Shuyun Xu, Yingni Sun, Jingjing Wei, Zengke Wei, Jie Yu, Meiqi Jiang, Chaofan Luo, Hui Zhang, Zhen Wang, Zijie Wang, Jie Wu, Xia Zhou
College of Animal Science and Technology, Shihezi University, Shihezi, 832003, Xinjiang, China.
Sydney School of Veterinary Science, The University of Sydney, Camperdown, Australia.
Sci Rep. 2025 Jul 1;15(1):22401. doi: 10.1038/s41598-025-05125-7.
To explore the antigenic characteristics of the NlpI protein. This study screened a potential antigen of Mannheimia haemolytica using NCBI Blast analysis and investigated its immunogenicity using online software. The recombinant NlpI protein (rNlpI) was expressed in prokaryotic cells, and its reactogenicity was detected by western blot. The transcription level of nlpI mRNA in M. haemolytica after various culture durations was determined by RT-qPCR. Mice were challenged with M. haemolytica strain 95 at an LD dose, and their serum was collected at various times. The rNlpI protein in M. haemolytica and western blot were used to detect the expression of antibodies specific to rNlpI. BALB/c mice were immunized with various protein doses and white oil adjuvant on days 0, 7, and 14, and the antibody titer was detected by indirect ELISA. Cytokines were detected by double antibody sandwich ELISA. Seven days after the third immunization, M. haemolytica strain 95 was injected intraperitoneally at 3 × 10 CFU/mouse. After 48 h, the surviving mice were dissected and the bacterial load in their lungs was assessed. NlpI is a hydrophilic and soluble protein with a transmembrane domain, 11 antigenic determinants, and 27 B cell epitopes. It was speculated that the protein has strong immunogenicity. The expressed NlpI protein could bind to bovine serum positive for M. haemolytica. The transcription level of the nlpI gene was the highest in the stable phase, followed in decreasing order by the logarithmic, slow, and decline phases. The expression levels in the various phases differed insignificantly. The IgG antibody titers of mice immunized with 10, 20, and 30 µg NlpI + white oil adjuvant and 30 µg NlpI were measured. The antibody level of the 30 µg NlpI + white oil adjuvant group was higher than that in the other groups (P < 0.001). The immune protection rates of the 10 µg rNlpI + white oil adjuvant and 30 µg rNlpI groups were under 30%, while they were 50% and 60%, respectively, in the 20 and 30 µg rNlpI + white oil adjuvant groups. The bacterial load of all immunized groups was 99.99% lower than in the PBS and white oil adjuvant control groups. The protection rates differed significantly among immunized groups (P < 0.001), with the 30 µg rNlpI + white oil adjuvant group showing the lowest bacterial load in the lungs. The rNlpI protein can react with bovine M. haemolytica positive blood with good reactogenicity, and can induce mice to produce high titers of specific IgG antibodies, effectively resisting homologous strain invasion. The results lay a good foundation for developing new M. haemolytica vaccines.
为探究NlpI蛋白的抗原特性。本研究利用NCBI Blast分析筛选溶血曼氏杆菌的一种潜在抗原，并使用在线软件研究其免疫原性。重组NlpI蛋白（rNlpI）在原核细胞中表达，通过蛋白质免疫印迹法检测其反应原性。采用逆转录定量聚合酶链反应（RT-qPCR）测定溶血曼氏杆菌在不同培养时间后nlpI mRNA的转录水平。用溶血曼氏杆菌95株以半数致死剂量攻击小鼠，并在不同时间点采集其血清。利用溶血曼氏杆菌中的rNlpI蛋白和蛋白质免疫印迹法检测rNlpI特异性抗体的表达。在第0、7和14天，用不同蛋白剂量和白油佐剂免疫BALB/c小鼠，通过间接酶联免疫吸附测定法（ELISA）检测抗体效价。采用双抗体夹心ELISA法检测细胞因子。第三次免疫7天后，以3×10 CFU/只的剂量腹腔注射溶血曼氏杆菌95株。48小时后，解剖存活小鼠并评估其肺内细菌载量。NlpI是一种具有跨膜结构域、11个抗原决定簇和27个B细胞表位的亲水性可溶性蛋白。推测该蛋白具有较强的免疫原性。表达的NlpI蛋白可与溶血曼氏杆菌阳性牛血清结合。nlpI基因转录水平在稳定期最高，其次依次为对数期、缓慢期和衰退期。各阶段表达水平差异不显著。测定了用10、20和30μg NlpI加白油佐剂以及30μg NlpI免疫的小鼠的IgG抗体效价。30μg NlpI加白油佐剂组的抗体水平高于其他组（P<0.001）。10μg rNlpI加白油佐剂组和30μg rNlpI组的免疫保护率低于30%，而20μg rNlpI加白油佐剂组和30μg rNlpI加白油佐剂组的免疫保护率分别为50%和60%。所有免疫组的细菌载量均比磷酸盐缓冲盐水（PBS）和白油佐剂对照组低99.99%。免疫组之间的保护率差异显著（P<0.001），30μg rNlpI加白油佐剂组肺内细菌载量最低。rNlpI蛋白可与溶血曼氏杆菌阳性牛血发生反应，反应原性良好，可诱导小鼠产生高滴度特异性IgG抗体，有效抵抗同源菌株入侵。这些结果为开发新型溶血曼氏杆菌疫苗奠定了良好基础。
