类鼻疽伯克霍尔德菌groEL基因的克隆、原核表达及其蛋白的生物信息学分析

doi:10.16431/j.cnki.1671-7236.2018.02.003

Abstract

Abstract:

This study was aimed to clone and express groEL gene of Burkholderia pseudomallei, and perform the bioinformatics analysis of groEL protein. The genome DNA of the bacteria was extracted as the template, a pair of primers were designed with reference to the groEL gene sequence of Burkholderia pseudomallei in GenBank. The groEL gene fragment of 1 641 bp was amplified by PCR, and ligated into pMD19-T vector to construct pMD19-T-groEL recombinant plasmid. The recombinant plasmid pET-28a(+)-groEL was constructed by digestion with BamHⅠand Hind Ⅲ. The correct pET-28a(+)-groEL was transformed into E.coli BL21 (DE3) competent cells and induced by IPTG. Protein was identified by SDS-PAGE and Western blotting. DNAMAN and BioEdit softwares were used to perform the bioinformatics analysis. The results showed that groEL gene of Burkholderia pseudomallei was successfully cloned and expressed. The fusion protein was about 64 ku. The formular of GroEL protein was C₄₅₁₀H₇₃₈₁N₁₆₄₁O₁₈₄₀S₅₂₁, the molecular mass was 15 893, the extinction coefficient was 32 500, the instability index was 40.31, and the average hydrophilicity was 0.901. The alpha helix (Hh), extended chain (Ee) and random coil (Cc) accounted for 48.71%, 13.19% and 38.10%,respectively. This results laid a foundation for the further study of molecular mechanism of Burkholderia pseudomallei groEL gene.

Key words: Burkholderia pseudomallei; groEL gene; cloning; prokaryotic expression; protein identification; bioinformatics analysis

CLC Number:

HUANG Haifeng, ZHANG Zhenxing, YANG Xiaojian, CAO Ruiyong, LI Baobao, NIE Xin, ZHU Shu, LI Guohua, PENG Dongmei, LI Yaying, WANG Fengyang, DU Li. Cloning, Prokaryotic Expression and Protein Bioinformatics Analysis of groEL Gene of Burkholderia pseudomallei[J]. , 2018, 45(2): 302-309.

References

[1] 方瑶. 类鼻疽伯克霍尔德菌全基因组测序及感染细胞模型的建立[D]. 重庆:第三军医大学, 2013.FANG Y. Whole genome sequencing and establishment of infected cell model of Burkholderia pseudomallei[D]. Chongqing:Third Military Medical University, 2013.(in Chinese)
[2] ABBINK F C, ORENDI J M, DE BEAUFORT A J. Mother-to-child transmission of Burkholderia pseudomallei[J]. New England Journal of Medicine, 2001, 344(15):1171-1172.
[3] PANOMKET P. Antimicrobial agents and Burkholderia pseudomallei:Perspectives from Thailand[J]. Asian Biomedicine, 2014, 8(2):167-172.
[4] LIMMATHUROTSAKUAL D, PEACOCK S J. Melioidosis:A clinical overview[J]. British Medical Bulletin, 2011, 99(1):125.
[5] 张经余, 赵志虎,蔡民华. groEL分子伴侣研究进展[J]. 生物技术通讯, 2001, 12(2):127-129.ZHANG J Y, ZHAO Z H, CAI M H. GroEL molecular chaperone research progress[J]. Biotechnology Communication, 2001, 12(2):127-129.(in Chinese)
[6] 陈彦江. Geobacillus sp. E263分子伴侣groEL及骨架蛋白MreB在高温噬菌体GVE2感染中的作用[D]. 杭州:浙江大学, 2013.CHEN Y J. Geobacillus sp. E263 chaperone groEL and skeletal protein MreB in high temperature phage GVE2 infection[D]. Hangzhou:Zhejiang University, 2013.(in Chinese)
[7] MCGOWAN J E, METCHOCK B, NOLTE F S. Laboratory diagnosis of tuberculosis:Past, present, and future[J]. Journal of the Medical Association of Georgia, 1995, 84(5):215-220.
[8] WOO P C, WOO G K, LAU S K, et al. Single gene target bacterial identification. groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis[J]. Diagnostic Microbiology and Infectious Disease, 2002, 44(2):143-149.
[9] 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 of LpxB gene and bioinformatics analysis of its protein[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2016, 43(7):1681-1687.(in Chinese)
[10] 徐开莲, 朱华培, 赵天靖, 等. 羊种布鲁氏菌Omp10基因的克隆及原核表达[J]. 中国畜牧兽医, 2014, 41(12):122-125.XU K L, ZHU H P, ZHAO T J, et al.Cloning and prokaryotic expression of Brucella Omp10 gene in sheep[J].Chinese Journal of Animal Science and Veterinary Medicine,2014, 41(12):122-125.(in Chinese)
[11] 毛旭虎,胡治强. 类鼻疽的实验室诊断方法及评价[J/OL]. 第三军医大学学报, 2016, 38(11):1200-1204.MAO X H,HU Z Q. The diagnostic methods and evaluation of the laboratory diagnosis of the nas-olar[J/OL].Journal of Third Military Medical University, 2016, 38(11):1200-1204.(in Chinese)
[12] PUMIRAT P, BROEK C V, JUNTAWIENG N, et al. Analysis of the prevalence,secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei[J]. PLoS One, 2014, 9(5):e96298.
[13] SHANKS J,BURTNICK M N,BRETT P J,et al. Burkholderia mallei tssM encodes a putative deubiquitinase that is secreted and expressed inside infected RAW 264.7 murine macrophages[J]. Infection & Immunity, 2009,77(4):1636.
[14] WIERSINGA W J, VAN D P T, WHITE N J, et al.Melioidosis:Insights into the pathogenicity of Burkholderia pseudomallei[J]. Nature Reviews Microbiology, 2006, 4(4):272-282.
[15] CURRIE B J. Advances and remaining uncertainties in the epidemiology of Burkholderia pseudomallei and melioidosis[J].Transactions of the Royal Society of Tropical Medicine & Hygiene,2008,102(3):225-227.
[16] AMORNRIT W, MUANGSOMBUT V, WANGTEERAPRASERT T, et al. Elevated intracellular levels of iron in host cells promotes Burkholderia pseudomallei infection[J]. Asian Biomedicine, 2012,6(3):465-471.
[17] 闫啸. 分子伴侣groEL/GroES介导重组蛋白可溶表达及折叠与组装初步研究[D]. 杭州:浙江大学,2012.YAN X. Molecular mate groEL/GroES mediated recombinant protein soluble expression and folding and assembly preliminary study[D]. Hangzhou:Zhejiang University, 2012.(in Chinese)
[18] BEARSS J J,HUNTER M,DANKMEYER J L, et al. Characterization of pathogenesis of and immune response to Burkholderia pseudomallei K96243 using both inhalational and intraperitoneal infection models in BALB/c and C57BL/6 mice[J]. PLoS One,2017,12(2):e0172627.
[19] DEMBEK Z F. Medical Aspects of Biological Warf-are[M]. Washington D C:Office of The Surgeon General, US Army Medical Department Center and School, Borden Institute,1997.
[20] 许峥, 陈小明, 郑卫东, 等. 大肠杆菌分子效伴侣groEL和GroES蛋白的高效表达、纯化与鉴定[J]. 南京大学学报(自然科学版),1997,33(1):71-79.XU Z, CHEN X M, ZHEN W D,et al. High-level expression, purification and identification of combed cheer and GroEs proteins in Escherichia coli[J]. Journal of Nanjing University (Natural Science), 1997, 33(1):71-79.(in Chinese)
[21] 王斯婷, 李晓娜, 王皎, 等. 代谢组学及其分析技术[J]. 药物分析杂志,2010,30(9):1792-1799.WANG S T, LI X N, WANG J, et al. Metabolomics and its analytical techniques[J]. Journal of Drug Analysis, 2010, 30(9):1792-1799. (in Chinese)
[22] 李霞, 李亦学, 廖飞. 生物信息学[M]. 北京:人民卫生出版社,2010.LI X, LI Y X, LIAO F. Bioinformatics[M]. Beijing:People's Health Publishing House, 2010.(in Chinese)
[23] FENTON W A, HORWICH A L. GroEL-mediated protein folding[J]. Protein Science,1997,6(4):743-760.

Cloning, Prokaryotic Expression and Protein Bioinformatics Analysis of groEL Gene of Burkholderia pseudomallei

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	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.
[2]	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.
[3]	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.
[4]	LIU Jialong, ZHANG Yiwen, ZHANG Yueran, SUN Qijuan, CHEN Jian, HE Lei, JIA Yanyan, DING Ke, YU Zuhua. Cloning,Prokaryotic Expression and Bioinformatics Analysis of ELAVL1 Gene in Chicken [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1807-1817.
[5]	ZHUO Na, ZHU Zhongwu, LI Jing, HU Shixiang, WANG Jianchang, LIN Hua, CHEN Chaolin, HAN Diangang, AI Jun, CHEN Peifu. Bioinformatics Analysis and Eukaryotic Expression of ORF2 Protein of Swine Hepatitis E Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1959-1970.
[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]	FAN Bingfeng, LI Wen, LIU Lixiang, ZHAO Xiangyuan, SHAO Jing, LIN Lefeng, SUN Huimin, ZHANG Xulin, XU Baozeng. Cloning and Bioinformatics Analysis of AANAT Gene in Mink [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1307-1318.
[8]	LIU Hongrun, ZHU Siran, FENG Lingli, ZHANG Kun, YAN Gang, WANG Yubin, ZHANG Shuai, JIANG Shan, XU Di, LAN Ganqiu, LIANG Jing. Cloning,Bioinformatics Analysis and Tissue Expression Localization of CDO1 Gene in Large White Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1352-1363.
[9]	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.
[10]	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.
[11]	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.
[12]	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.
[13]	ZHANG Yongjie, SUI Zhiyuan, ZHANG Zhishuai, WANG Chenguang, LI Xiaojun, XING Feng. Cloning and Bioinformatics Analysis of NPTX1 Gene and Its Tissues Expression at Puberty in Dolang Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1048-1058.
[14]	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.
[15]	WEI Yirong, ZHENG Zihua, ZHANG Sanbao, SONG Ying, JIANG Haiyu, SUN Wenyue, CHENG Pengfei, LIU Yufan, ZOU Jianwei, HUANG Yanna, PAN Yan, JIANG Qinyang. Cloning,Bioinformatics Analysis and Tissue Expression of UCP1 Gene in Nubian Goats [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 440-450.