布鲁氏菌BtpA蛋白亚单位疫苗的制备及其对小鼠的免疫效果评价

doi:10.11843/j.issn.0366-6964.2025.11.028

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (11): 5697-5705.doi: 10.11843/j.issn.0366-6964.2025.11.028

布鲁氏菌BtpA蛋白亚单位疫苗的制备及其对小鼠的免疫效果评价

任文浩¹^,²(), 姚梦欣¹^,², 张乾艺¹^,², 徐艺玫³, 郭伟⁴, 陈创夫¹^,², 马忠臣¹^,²^,*(), 王勇¹^,²^,*()

1. 石河子大学动物科技学院, 石河子 832000
2. 动物健康养殖国际联合研究中心, 石河子 832000
3. 新疆维吾尔自治区疾病预防控制中心, 乌鲁木齐 830000
4. 福建圣维生物科技有限公司, 南平 350000

收稿日期:2024-10-08 出版日期:2025-11-23 发布日期:2025-11-27
通讯作者: 马忠臣,王勇 E-mail:r15254362709@163.com;zhongchen_ma@163.com;yongwang@shzu.edu.cn
作者简介:任文浩(1998-)，男，山东无棣人，硕士生，主要从事人兽共患传染病免疫机制研究，E-mail：r15254362709@163.com
基金资助:
新疆维吾尔自治区“天池英才(青年博士)”计划；国家自然科学基金(32060789)

Preparation of Brucella BtpA Protein Subunit Vaccine and Evaluation of Its Immune Efficacy in Mice

REN Wenhao¹^,²(), YAO Mengxin¹^,², ZHANG Qianyi¹^,², XU Yimei³, GUO Wei⁴, CHEN Chuangfu¹^,², MA Zhongchen¹^,²^,*(), WANG Yong¹^,²^,*()

1. School of Animal Science and Technology, Shihezi University, Shihezi 832000, China
2. International Joint Research Center for Animal Health Breeding, Shihezi 832000, China
3. Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention, Urumqi 830000, China
4. Fujian Shengwei Biotechnology Co., Ltd., Nanping 350000, China

Received:2024-10-08 Online:2025-11-23 Published:2025-11-27
Contact: MA Zhongchen, WANG Yong E-mail:r15254362709@163.com;zhongchen_ma@163.com;yongwang@shzu.edu.cn

摘要/Abstract

摘要：

旨在研究布鲁氏菌抗原btpA的免疫原性和对小鼠的免疫保护效果。试验通过PCR扩增布鲁氏菌btpA基因，克隆到pET-22b原核表达载体中，获得重组质粒pET-22b-BtpA，双酶切鉴定后在E.coli BL21(DE3)中用IPTG诱导目的蛋白表达并纯化后通过SDS-PAGE与Western blot鉴定。腹腔注射100 μg·只^-1 rbtpA蛋白到6周龄SPF级BALB/c小鼠中，14 d以50 μg·只^-1加强免疫。分别在7 d、14 d、21 d和28 d对小鼠尾部静脉采血。对小鼠血清中的布鲁氏菌特异性IgG抗体和抗体亚型(IgG1和IgG2a)使用间接ELISA进行检测。使用ELISpot方法检测在小鼠脾中释放IFN-γ的淋巴细胞数量。免疫28 d攻毒布鲁氏菌流行株(含2×10⁵ CFU·只^-1)，15 d后剖检，根据小鼠脾中的布鲁氏菌载菌量评价亚单位疫苗的保护效力。结果表明，获得30 ku纯化的重组BtpA蛋白(rBtpA)；rBtpA免疫7 d后小鼠产生布鲁氏菌特异性抗体，28 d水平最高，而PBS组未检测到特异性抗体；rBtpA免疫小鼠14 d和28 d后IgG2a/IgG1值都大于1，28 d更加显著，说明rBtpA诱导小鼠产生Th1偏向型的免疫应答反应。28 d内，小鼠释放IFN-γ的脾淋巴细胞数量明显超过PBS对照组，表明在rBtpA免疫后小鼠可以产生较高水平的Th1型细胞免疫应答反应。rBtpA组小鼠脾指数SI显著低于PBS组(P < 0.001)，脾载菌量也显著低于PBS组(P < 0.01)，表明rBtpA亚单位疫苗组的小鼠脾脏指数和脾载菌量更低。上述研究证明，rBtpA亚单位疫苗有作为布鲁氏菌病候选疫苗的潜质，本试验位研发布鲁氏菌rBtpA亚单位疫苗奠定了基础。

关键词: 布鲁氏菌, BtpA蛋白, 亚单位疫苗, 保护效果, ELISpot

Abstract:

This experiment aims to study the immunogenicity of Brucella antigen btpA and its immune protective effect on mice. In the experiment, the btpA gene of Brucella strain was amplified by PCR and cloned into the pET-22b prokaryotic expression vector to obtain the recombinant plasmid pET-22b-BtpA. After double enzyme digestion and identification, it was induced with IPTG in E.coli BL21 (DE3). The target protein was expressed and purified and then identified by SDS-PAGE and Western blot. 100 μg per mouse of rbtpA protein was intraperitoneally injected into 6-week-old SPF grade BALB/c mice, and the mice were boosted with 50 μg per mouse on the 14th day. Blood was collected from the tail vein of mice on 7 d, 14 d, 21 d and 28 d, respectively. Brucella-specific IgG antibodies and antibody subtypes (IgG1 and IgG2a) in mouse sera were detected using an indirect ELISA. The number of IFN-γ-releasing lymphocytes in mouse spleens was detected using the ELISpot method. The mice were challenged with the Brucella epidemic strain (including 2×10⁵ CFU per mouse) for 28 days, and necropsy was performed 15 days later. The protective efficacy of the subunit vaccine was evaluated based on the Brucella load in the spleen of the mice.The results showed that 30 ku purified recombinant BtpA protein (rBtpA) was obtained; Mice produced Brucella-specific antibodies 7 days after rBtpA immunization, with the highest levels at 28 days, while undetectable in the PBS group; After rBtpA-immunized mice 14 days and 28 days, the IgG2a/IgG1 values were all greater than 1, and were more significant at 28 days, indicating that rBtpA induced a Th1-biased immune response in mice. Within 28 days, the number of IFN-γ-releasing splenic lymphocytes in mice was significantly higher than that in the PBS control group, indicating that mice can produce a higher level of Th1-type cellular immune response after rBtpA immunization. The spleen index SI of mice in the rBtpA group was significantly lower than that of the PBS group (P < 0.001), and the spleen bacterial load was also significantly lower than that of the PBS group (P < 0.01), indicating that the spleen index and spleen bacterial load of mice in the rBtpA subunit vaccine group were much lower. The results indicate that the rBtpA subunit vaccine has the potential to be a candidate vaccine for brucellosis. This experiment laid the foundation for the development of Brucella rBtpA subunit vaccine.

Key words: Brucella, BtpA protein, subunit vaccine, protection effect, ELISpot

中图分类号:

S852.614

任文浩, 姚梦欣, 张乾艺, 徐艺玫, 郭伟, 陈创夫, 马忠臣, 王勇. 布鲁氏菌BtpA蛋白亚单位疫苗的制备及其对小鼠的免疫效果评价[J]. 畜牧兽医学报, 2025, 56(11): 5697-5705.

REN Wenhao, YAO Mengxin, ZHANG Qianyi, XU Yimei, GUO Wei, CHEN Chuangfu, MA Zhongchen, WANG Yong. Preparation of Brucella BtpA Protein Subunit Vaccine and Evaluation of Its Immune Efficacy in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5697-5705.

图/表 5

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布鲁氏菌BtpA蛋白亚单位疫苗的制备及其对小鼠的免疫效果评价

Preparation of Brucella BtpA Protein Subunit Vaccine and Evaluation of Its Immune Efficacy in Mice

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