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

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

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