis S2 strain[J].Progress in Veterinary Medicine,2018,39(12):10-14.(in Chinese)
[19] 康彪,高闪电,独军政,等.牛病毒性腹泻病毒E1蛋白的原核表达及多克隆抗体的制备[J].动物医学进展,2018,39(11):34-38. KANG B,GAO S D,DU J Z,et al.Prokaryotic expression and poilclonal antibody preparation of E1 protein of bovine viral diarrhea virus[J].Progress in Veterinary Medicine,2018,39(11):34-38.(in Chinese)
[20] 马忠臣,胡尊楠,王震,等.布鲁氏菌分泌蛋白BspJ多克隆抗体的制备[J].中国动物传染病学报,2020,28(3):61-67. MA Z C,HU Z N,WANG Z,et al.Preparation of polyclonal antibody against brucellosis protein BspJ[J].Chinese Journal of Zoonological Infectious Diseases,2020,28(3):61-67.(in Chinese)
[21] 马忠臣,胡尊楠,张超,等.布鲁氏菌分泌蛋白BspG的亚细胞定位分析[J].中国动物传染病学报,2020,2:1-14. MA Z C,HU Z N,ZHANG C,et al.Subcellular localization analysis of Brucella secreted protein BspG[J].Chinese Journal of Zoonological Infectious Diseases,2020,2:1-14.(in Chinese)

布鲁氏菌分泌蛋白BspD多克隆抗体制备及其生物信息学分析

Polyclonal Antibody Preparation and Bioinformatics Analysis of Brucella Secreted Protein BspD

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]	刘佳隆, 张译文, 张悦然, 孙奇娟, 陈建, 何雷, 贾艳艳, 丁轲, 余祖华. 鸡ELAVL1基因克隆、原核表达及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(5): 1807-1817.
[4]	卓娜, 朱忠武, 李静, 胡世享, 王建昌, 林华, 陈朝林, 韩佃刚, 艾军, 陈培富. 猪源戊型肝炎病毒ORF2蛋白生物信息学分析及真核表达研究[J]. 中国畜牧兽医, 2023, 50(5): 1959-1970.
[5]	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.
[6]	范冰峰, 李文, 刘理想, 赵向远, 邵静, 林乐丰, 孙慧敏, 张旭林, 许保增. 水貂AANAT基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(4): 1307-1318.
[7]	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.
[8]	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.
[9]	贾新月, 马静, 张艳艳, 马勋, 王正荣, 薄新文. 细粒棘球绦虫EGR-07243和EGR-07244基因生物信息学分析及原核表达[J]. 中国畜牧兽医, 2023, 50(3): 847-858.
[10]	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.
[11]	韦义荣, 郑自华, 张叁保, 宋颖, 蒋海玉, 孙雯玥, 程鹏飞, 刘雨帆, 邹剑伟, 黄艳娜, 潘艳, 蒋钦杨. 努比亚山羊UCP1基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2023, 50(2): 440-450.
[12]	甘露, 郑会珍, 诺明达来, 刘燕, 金敏, 何文文, 温丽翠, 李永畅, 巴音查汗·盖力克. 伊氏锥虫伊犁株扩繁及其PFR基因克隆表达和生物信息学分析[J]. 中国畜牧兽医, 2023, 50(2): 469-478.
[13]	魏春燕, 郭嘉, 朱德馨, 张伟, 朱嘉乐, 邓兴梅, 贾思锋, 刘良波, 张辉. 布鲁氏菌BPE159基因缺失株的构建及BPE159蛋白对细胞自噬因子表达的影响[J]. 中国畜牧兽医, 2023, 50(1): 26-36.
[14]	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.
[15]	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.