布鲁氏菌核糖体L7/L12蛋白的原核表达及对鼠源树突状细胞的作用

doi:10.3969/j.issn.1004-1524.2022.03.09

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (3): 489-497.DOI: 10.3969/j.issn.1004-1524.2022.03.09

布鲁氏菌核糖体L7/L12蛋白的原核表达及对鼠源树突状细胞的作用

张广林¹^,²(), 徐龙¹^,², 高云艳², 李玲霞², 尚佑军², 张勇¹, 曹小安^*(), 赵兴绪^*()

1.甘肃农业大学动物医学院,甘肃兰州 730070
2.中国农业科学院兰州兽医研究所,甘肃兰州 730000

收稿日期:2020-11-11 出版日期:2022-03-25 发布日期:2022-03-30
通讯作者: 曹小安,赵兴绪
作者简介:*赵兴绪,E-mail: zhaoxx@gsau.edu.cn;曹小安,E-mail: caoxiaoan@caas.cn
张广林(1992—),男,甘肃武都人,硕士研究生,研究方向为动物传染病与病原分子生物学。E-mail: 1311758595@qq.com
基金资助:
国家重点研发计划(2018YFDO5OO9OO);国家重点研发计划(2018YFD0502000)

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

摘要/Abstract

摘要：

为了研究和探讨布鲁氏菌核糖体L7/L12蛋白对鼠源树突状细胞(BM-DCs)分化和成熟的影响,用布鲁氏菌S2疫苗株为模板扩增L7/L12基因,构建重组质粒pET30a-L7/L12,用大肠埃希菌原核表达系统进行诱导表达,并用Ni柱对表达的蛋白进行纯化。用IL-4和GM-CSF诱导培养鼠源DCs,用脂多糖(LPS)和L7/L12蛋白刺激DCs后检测其表面共刺激分子和炎症因子的变化。结果表明,重组蛋白在1 mmol·L^-1的IPTG、16 ℃过夜的条件下以可溶性形式高效表达,其大小为18 ku,纯度达到93%以上并有一定的反应原性。流式细胞仪检测被刺激的BM-DCs细胞表面CD40、CD80等抗原分子的表达显著(P<0.05)高于空白对照组。qPCR结果显示,炎性细胞因子TNF-β、IL-1β、IL-12极显著(P<0.01)高于空白对照组。重组L7/L12蛋白具有刺激DCs细胞分化、成熟和促进炎症因子释放的功能。

关键词: 布鲁氏菌, 树突状细胞, 免疫细胞活化

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

中图分类号:

S852

张广林, 徐龙, 高云艳, 李玲霞, 尚佑军, 张勇, 曹小安, 赵兴绪. 布鲁氏菌核糖体L7/L12蛋白的原核表达及对鼠源树突状细胞的作用[J]. 浙江农业学报, 2022, 34(3): 489-497.

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.

图/表 12

图1 L7/L12基因表达片段PCR扩增的电泳 M,标准DNA分子量;1~5,PCR扩增样品。

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

图2 克隆载体菌液PCR鉴定 M,标准DNA分子量;1~3,单克隆菌液PCR扩增;4,阴性对照。

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

图3 重组pET30a-L7/L12质粒双酶切鉴定 M,标准DNA分子量;1,双酶切重组pET30a-L7/L12质粒。

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.

图4 重组蛋白L7/L12诱导表达结果电泳 M,标准蛋白质分子量;1,破碎前菌体;2,破碎后沉淀;3,破碎后上清。

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

图5 L7/L12蛋白最佳诱导表达条件筛选电泳 M,标准蛋白质分子量;1~3,16 ℃条件下IPTG浓度分别为0.25、0.5、1 mmol·L-1;4~6,28 ℃条件下IPTG浓度分别为0.25、0.5、1 mmol·L-1;7~9,37 ℃条件下IPTG 浓度分别为0.25、0.5、1 mmol·L-1。

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.

图6 L7/L12蛋白纯化电泳检测 M,标准蛋白质分子量;1,L7/L12流穿液;2,LE Buffer 清洗液;3,含50 mmol·L-1咪唑LE Buffer清洗结果;4,含70 mmol·L-1咪唑LE Buffer清洗结果;5,含85 mmol·L-1咪唑LE Buffer清洗结果;6,含100 mmol·L-1咪唑LE Buffer清洗结果;7,含160 mmol·L-1咪唑LE Buffer清洗结果;8,含250 mmol·L-1咪唑LE Buffer清洗结果;9,含300 mmol·L-1咪唑LE Buffer清洗结果。

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.

图7 基于Western blot的L7/L12蛋白鉴定 M,标准蛋白质分子量;1~2,L7/L12 蛋白。

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

图8 小鼠髓源树突状细胞体外诱导培养 A,诱导培养第2天(×200);B,诱导培养第3天(×200);C,诱导培养第5天(×200);D,诱导培养第5天(×400);E,诱导培养第7天(×200);F,诱导培养第7天(×400)。

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).

图9 DCs活性检测

Fig.9 Activity detection of dendritic cells

图10 处理前后DCs表面共刺激分子的变化

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

图11 DC细胞表面共刺激分子的表达差异 DC,未处理的DCs细胞(空白对照);LPS,脂多糖(阳性对照);L7/L12,L7/L12重组蛋白(实验组);***, P<0.01。下同。

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

图12 L7/L12蛋白和LPS处理后DCs炎性因子的表达 IL-12,白介素12;TNF,肿瘤坏死因子;IL-1β,白介素1β。

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..

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布鲁氏菌核糖体L7/L12蛋白的原核表达及对鼠源树突状细胞的作用

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

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