布鲁氏菌P39基因的克隆、原核表达及其糖基化修饰

doi:10.16431/j.cnki.1671-7236.2018.11.003

Abstract

Abstract:

In order to investigate the effect of glycosylation modification on the immunogenicity of Brucella P39 protein,the expression and purification of Brucella P39 protein were carried out in this experiment,and the expressed protein was modified with mannose hexasaccharide.The primers were designed according to the Brucella P39 gene sequence published in GenBank,and the P39 gene fragment was cloned from the Brucella 16M genome and ligated into pMD19-T vector to transform E.coli DH5α competent cells.The positive strain plasmid was extracted and identified by enzyme digestion.After correct identification,the recombinant plasmid pGEX-6P-1-P39 was constructed,and the induction expression and optimization of recombinant protein were analyzed.The target protein induced was analyzed by SDS-PAGE and Western blotting,and the correct protein was purified by GST affinity chromatography.The purified P39 protein was modified by mannose hexasaccharide using EDC/NHS method to study the effect of the target protein on the phagocytosis of macrophages after modification of mannose hexose.The results showed that the target gene with a fragment size of 1 206 bp was successfully cloned,and the prokaryotic expression vector pGEX-6P-1-P39 was constructed.The P39 protein was successfully expressed in E.coli,and the protein was mainly soluble.Western blotting results showed a specific band at approximately 65 ku.After purification,the target protein size was 43 ku,the target protein P39 was successfully glycosylated,and the molar ratio of the modified product to the protein was 2.3:1.In addition,glycosylation modification could significantly advance the phagocytosis time of mouse macrophages by protein activation.This study could provide a reference for research the mechanism of mannose hexose modification affecting the immunogenicity of P39 protein.

Key words: Brucella; P39 gene; prokaryotic expression; purification; glycosylation; phagocytosis

CLC Number:

S852.61

HU Xiangkun, LU Tiancheng, LIU Siyang, FU Yuqin, WANG Mingyu, ZHENG Hanxu, WANG Yan, WANG Xiuran. Cloning, Prokaryotic Expression and Glycosylation of P39 Gene from Brucella[J]. , 2018, 45(11): 2996-3002.

References

[1] ANNA S D,LISA G,HELENA G,et al.Global burden of human brucellosis:A systematic review of disease frequency[J].Public Library of Science Neglected Tropical Diseases,2012,6(10):e1865.
[2] ERIC D A,AHIDÉ L M,NAMMALWAR S,et al.A history of the development of Brucella vaccines[J].Biomed Research Internation,2013,2013:743509.
[3] OLSEN S C,PALMER M V.Advancement of knowledge of Brucella over the past 50 years[J].Veterinary Pathology,2014,51(6):1076-1089.
[4] 李青上,王全楚.布鲁菌病19例误诊原因分析[J].临床误诊误治,2016,29(1):48-49. LI Q S,WANG Q C.Clinical analysis of misdiagnosis of 19 caese of brucellosis[J].Clinical Misdiagnosis & Mistherapy,2016,29(1):48-49.(in Chinese)
[5] MARTIROSYAN A,GORVEL J P.Brucella evasion of adaptive immunity[J].Future Microbiol,2013,8(2):147-154.
[6] PAULD F,THOMAS A F,ALLISONR F,et.al.Pathogenesis and immunobiology of brucellosis review of Brucellae host interactions[J].The American Journal of Pathology,2015,185(6):1508-1517.
[7] 包红红,孟巧玲,韩俊锋.灰色模型预测河南省安阳市人布鲁菌病发病趋势[J].中华地方病学杂志,2017,36(10):706-708. BAO H H,MENG Q L,HAN J F.Application of gray model in predication of brecellosis incidence in Anyang city,Henan province[J].Chinese Journal of Endemiology,2017,36(10):706-708.(in Chinese)
[8] CHRISTOPHER S,UMAPATHY B L,RAVIKUMAR K L.Brucellosis:Review on the recent trends in pathogenicity and laboratory diagnosis[J].Journal of Laboratory Physicians,2010,2(2):55-60.
[9] 潘超,朱力,王恒裸.病原细菌多糖疫苗和多糖结合疫苗研究进展[J].生物技术通讯,2013,24(5):705-709. PAN C,ZHU L,WANG H L.Progress on polysaccharide vaccines and glycoconjugate vaccines of bacteria[J].Letters in Biotechnoligy,2013,24(5):705-709.(in Chinese)
[10] 董岩.肠道细菌O多糖结合疫苗生物合成研究[D].北京:中国食品药品检定研究院,2015. DONG Y.The study of enterobacterial o-specific polysaccharides bio-conjugate vaccines[D].Beijing:National Institutes for Food and Drug Control,2015.(in Chinese)
[11] 池源,王丽波.苯酚-硫酸法测定南瓜籽多糖含量的条件优化[J].食品与机械,2014,30(1):89-92. CHI Y,WANG L B.Study on phenol-sulfuric acid method for determination of polysaccharide content in pumpkin seeds[J].Food & Machinery,2014,30(1):89-92.(in Chinese)
[12] 王宇,沈佳胤,罗兰,等.FITC标记的GSTP1蛋白在鼠巨噬细胞中的定位[J].南京师大学报(自然科学版),2008,31(2):105-110. WANG Y,SHEN J Y,LUO L,et al.The location of FITC-GSTP1 in the murine macrophages[J].Journal of Nanjing Normal University (Natural Science Edition),2008,31(2):105-110.(in Chinese)
[13] HOP H T,ARAYAN L T,HUY T X N,et al.Lipocalin 2(Lcn2) interferes with iron uptake by Brucella abortus and dampens immunoregulation during infection of RAW 264.7 macrophages[J].Cell Microbiol,2018,20(3):e12813.
[14] 易继海,张俊波,李爽,等.NF-κB信号通路调控布鲁氏菌胞内存活分子机制的初步研究[J].中国畜牧兽医,2016,43(3):592-600. YI J H,ZHANG J B,LI S,et al.Preliminary study on NF-κB signaling pathway regulating Brucella intracellular survival molecular mechanisms[J].China Animal Husbandry & Veterinary Medicine,2016,43(3):592-600.(in Chinese)
[15] 聂鑫,赵天靖,曹瑞勇,等.羊种布鲁氏菌LpxB基因的克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2016,43(7):1681-1687. NIE X,ZHAO T J,CAO R Y,et al.Cloning,prokaryotic expression and bioinformatics analysis of LpxB gene of Brucella melitensis[J].China Animal Husbandry & Veterinary Medicine,2016,43(7):1681-1687.(in Chinese)
[16] 李宝宝,聂鑫,杨小健,等.羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(7):1947-1953. LI B B,NIE X,YANG X J,et al.Cloning,prokaryotic expression and bioinformatics analysis of dhbC gene of Brucella melitensis[J].China Animal Husbandry & Veterinary Medicine,2017,44(7):1947-1953.(in Chinese)
[17] 刘小波.布鲁氏菌优势抗原表位基因的筛选与抗原免疫特性研究[D].包头:内蒙古科技大学,2015. LIU X B.The study of antigen immunological properties and screening of advantaged epitope gene of Brucella[D].Baotou:Inner Mongolia University of Science and Technology,2015.(in Chinese)
[18] 何作萍.羊种布鲁氏菌外膜蛋白诱导的细胞凋亡分子机制[D].广州:南方医科大学,2016. HE Z P.Molecular mechanism of apoptosis induced by the outer membrane proteins of Brucella melitensis[D].Guangzhou:Southern Medical University,2016.(in Chinese)
[19] 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.Screening and identification of recombinant proteins as stimulants in brucelosis gamma interferon release assay for cattle[D].Yangzhou:Yangzhou University,2015.(in Chinese)
[20] 段小宇.布鲁氏菌分子标记疫苗株的构建及鉴别诊断方法的建立[D].长春:吉林大学,2007. DUAN X Y.Construction of labelling vaccines of Brucella and the establishment of differential diagnosis[D].Changchun:Jilin University,2007.
[21] TADEPALLI G,KONDURU B,MURALI H S,et al.Intraperitoneal administration of a novel chimeric immunogen (rOP) elicits IFN-γ and IL-12p70 protective immune response in BALB/c mice against virulent Brucella[J].Immunology Letters,2017,192:79-87.(in Chinese)
[22] 佟春玉.甘露聚糖修饰布鲁氏菌多肽靶向树突细胞增加免疫机制的研究[D].长春:吉林大学,2015. TONG C Y.Enhance immunity mechanism by mannosylated Brucella polypeptide targeting dendritic cell[D].Changchun:Jilin University,2015.(in Chinese)
[23] 许春晓.糖基化修饰对蛋白Rsα、AhcY免疫原性影响的研究[D].长春:吉林农业大学,2016. XU C X.Study the effect of glycosylation on immunogenicity of protein Rsα and AhcY[D].Changchun:Jilin Agricultural University,2016.(in Chinese)
[24] 王晓龙.甘露寡糖修饰对布鲁氏菌AhcY蛋白免疫原性的影响[D].长春:吉林农业大学,2017. WANG X L.The effect of glycosylation on the characteristics of the protein AhcY[D].Changchun:Jilin Agricultural University,2017.(in Chinese)
[25] 付景丽.靶向化布鲁氏菌树突状细胞疫苗的试制研究[D].保定:河北农业大学,2011. FU J L.Research and development of targeted dendritic cells vaccine for brucellosis[D].Baoding:Hebei Agricultural University,2011.(in Chinese)
[26] 陈师涛.牛布鲁氏菌和牛分支杆菌抗原的基因克隆、表达和抗原性鉴定[D].广州:暨南大学,2013. CHEN S T.Gene cloning,expression and antigenicity identification of Brucella abortus and Mycobacterium bovis antigen[D].Guangzhou:Jinan University,2013.(in Chinese)

Cloning, Prokaryotic Expression and Glycosylation of P39 Gene from Brucella

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WU Xuan, ZENG Shenming. Research Progress on the Function,Application and Purification Technology in Serum Albumin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2265-2275.
[2]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[3]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[4]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[5]	LIU Peng, LI Jingjing, QIAO Yanliang, ZHU Lianqin, ZHU Fenghua. Extraction and Structure Characterization of Scutellaria baicalensis Polysaccharide [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2518-2530.
[6]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[7]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[8]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[9]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[10]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[11]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[12]	CHENG Longfei, LIAO Weilian, CHEN Hongmei, JIANG Nansong, FU Qiuling, WAN Chunhe, LIU Rongchang, SHI Shaohua, HUANG Yu, FU Guanghua. Screening and Biological Characteristics of Broad-spectrum Riemerella anatipestifer Phage [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1169-1176.
[13]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[14]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.
[15]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.