Acta Veterinaria et Zootechnica Sinica ›› 2025, Vol. 56 ›› Issue (11): 5697-5705.doi: 10.11843/j.issn.0366-6964.2025.11.028

• Preventive Veterinary Medicine • Previous Articles     Next Articles

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. 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

RichHTML

1

PDF (PC)

13

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

CLC Number: