Expression of Brucella ribosomal L7/L12 protein and its effect on mouse derived dendritic cells

doi:10.3969/j.issn.1004-1524.2022.03.09

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (3): 489-497.DOI: 10.3969/j.issn.1004-1524.2022.03.09

• Animal Science • Previous Articles Next Articles

Expression of Brucella ribosomal L7/L12 protein and its effect on mouse derived dendritic cells

ZHANG Guanglin¹^,²(), XU Long¹^,², GAO Yunyan², LI Lingxia², SHANG Youjun², ZHANG Yong¹, CAO Xiao’an2^*(), ZHAO Xingxu

1. School of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
2. Lanzhou Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China

Received:2020-11-11 Online:2022-03-25 Published:2022-03-30
Contact: CAO Xiao’an2

Abstract

Abstract:

In order to study the effect of ribosomal L7/L12 protein of Brucella on the differentiation and maturation of mouse dendritic cells (BM-DCs), the L7/L12 gene was amplified using Brucella S2 vaccine strain as template,and the recombinant plasmid pET30a-L7/L12 was constructed. The recombinant plasmid pET30a-L7/L12 was induced in E. coli prokaryotic expression system, and the expressed protein was purified by Ni column. Mouse-derived DCs was induced by IL-4 and GM-CSF, and the changes of costimulatory molecules and inflammatory factors on the surface of DCs were detected after DCs was stimulated by LPS and L7/L12 proteins. The results showed that the recombinant protein was highly expressed in soluble form under the condition of 1 mmol·L^-1 IPTG and overnight at 16 ℃. The size of the recombinant protein was 18 ku, the purity was more than 93% and had certain reactivity. Flow cytometry showed that the expression of CD40, CD80 and other antigen molecules on the surface of stimulated BM-DCs cells was significantly (P<0.05) higher than that of the blank control group. The results of qPCR showed that the levels of inflammatory cytokines TNF-β, IL-1 β and IL-12 in the control group were significantly (P<0.01) higher than those in the control group. Recombinant L7/L12 protein can stimulate the differentiation and maturation of DCs cells and promote the release of inflammatory factors.

Key words: Brucella, murine dendritic cells, immune cell activation

CLC Number:

S852

ZHANG Guanglin, XU Long, GAO Yunyan, LI Lingxia, SHANG Youjun, ZHANG Yong, CAO Xiao’an2, ZHAO Xingxu. Expression of Brucella ribosomal L7/L12 protein and its effect on mouse derived dendritic cells[J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 489-497.

Figures/Tables 12

Fig.1 Electrophoresis of expression fragment of L7/L12 gene by PCR amplification M, DNA Marker; 1-5, Product of PCR amplification.

Fig.2 Identification of clonal vector bacteria by PCR electrophoresis M, DNA Marker; 1-3, PCR amplification of monoclonal bacteria solution; 4, Negative control.

Fig.3 Identification of recombinant plasmid pET30a-L7/L12 by double enzyme digestion M, DNA Marker; 1, Recombinant PET30A-L7/L12 plasmid double enzyme digestion.

Fig.4 Induced expression of recombinant protein L7/L12 M, Standard protein marker; 1, Whole bacterial protein; 2, Lytic precipitation; 3, Lytic supernatant.

Fig.5 Selection of optimal inducible expression conditions of L7/L12 protein M, Standard protein marker; 1-3, At 16 ℃, 0.25, 0.5, 1 mmol·L-1 IPTG respectively; 4-6, At 28 ℃, 0.25, 0.5, 1 mmol·L-1 IPTG respectively; 7-9, At 37 ℃, 0.25, 0.5, 1 mmol·L-1 IPTG respectively.

Fig.6 L7/L12 protein purification by electrophoresis M, Standard protein marker; 1, L7/L12 flow-through solution; 2, LE Buffer cleaning solution; 3, LE Buffer containing 50 mmol·L-1 imidazole cleaning results; 4, LE Buffer containing 70 mmol·L-1 imidazole cleaning results; 5, LE Buffer 85 mmol·L-1 imidazole cleaning results; 6, LE Buffer containing 100 mmol·L-1 imidazole cleaning results; 7, LE Buffer containing 160 mmol·L-1 imidazoler cleaning results; 8, LE Buffe containing 250 mmol·L-1 imidazole cleaning results; 9, LE Buffer containing 300 mmol·L-1 imidazole cleaning results.

Fig.7 Identification of L7/L12 protein by Western blot M, Standard protein marker; 1-2, Protein L7/L12.

Fig.8 Mouse myeloid dendritic cells cultured in vitro A, Induction culturing day 2 (×200); B, Induction culturing day 3 (×200); C, Induction culturing day 5 (×200); D, Induction culturing day 5 (×400); E, Induction culturing day 7 (×200); F, Induction culturing day 7 (×400).

Fig.9 Activity detection of dendritic cells

Fig.10 Changes of co-stimulating molecules on DCs surface

Fig.11 Difference in expression of costimulatory molecules on the surface of DC cells

Fig.12 Changes of expression of inflammatory factors in DCs treated with L7/L12 protein and LPS IL-12, Interleukin12; TNF, Tumor necrosis factor; IL-1β, Interleukin 1β..

References 19

[1]	李文桂, 陈雅棠. 载体介导的布鲁氏菌L7/L12疫苗研制现状[J]. 中国病原生物学杂志, 2019, 14(7): 864-866.
	LI W G, CHEN Y T. The status of research on a Brucella L7/L12 vaccine mediated by vectors[J]. Journal of Pathogen Biology, 2019, 14(7): 864-866. (in Chinese with English abstract)
[2]	ADONE R, FRANCIA M, CIUCHINI F. Brucella melitensis B115-based complement fixation test to detect antibodies induced by Brucella rough strains[J]. Journal of Applied Microbiology, 2008, 105(2): 567-574. DOI URL
[3]	司瑞, 白文涛, 张芳琳, 等. 布鲁菌核糖体蛋白L7/L12的表达纯化及生物活性鉴定[J]. 生物技术通讯, 2006, 17(6): 872-874.
	SI R, BAI W T, ZHANG F L, et al. Expression and purification of ribosomal L7/L12 protein of Brucella melitensis and its characterization of bioactivity[J]. Letters in Biotechnology, 2006, 17(6): 872-874. (in Chinese with English abstract)
[4]	曾政, 王英, 赵光宇, 等. 布氏杆菌pCDNA3.1-L7L12核酸疫苗的构建及其免疫学评价[J]. 免疫学杂志, 2004, 20(3): 208-212.
	ZENG Z, WANG Y, ZHAO G Y, et al. Construction of DNA vaccine carrying Brucella abortus ribosomal L7/L12 gene and evaluation of its immune response in vaccinated mice[J]. Immunological Journal, 2004, 20(3): 208-212. (in Chinese with English abstract)
[5]	STEINMAN R M. Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation[J]. The Mount Sinai Journal of Medicine, New York, 2001, 68(3): 160-166.
[6]	FLÓREZ-GRAU G, ZUBIZARRETA I, CABEZÓN R, et al. Tolerogenic dendritic cells as a promising antigen-specific therapy in the treatment of multiple sclerosis and neuromyelitis optica from preclinical to clinical trials[J]. Frontiers in Immunology, 2018, 9: 1169. DOI URL
[7]	FOULON E, FOUCRAS G. Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression[J]. Journal of Immunological Methods, 2008, 339(1): 1-10. DOI URL
[8]	FRIEBE A, FRIEDERICHS S, SCHOLZ K, et al. Characterization of immunostimulatory components of orf virus (Parapoxvirus ovis)[J]. The Journal of General Virology, 2011, 92(Pt 7): 1571-1584. DOI URL
[9]	OLIVEIRA S C, SPLITTER G A. Immunization of mice with recombinant L7L12 ribosomal protein confers protection against Brucella abortus infection[J]. Vaccine, 1996, 14(10): 959-962. DOI URL
[10]	OLIVEIRA S C, ZHU Y, SPLITTER G A. Recombinant L7/L12 ribosomal protein and gamma-irradiated Brucella abortus induce a T-helper 1 subset response from murine CD4+T cells[J]. Immunology, 1994, 83(4): 659-664.
[11]	KURAR E, SPLITTER G A. Nucleic acid vaccination of Brucella abortus ribosomal L7L12 gene elicits immune response[J]. Vaccine, 1997, 15(17/18): 1851-1857. DOI URL
[12]	孙建聪. 转染SEA基因的肝癌-树突状细胞融合瘤苗的体外抗肿瘤研究[D]. 广州: 广州医学院, 2006.
	SUN J C. In vitro antitumor study of hepatocarcinoma-dendritic cell fusion tumor vaccine transfected with SEA gene[D]. Guangzhou: Guangzhou Medical College, 2006. (in Chinese with English abstract)
[13]	曹雪涛. 树突状细胞的基础与临床研究新进展[J]. 中国免疫学杂志, 1998, 14(3): 1-8.
	CAO X T. New progress in basic and clinical research on dendritic cells[J]. Chinese Journal of Immunology, 1998, 14(3): 1-8. (in Chinese)
[14]	FITZGERALD-BOCARSLY P, DAI J H, SINGH S. Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history[J]. Cytokine & Growth Factor Reviews, 2008, 19(1): 3-19.
[15]	KAPSENBERG M L. Dendritic-cell control of pathogen-driven T-cell polarization[J]. Nature Reviews Immunology, 2003, 3(12): 984-993. DOI URL
[16]	汪志文, 张海艳, 黄瑜, 等. 尼罗罗非鱼CD80基因的分子克隆与mRNA表达分析[J]. 基因组学与应用生物学, 2017, 36(1): 190-200.
	WANG Z W, ZHANG H Y, HUANG Y, et al. Molecular cloning and m RNA expression analysis of CD80 gene in Nile tilapia (Oreochromis niloticus)[J]. Genomics and Applied Biology, 2017, 36(1): 190-200. (in Chinese with English abstract)
[17]	DUPASQUIER M. Dendritic cells in vivo and in vitro[J]. Cellular & Molecular Immunology, 2005, 2(1): 28-35.
[18]	朱诗语, 王春凤, 杨桂连. 树突状细胞在病毒感染动物免疫应答中的作用[J]. 畜牧与兽医, 2014, 46(12): 106-108.
	ZHU S Y, WANG C F, YANG G L. The role of dendritic cells in the immune response of virus-infected animals[J]. Animal Husbandry & Veterinary Medicine, 2014, 46(12): 106-108. (in Chinese)
[19]	CHRISTOPHER-HENNINGS J. The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) in the boar[J]. Veterinary Research, 2000, 31(1): 57-58. DOI URL

Expression of Brucella ribosomal L7/L12 protein and its effect on mouse derived dendritic cells

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 12

References 19

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	MOU Hongye, ZHOU Xiaojie, YANG Yongchun, WANG Xiaodu, ZHOU Yingshan, SONG Houhui. Establishment of a duplex fluorescent quantitative PCR assay for detection of porcine circovirus type 2 and type 3 [J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 457-463.
[2]	TAN Tianyu, CAI Dongjie, WANG Zhisheng, ZUO Zhicai. Effect of glucose concentrations on proinflammatory cytokine released from bovine alveolar macrophages [J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 464-470.
[3]	XIA Qing, CAI Dongjie, FANG Jing, WANG Zhisheng, DENG Junliang, YI Jun, ZUO Zhicai. Effect of beta-hydroxybutyrate on proinflammatory cytokine production in bovine alveolar macrophages [J]. Acta Agriculturae Zhejiangensis, 2022, 34(2): 293-299.
[4]	XIA Jiangying, YANG Ju, SONG Tianhao, PANG Lianfeng, YE Ting, REN Zhihua, DENG Junliang. Protective effect of vitamin C in piglet intestinal epithelial cells inflammatory injury induced by β-conglycinin [J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2017-2025.
[5]	YANG Ju, DENG Junliang, XIA Jiangying, SONG Tianhao, PANG Lianfeng, REN Zhihua. Effect of vitamin A on barrier function damage of piglet intestinal epithelial cells induced by soybean 7S globulin [J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2026-2033.
[6]	MA Zhiyu, HUANG Fangyuan, WANG Wei, YI Jun, ZUO Zhicai. Isolation, identification and sensitive drug screening of a pathogenic Proteus cibarius strain from beef cattle [J]. Acta Agriculturae Zhejiangensis, 2021, 33(10): 1836-1843.
[7]	GUO Jia, MEN Xiaoming, DENG Bo, XU Ziwei. Advances of function, expression of animal selenoproteins and their regulation mechanism on meat quality [J]. Acta Agriculturae Zhejiangensis, 2021, 33(9): 1779-1788.
[8]	TU Teng, YIN Qingqing, ZHANG Pengfei, WANG Yin, YANG Zexiao, YAO Xueping, LUO Yan. Establishment and application of multiplex PCR-capillary electrophoresis for 7 swine diseases [J]. Acta Agriculturae Zhejiangensis, 2021, 33(4): 618-631.
[9]	FAN Lihong, GUO Hongrui, WU Jiang, YI Jun, MA Xiaoping, GOU Liping, XIE Yue, YE Gang, ZUO Zhicai. Pathogenicity of Acinetobacter pittii from beef cattle in mice [J]. Acta Agriculturae Zhejiangensis, 2021, 33(2): 230-238.
[10]	TANG Biao, CHANG Jiang, HU Ji, QIAN Mingrong, XIA Xiaodong, YANG Hua. Structures and characteristics of 103 plasmids carrying mcr gene [J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 43-51.
[11]	TANG Biao, CHEN Lingyun, LUO Yi, SHI Xingfen, HU Ji, CHEN Yifei, QIAN Mingrong, YANG Hua. Antimicrobial resistance in Salmonella and Campylobacter isolated from Zhejiang and Fujian Province in 2019 [J]. Acta Agriculturae Zhejiangensis, 2020, 32(12): 2218-2225.
[12]	XU Lihua, LAN Shengzhi, YU Bin, LI Junxing, ZHANG Pengchao, LI Baochen, SU Fei, YUAN Xiufang. Detection and genetic variation analysis of prevalent porcine circovirus type 2 strains in Zhejiang Province [J]. , 2020, 32(11): 1970-1977.
[13]	WANG Zhekan, ZHOU Haolan, LI Danlei, ZHAO Ayong. Investigation and genotyping of Giardia duodenalis infection in South China Sea [J]. , 2020, 32(8): 1363-1368.
[14]	YUN Tao, HUA Jionggang, YE Weicheng, NI Zheng, CHEN Liu, ZHANG Cun. Development and application of a one-step real-time TaqMan-MGB RT-PCR assay for detection of novel duck reovirus [J]. , 2020, 32(4): 571-576.
[15]	AYIDING Aerzuguli, LU Meilin, LI Ziliang, HE Dan, QIAO Zilin, ABUDUREYIMU Aymuguri. Study on subculture stability and serum-free acclimation of Marc-145 cells in low serum adaptation culture [J]. , 2020, 32(3): 415-420.