布鲁氏菌Omp19基因的生物信息学分析、克隆表达和ELISA方法的初步建立

doi:10.16431/j.cnki.1671-7236.2020.10.005

Abstract

Abstract: This study was aimed to further investigate the structure and function of Brucella outer membrane protein 19 (OMP19),obtain the recombinant protein of OMP19 with reactivity,and establish the new method for diagnosing brucellosis based on indirect ELISA.The amino acid sequence of OMP19 protein was analyzed by bioinformatics software,Omp19 gene was amplified by PCR,and the recombinant expression vector pET-32a-Omp19 was constructed by seamless cloning method,it was transformed into E.coli BL21 (DE3) competent cells,induced and purified OMP19 protein.The immunogenicity of OMP19 protein was detected by Western blotting and ELISA,respectively,to establish an indirect ELISA method based on the protein.The results showed that there were 19 signal peptide sequences in the N terminal of OMP19 protein,and the secondary structure was random coil,and OMP19 protein contained dominant antigenic epitopes.The prokaryotic expression vector pET-32a-Omp19 was successfully constructed by PCR and seamless cloning method,the OMP19 fusion protein was successfully induced,expressed and purified,the size of protein was about 35 ku,which was consistent with the expect size.The reactivity of OMP19 was identified by Western blotting and ELISA,the results showed that the fusion protein had good reactivity,and the coating concentration was 10 μg/mL.The dilution ratio of the first antibody was 1：50 and the second antibody dilution ratio was 1：8 000,the P/N value reach the highest at 2.80.The results laid a foundation for the establishment of rapid diagnosis of brucellosis and the development of new vaccine.

Key words: Brucella; Omp19 gene; bioinformatics analysis; reactivity

CLC Number:

YANG Ningning, WANG Zhen, YANG Yajun, ZHANG Qian, SONG Shengnan, ZHANG Huan, CHEN Chuangfu, LIU Mingjun. Bioinformatics Analysis,Cloning and Expression of Omp19 Gene of Brucella and Preliminary Establishment of ELISA Method[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3078-3087.

References 25

[1]	徐晓阳.布鲁氏菌胞内生存相关sRNA的筛选及非编码小RNA BSR0441功能的初步研究[D].雅安:四川农业大学,2016. XU X Y.Screening of sRNAs related to Brucella intracellular and function study of BSR0441[D].Ya'an:Sichuan Agricultural University,2016.(in Chinese)
[2]	薛月鹏,刘亚军,张东,等.动物布鲁氏菌病及其防控策略[J].中国畜牧兽医文摘,2015,4:82. XUE Y P,LIU Y J,ZHANG D,et al.Brucellosis in animals and its control strategy[J] Chinese Animal Husbandry and Veterinary Abstracts,2015,4:82.(in Chinese)
[3]	程建国,张富荣,李小东,等.动物布鲁氏菌病实验室诊断与检测技术进展[J].兽医导刊,2014,8:26-28. CHENG J G,ZHANG F R,LI X D,et al.Progress in laboratory diagnosis and detection of brucellosis in animals[J].Veterinary Orientation,2014,8:26-28.(in Chinese)
[4]	VALGUARNERA E,SPERA J M,CZIBENER C,et al.RomA,a periplasmic protein involved in the synthesis of the lipopolysaccharide tunes down the inflammatory response triggered by Brucella[J].Journal of Infectious Diseases,2018,217(8):1257-1266.
[5]	VERDIGUEL-FERNÁNDEZ L,OROPEZA-NAVARRO R,BASURTO-ALCÁNTRARA F J,et al.Omp31 plays an important role on outer membrane properties and intracellular survival of Brucella melitensis in murine macrophages and HeLa cells[J].Archives of Microbiology,2017,199(7):971-978.
[6]	罗小毛.布鲁氏菌外膜蛋白Omp25对巨噬细胞产生TNF-α的调控作用及机制研究[D].杨凌:西北农林科技大学,2018. LUO X M.The effects and regulatory mechanism of Brucella outer membrane protein Omp25 on TNF-α production in macrophages[D].Yangling:Northwest A&F University,2018.(in Chinese)
[7]	CLOECKAERT A,VIZCAINO N,PAQUET J Y,et al.Major outer membrane proteins of Brucella spp.past,present and future[J].Veterinary Microbiology,2002,90(1):229-247.
[8]	DING X Z,BHATTACHARJEE A,NIKOLICH M P,et al.Cloning,expression,and purification of Brucella suis outer membrane proteins[J].Protein Expression & Purification,2005,40(1):134-141.
[9]	CASSATARO J,ESTEIN S M,PASQUEVICH K A,et al.Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+ T helper 1 response that protects against Brucella melitensis infection[J].Infection and Immunity,2005,73:8079-8088.
[10]	VISHNU U S,SANKARASUBRAMANIAN J,GUNASEKARAN P,et al.Identification of potential antigens from non-classically secreted proteins and designing novel multitope peptide vaccine candidate against,Brucella melitensis,through reverse vaccinology and immunoinformatics approach[J].Infection,Genetics and Evolution,2017,55:151-158.
[11]	ALVES D S F J,MARTINS V D P,CAMPOS P C,et al.Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice[J].Infection and Immunity,2015,83(4):1458-1464.
[12]	VATANKHAH M,BEHESHTI N,MIRKALANTARI S,et al.Recombinant Omp2b antigen-based ELISA is an efficient tool for specific serodiagnosis of animal brucellosis[J].Brazilian Journal of Microbiology,2019,50(4):979-984.
[13]	KOYUNCU I,KOCYIGIT A,OZER A,et al.Brucella melitensis diagnostic potential of Rev1 native Omp28 precursor in human brucellosis[J].Central European Journal Immunology,2018,43(1):81-89.
[14]	马长胜.布鲁氏菌三个外膜脂蛋白的原核表达、纯化及免疫原性实验[D].泰安:山东农业大学,2012. MA C S.Prokaryotic expression,purification of three lipoproteins of Brucella and immunogenicity of the expressed products[D].Tai'an:Shandong Agricultural University,2012.(in Chinese)
[15]	印双红,张俊波,王浩,等.布鲁氏菌Omp19基因真核载体的构建及对细胞免疫的影响[J].中国病原生物学杂志,2016,11(2):122-125. YIN S H,ZHANG J B,WANG H,et al.Construc-tion of a eukaryotic expression vector to express the Omp19 gene of the 16M strain of Brucella melitensis[J].Journal of Pathogen Biology,2016,11(2):122-125.(in Chinese)
[16]	孙丽媛.吉林省布鲁氏菌流行株种属鉴定及其耐药分子机制的研究[D].长春:吉林大学,2011. SUN L Y.Identification of Brucella isolates and the molecular resistance mechanism from Jilin regions[D].Changchun:Jilin University,2011.(in Chinese)
[17]	YANG X,SKYBERG J A,CAO L,et al.Progress in Brucella vaccine development[J].Frontiers in Biology,2013,8(1):60-77.
[18]	IM Y B,SHIM S,SOH S H,et al.Cytokines production and Toll-like receptors expression in human leukemic monocyte cells,THP-1,stimulated with Brucella abortus cellular antigens[J].Microbial Pathogenesis,2018,122:7-12.
[19]	王羽.布鲁氏菌疫苗的研究进展[J].兽医导刊,2010,7:44-45. WANG Y.Research progress of Brucella vaccine[J].Veterinary Orientation,2010,7:44-45.(in Chinese)
[20]	PASQUEVICH K A,ESTEIN S S C.Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus infection[J].Infection & Immunity,2009,77(1):436-445.
[21]	王娜.布鲁氏菌Omp19抗原性研究及内蒙古人间布鲁氏菌病横断面调查[D].呼和浩特:内蒙古农业大学,2014. WANG N.The research on Omp19 antigenicity of Brucella and cross-sectional survey of human brucellosis in Inner Mongolia[D].Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)
[22]	LI Z W,ZHANG F B,ZHANG C T,et al.Immunoinformatics prediction of OMP2b and BCSP31 for designing multi-epitope vaccine against Brucella[J].Molecular Immunology,2019,114:651-660.
[23]	SHOJAEI M,TAHMOORESPUR M,SOLTANI M,et al.Brucella melitensis immunogenicity evaluation of plasmids encoding Omp25 and Omp31 antigens in BALB/c mice[J].Iranian Journal of Basic Medical Sciences,2018,21(9):957-964.
[24]	HOU H H,LIU X F,PENG Q S.The advances in brucellosis vaccines[J].Vaccine,2019,37(30):3981-3988.
[25]	MENZIES P I.Vaccination programs for reproductive disorders of small ruminants[J].Animal Reproduction Sciences,2012,130(3-4):162-172.

Bioinformatics Analysis,Cloning and Expression of Omp19 Gene of Brucella and Preliminary Establishment of ELISA Method

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