布鲁氏菌热休克蛋白GroEL基因缺失及过表达菌株的构建及生长特性鉴定

doi:10.16431/j.cnki.1671-7236.2025.12.030

摘要/Abstract

摘要： 【目的】探究热休克蛋白GroEL对布鲁氏菌体外生长的影响及其在巨噬细胞定位情况，为研究GroEL基因的生物学功能奠定基础。【方法】采用同源重组及SacB反向筛选技术，以牛种布鲁氏菌(Brucella abortus)A19株为研究对象，构建牛种布鲁氏菌A19株GroEL基因缺失株(A19ΔGroEL)和过表达株(A19-HA/GroEL)；绘制GroEL基因缺失菌株及过表达菌株的体外生长曲线，检测GroEL对布鲁氏菌体外生长的影响，采用间接免疫荧光试验检测GroEL在RAW264.7巨噬细胞中的定位，并通过免疫电镜观察GroEL在布鲁氏菌中定位。【结果】PCR结果显示，扩增出了大小为1 520 bp的特异性条带，表明GroEL基因缺失株A19ΔGroEL构建成功。Western blotting结果显示，获得了大小为60.2 ku的目的条带，表明过表达株A19-HA/GroEL构建成功。细菌生长曲线结果显示，牛种布鲁氏菌A19、A19ΔGroEL和A19-HA/GroEL的体外生长趋势无显著差异(P＞0.05)。间接免疫荧光试验结果显示，GroEL主要定位于RAW264.7巨噬细胞的胞质中。免疫电镜观察结果显示，GroEL主要定位于布鲁氏菌的外膜和内膜。【结论】本研究成功构建了布鲁氏菌GroEL基因缺失株和过表达株，揭示了GroEL基因的缺失不影响布鲁氏菌的体外生长，且GroEL主要定位于RAW264.7巨噬细胞的细胞质和布鲁氏菌的外膜和内膜，为深入探究GroEL的生物学功能提供了理论基础和数据支持。

关键词: 布鲁氏菌; 热休克蛋白; GroEL基因; 缺失株; 过表达株

Abstract: 【Objective】 This study was conducted to investigate the effect of heat shock protein GroEL on the growth of Brucella in vitro and its cellular localization in macrophages. 【Method】 In this study,with Brucella abortus strain A19 as the research object,homologous recombination and SacB reverse screening technology were used to construct GroEL gene deletion strain (A19ΔGroEL) and overexpression strain (A19-HA/GroEL) of Brucella abortus strain A19.The in vitro growth curves of GroEL gene deletion and overexpression strains were plotted to detect the effect of GroEL on the in vitro growth of Brucella.Indirect immunofluorescence assay was used to detect the localization of GroEL in RAW264.7 macrophages.And the localization of GroEL in Brucella was observed by immunoelectron microscopy. 【Result】 PCR results showed that a specific band with a size of 1 520 bp was amplified,indicating that the GroEL gene deletion strain A19ΔGroEL was successfully constructed.Western blotting results showed that the target band with size of 60.2 ku was obtained,indicating that overexpression strain A19-HA/GroEL was successfully constructed.The results of the bacterial growth curve showed that there was no significant difference in the in vitro growth trends among Brucella abortus A19,A19ΔGroEL and A19-HA/GroEL (P＞0.05).The results of indirect immunofluorescence assay showed that the GroEL was mainly localized in the cytoplasm of RAW264.7 macrophages.The results of immunoelectron microscopy observation showed that GroEL was mainly located in the outer and inner membranes of Brucella. 【Conclusion】 The results showed that the GroEL gene deletion and overexpression strains of Brucella abortus were successfully constructed in this study,which initially revealed that the deletion of GroEL gene did not affect the growth of Brucella in vitro,and GroEL was mainly localized in the cytoplasm of RAW264.7 macrophages and the outer and inner membranes of Brucella,which provided the theoretical basis and data support for the in-depth investigation of the biological function of GroEL.

Key words: Brucella; heat shock protein; GroEL gene; deletion strain; overexpression strain

中图分类号:

S852.61

张瑜瑜, 张粟子, 李娜, 牟雪梅, 支飞杰, 冉多良, 储岳峰, 李斌, 许健. 布鲁氏菌热休克蛋白GroEL基因缺失及过表达菌株的构建及生长特性鉴定[J]. 中国畜牧兽医, 2025, 52(12): 5859-5868.

ZHANG Yuyu, ZHANG Suzi, LI Na, MOU Xuemei, ZHI Feijie, RAN Duoliang, CHU Yuefeng, LI Bin, XU Jian. Construction and Identification of Growth Characteristics of Brucella Heat Shock Protein GroEL Gene Deletion and Overexpression Strains[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5859-5868.

参考文献

[1] MORIYÓN I,BLASCO J M,LETESSON J J,et al.Brucellosis and one health:Inherited and future challenges[J].Microorganisms,2023,11(8):2070.
[2] MATLE I,LEDWABA B,MADIBA K,et al.Characterisation of Brucella species and biovars in South Africa between 2008 and 2018 using laboratory diagnostic data[J].Veterinary Medicine and Science,2021,7(4):1245-1253.
[3] JIA W,CHEN S,CHI S,et al.Recent progress on tick-borne animal diseases of veterinary and public health significance in China[J].Viruses,2022,14(2):355.
[4] LAINE C G,JOHNSON V E,SCOTT H M,et al.Global estimate of human brucellosis incidence[J].Emerging Infectious Diseases,2023,29(9):1789-1797.
[5] DE FIGUEIREDO P,FICHT T A,RICE-FICHT A,et al.Pathogenesis and immunobiology of brucellosis:Review of Brucella-host interactions[J].The American Journal of Pathology,2015,185(6):1505-1517.
[6] KHAIRULLAH A R,KURNIAWAN S C,PUSPITASARI Y,et al.Brucellosis:Unveiling the complexities of a pervasive zoonotic disease and its global impacts[J]. Open Veterinary Journal,2024,14(5):1081-1097.
[7] TULU D.Bovine brucellosis:Epidemiology,public health implications,and status of brucellosis in Ethiopia[J].Veterinary Medicine (Auckland,N.Z.),2022,13:21-30.
[8] ZHAI Y,WANG H,SUN K,et al.Enhancing host defense against Brucella:The immune effect exerted by anti-OMP16 monoclonal antibody[J].International Immunopharmacology, 2025,148:114142.
[9] SELEEM M N,BOYLE S M,SRIRANGANATHAN N.Brucella:A pathogen without classic virulence genes[J].Veterinary Microbiology, 2008,129(1-2):1-14.
[10] GORVEL J P,MORENO E.Brucella intracellular life:From invasion to intracellular replication[J].Veterinary Microbiology,2002,90(1-4):281-297.
[11] CELLI J.Surviving inside a macrophage:The many ways of Brucella[J].Research in Microbiology,2006,157(2):93-98.
[12] HE J,YIN S,DENG X,et al.The effector protein BspE affects Brucella survival by regulating the inflammatory response and apoptosis[J].International Immunopharmacology,2025,144:113576.
[13] 杜强.牛布鲁氏菌抗原蛋白的克隆表达及其刺激能力的评价[D].扬州：扬州大学,2015.
DU Q.Cloning and expression of antigen protein of Brucella abortus and evaluation of its stimulating ability[D].Yangzhou:Yangzhou University,2015.(in Chinese)
[14] MINHAS P,SUNIL K B,VERMA R.Evaluation of immuno-modulating effect of recombinant heat shock protein 40 of Brucella abortus in mice[J].3 Biotech,2019,9(10):366.
[15] ABBASSI-DALOII T,YOUSEFI S,SEKHAVATI M H,et al.Impact of heat shock protein 60KD in combination with outer membrane proteins on immune response against Brucella melitensis[J].Acta Pathologica,Microbiologica,et Immunologica Scandinavica,2018,126(1):65-75.
[16] 徐正中,杜强,孟闯,等.布鲁菌抗原蛋白的原核表达、纯化与鉴定[J].扬州大学学报(农业与生命科学版),2017,38(2):1-4.
XU Z Z,DU Q,MENG C,et al.Prokaryotic expression,purification and identification of Brucella antigen protein[J].Journal of Yangzhou University (Agriculture and Life Sciences),2017,38(2):1-4.(in Chinese)
[17] WASHBURN A,ABDEEN S,OVECHKINA Y,et al.Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors:A polypharmacology strategy for treating Mycobacterium tuberculosis infections[J].Bioorganic & Medicinal Chemistry Letters,2019,29(13):1665-1672.
[18] GARDUÑO R A,FAULKNER G,TREVORS M A,et al.Immunolocalization of Hsp60 in Legionella pneumophila[J].Journal of Bacteriology,1998,180(3):505-513.
[19] TIAN M,BAO Y,LI P,et al.The putative amino acid ABC transporter substrate-binding protein AapJ2 is necessary for Brucella virulence at the early stage of infection in a mouse model[J].Veterinary Research,2018,49(1):32.
[20] SEKHAVATI M H,HERAVI R M,TAHMOORESPUR M,et al.Cloning,molecular analysis and epitopics prediction of a new chaperone GroEL Brucella melitensis antigen[J].Iranian Journal of Basic Medical Sciences,2015,18(5):499-505.
[21] FOURIE K R,WILSON H L.Understanding GroEL and DnaK stress response proteins as antigens for bacterial diseases[J].Vaccines (Basel),2020,8(4)：773.
[22] HOFFMAN P S,GARDUNO R A.Surface-associated heat shock proteins of Legionella pneumophila and Helicobacter pylori:Roles in pathogenesis and immunity[J].Infectious Diseases in Obstetrics and Gynecology,1999,7(1-2):58-63.
[23] 刘辉.新疆绵羊种布鲁氏菌HtrA基因和GroEL基因的克隆及原核表达[D].石河子：石河子大学,2006.
LIU H.Cloning and prokaryotic expression of HtrA gene and GroEL gene of Brucella melitensis in Xinjiang[D].Shihezi:Shihezi University,2006.(in Chinese)
[24] 易新萍,叶锋,姚刚,等.牛布鲁氏菌A19突变株的构建及在BALB/c鼠中的免疫保护评估[J].微生物学报,2013,53(11):1213-1220.
YI X P,YE F,YAO G,et al.Construction of Brucella abortus A19-delta VirB12 mutant and evaluation of its protective efficacy against 2308 strain challenge in BALB/c mice[J].Acta Microbiologica Sinica, 2013,53(11):1213-1220.(in Chinese)
[25] 潘文,王佳莹,赵明秋,等.布鲁氏菌bp26基因缺失株的构建[J].中国兽医科学,2011,41(3):280-286.
PAN W,WANG J Y,ZHAO M Q,et al.Construction of Brucella bp26 gene deletion strain[J].Chinese Veterinary Science,2011,41(3):280-286.(in Chinese)
[26] 承潇潇,任文浩,郑炜,等.布鲁氏菌T4SS效应蛋白BspJ的生物学功能研究[J].中国预防兽医学报,2024,46(9):895-901.
CHENG X X,REN W H,ZHENG W,et al.Study on the biological function of Brucella T4SS effector protein BspJ[J].Chinese Journal of Preventive Veterinary Medicine, 2024,46(9):895-901.(in Chinese)
[27] 徐朕宇,邓肖玉,王月丽,等.牛种布鲁氏菌A19ΔBtpA缺失株生物学特性及其免疫原性研究[J].畜牧兽医学报,2024,55(5):2135-2145.
XU Z Y,DENG X Y,WANG Y L,et al.Biological characteristics and immunogenicity of Brucella abortus A19ΔBtpA deletion strain[J].Acta Veterinaria et Zootechnica Sinica,2024,55(5):2135-2145.(in Chinese)
[28] 宋甲宝,许达,姜利英,等.马耳他布鲁氏菌OmpR基因缺失株的构建及其对布鲁氏菌生长与复制的影响[J].中国预防兽医学报,2018,40(12):1105-1110.
SONG J B,XU D,JIANG L Y,et al.Construction of Brucella melitensis OmpR gene deletion strain and its effect on the growth and replication of Brucella[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(12):1105-1110.(in Chinese)
[29] URBAN-CHMIEL R,DEC M,PUCHALSKI A,et al.Characterization of heat-shock proteins in Escherichia coli strains under thermal stress in vitro[J].Journal of Medical Microbiology,2013,62(Pt 12):1897-1901.
[30] GOULHEN F,HAFEZI A,UITTO V J,et al.Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans[J].Infection and Immunity,1998,66(11):5307-5313.