布鲁氏菌分泌蛋白BspI生物信息学分析及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2022.08.027

摘要/Abstract

摘要： 【目的】试验旨在对布鲁氏菌分泌蛋白BspI进行生物信息学分析,构建原核表达载体,获得BspI蛋白并制备其多克隆抗体,为后续研究该蛋白的生物学功能提供材料。【方法】使用在线软件对BspI蛋白的氨基酸序列进行生物信息学分析,参照布鲁氏菌16M株BspI基因序列(GenBank登录号:DK63_1233)设计引物,PCR扩增目的基因后连接至pMD19-T克隆载体并筛选阳性克隆,将目的基因连接到pET-28a表达载体上,转化大肠杆菌BL21(DE3)感受态细胞,加入IPTG诱导蛋白表达,并进行SDS-PAGE分析,用镍柱亲和层析方法纯化蛋白,纯化后的蛋白与弗氏佐剂混合免疫试验兔,采血分离血清,通过Western blotting、间接ELISA法进行多克隆抗体特异性及抗体效价分析。【结果】BspI蛋白为不稳定亲水性蛋白,存在跨膜结构,无信号肽区域,有13个磷酸化位点和7个抗原决定簇,二级结构主要由α-螺旋、延伸链和无规则卷曲组成。PCR成功扩增出675 bp的BspI基因,成功构建了pET-28a-BspI表达载体。SDS-PAGE和Western blotting分析结果表明,成功表达出25.3 ku的蛋白,纯化后无明显杂带,制备的多克隆抗体能够特异性地与BspI蛋白结合。间接ELISA结果显示,BspI蛋白多克隆抗体效价为1∶409 600。【结论】制备的兔源BspI蛋白多克隆抗体可以特异性识别BspI蛋白,该蛋白具有较好的反应原性。试验结果为进一步研究BspI蛋白在布鲁氏菌的胞内寄生中发挥的作用提供了参考。

关键词: 布鲁氏菌; 生物信息分析; 分泌蛋白BspI; 多克隆抗体

Abstract: 【Objective】 The purpose of this study was to analyze the Brucella secreted protein BspI by bioinformatics,construct prokaryotic expression vector,and obtain BspI protein and prepare its polyclonal antibody,so as to provide materials for the follow-up study of the biological function of BspI protein.【Method】 Bioinformatics analysis of the amino acid sequence of BspI protein was performed using online software,referring to the BspI gene sequence (GenBank accession No.:DK63_1233) of Brucella 16M strain,after PCR amplification,BspI gene was ligated into pMD19-T cloning vector and screened for positive clones.The target gene was ligated into pET-28a expression vector,transformed into Escherichia coli BL21(DE3) competent cells,induced protein expression by IPTG,and analyzed by SDS-PAGE.The protein was purified by nickel column affinity chromatography, the purified protein was mixed with Freund's adjuvant to immunize rabbits,and blood was collected to isolate serum for analysis of polyclonal antibody specificity and antibody titer by Western blotting and indirect ELISA,respectively.【Result】 BspI protein was unstable hydrophilic protein,presented a transmembrane structure with no signal peptide region,had 13 phosphorylation sites and 7 antigenic determinants,and the secondary structure of mainly contained alpha helix,extended chain,and random coil.The 675 bp of BspI target gene was successfully amplified by PCR,and the pET-28a-BspI expression vector was successfully constructed.SDS-PAGE and Western blotting analysis results showed that the 25.3 ku protein was successfully expressed with no obvious heterobands after purification,and the prepared polyclonal antibody was able to specifically bind to BspI protein.The indirect ELISA results represented the polyclonal antibody titer of BspI protein was 1:409 600.【Conclusion】 The prepared polyclonal antibody against BspI protein of rabbit origin could specifically recognize BspI protein,and the protein was relatively reactogenic,which provided a reference for further research on the role played by BspI protein in the endoparasitism of Brucella.

Key words: Brucella; bioinformatic analysis; secreted protein BspI; polyclonal antibody

中图分类号:

S852.61⁺4

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.

XIAO Yangyang, MA Zhongchen, LI Ruirui, CHEN Chuangfu, ZHENG Wei, WANG Yong, WANG Pengyan. Bioinformatic Analysis and Polyclonal Antibody Preparation of Brucella Secreted Protein BspI[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3112-3121.

参考文献

[1] ZHOU K,WU B B,PAN H,et al.One health approach to address zoonotic brucellosis:A spatiotemporal associations study between animals and humans[J].Frontiers in Veterinary Science,2020,7:521.
[2] 李芮芮,马忠臣,张弘扬,等.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 R R,MA Z C,ZHANG H Y,et al.Preparation and bioinformatics analysis of polyclonal antibody against Brucella secretory protein BspD[J].China Animal Husbandry&Veterinary Medicine,2021,48(2):676-684.(in Chinese)
[3] ZHENG R J,XIE S S,LU X B,et al.A systematic review and meta-analysis of epidemiology and clinical manifestations of human brucellosis in China[J].Biomed Research International,2018,2018:5712920.
[4] 杨江流,乔军伟,宋青山,等.布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析[J].中国畜牧兽医,2021,48(8):2746-2753. YANG J L,QIAO J W,SONG Q S,et al.Prokaryotic expression and bioinformatics analysis of virulence gene BvfA of Brucella S19[J].China Animal Husbandry&Veterinary Medicine,2021,48(8):2746-2753.(in Chinese)
[5] 孙天浩,马忠臣,张欢,等.家畜布鲁氏菌病相关疫苗的研究进展[J].现代畜牧兽医,2021,7:80-86. SUN T H,MA Z C,ZHANG H,et al.Progress of research on brucellosis vaccine for livestock[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2021,7:80-86.(in Chinese)
[6] COMERCI D J,MARTINEZ-LORENZO M J,SIEIRA R,et al.Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole[J].Cellular Microbiology,2001,3(3):159-168.
[7] CELLI J,DE C C,FRANCHINI D,et al.Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum[J].Joural of Experimental Medicine,2003,198(4):545-556.
[8] GALINSKA E M,ZAGORSKI J.Brucellosis in humans——Etiology,diagnostics,clinical forms[J].Annals of Agricultural&Environmental Medicine,2013,20(2):233-238.
[9] SEDZICKI J,TSHON T,LOW S H,et al.3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum[J].Journal of Cell Science,2018,131(4):jcs210799.
[10] MARTIROSYAN A,MORENO E,GORVEL J.An evolutionary strategy for a stealthy intracellular Brucella pathogen[J].Immunological Reviews,2011,240(1):211-234.
[11] QIN Q M,PEI J W,ANCONA V,et al.RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication[J].PLoS Pathogens,2008,4(7):e1000110.
[12] CELLI J,TSOLIS R M.Bacteria,the endoplasmic reticulum and the unfolded protein response:Friends or foes?[J].Nature Reviews Microbiology,2015,13(2):71-82.
[13] SALCEDO S P,MARCHESINI M I,DEGOS C,et al.BtpB,a novel Brucella TIR-containing effector protein with immune modulatory functions[J].Frontiers in Cellular&Infection Microbiology,2013,3:28.
[14] DOHMER O H,VALGUARNERA E,CZIBENER C,et al.Identification of a type Ⅳ secretion substrate of Brucella abortus that participates in the early stages of intracellular survival[J].Cellular Microbiology,2014,16(3):396-410.
[15] 任晶晶.布鲁氏菌Ⅳ型分泌系统非依赖型效应蛋白BspI诱导巨噬细胞内质网应激的研究[D].长春:吉林大学,2021. REN J J.Brucella type Ⅳ secretory system independent effector BspI induces ER stress in macrophages[D].Changchun:Jilin University,2021.(in Chinese)
[16] MARTIROSYAN A,PEREZ-GUTIERREZ C,BANCHEREAU R,et al.Brucella beta 1,2 cyclic glucan is an activator of human and mouse dendritic cells[J].PLoS Pathogens,2012,8(11):e1002983.
[17] 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.
[18] MA Z C,YU S F,CHENG K J,et al.Brucella abortus nucleomodul in BspJ as an effector promotes the colonization of in the host[J].Journal of Vetrinary Sciense,2022,23(1):e8.
[19] 焉鑫,李干武,胡森,等.布鲁氏菌分泌系统及其底物蛋白的研究进展[J].中国预防兽医学报,2021,43(9):1019-1024. YAN X,LI G W,HU S,et al.Research progress of Brucella secretory system and its substrate protein[J].Chinese Journal of Preventive Veterinary Medicine,2021,43(9):1019-1024.(in Chinese)
[20] 马忠臣,胡瑞瑞,李芮芮,等.布鲁氏菌的胞内生存机制研究进展[J].动物医学进展,2021,42(6):71-78. MA Z C,HU R R,LI R R,et al.Research progress on intracellular survival mechanism of Brucella[J].Progress in Veterinary Medicine,2021,42(6):71-78.(in Chinese)
[21] 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.
[22] 任晶晶,李晨,彭其胜.布鲁菌Ⅳ型分泌系统非依赖型效应蛋白BspI诱导巨噬细胞内质网应激[J].中国兽医学报,2021,41(5):950-956. REN J J,LI C,PENG Q S.Brucella type Ⅳ secretory system independent effector BspI induces ER stress in macrophages[J].Chinese Journal of Veterinary Medicine,2021,41(5):950-956.(in Chinese)
[23] 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.
[24] 马忠臣,胡尊楠,杨亚军,等.布鲁氏菌分泌蛋白BspJ亚细胞定位分析[J].生物技术,2018,28(6):572-578. MA Z C,HU Z N,YANG Y J,et al.Subcellular localization analysis of Brucella secreted protein BspJ[J].Biotechnology,2018,28(6):572-578.(in Chinese)
[25] ASCHTGEN M,LYNCH J B,KOCH E,et al.Rotation of Vibrio fischeri flagella produces outer membrane vesicles that induce host development[J].Journal of Bacteriology,2016,198(16):2156-2165.
[26] 路殿英,赵鸿雁,朴东日,等.布鲁氏菌鞭毛研究进展[J].中国人兽共患病学报,2019,35(1):66-69. LU D Y,ZHAO H Y,PIAO D R,et al.Research progress of Brucellosis flagella[J].Chinese Journal of Zoonoses,2019,35(1):66-69.(in Chinese)