Department of Proteomics, Center for Genetic Engineering and Biotechnology (CIGB), Avenida 31, e/ 158 y 190, Cubanacán, Playa, 10600, Havana, Cuba.
Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), Avenida 31, e/ 158 y 190, Cubanacán, Playa, 10600, Havana, Cuba.
Anal Bioanal Chem. 2021 Sep;413(23):5885-5900. doi: 10.1007/s00216-021-03569-0. Epub 2021 Aug 3.
A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-CyspP0 and p64K-βAlapP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli, the same cross-linking reagent, and two analogues of pP0. The SDS-PAGE analysis of p64K-CyspP0 showed a heterogeneous conjugate compared to p64K-βAlapP0 that was detected as a protein band at 91kDa. The pP0/p64K ratio determined by MALDI-MS for p64K-CyspP0 ranged from 1 to 8, being 3-5 the predominant ratio, while in the case of p64K-βAlapP0 this ratio was 5-7. CyspP0 was partially linked to 35 out of 39 Lys residues and the N-terminal end, while βAlapP0 was mostly linked to the six free cysteine residues, to the N-terminal end, and, in a lesser extent, to Lys residues. The assignment of the conjugation sites and side reactions were based on the identification of type 2 peptides. Rabbit immunizations showed the best anti-pP0 titers and the highest efficacy against Rhipicephalus sanguineus ticks when the p64K-CyspP0 was used as vaccine antigen. The presence of high molecular mass aggregates observed in the SDS-PAGE analysis of p64K-CyspP0 could be responsible for a better immune response against pP0 and consequently for its better efficacy as an anti-tick vaccine. Graphical abstract.
蜱酸性核糖体蛋白 P0 肽（pP0）与来自巨蛤 Megathura crenulata 的血蓝蛋白连接物，当用于宿主疫苗接种时，已被证明对不同的蜱种有效。将这种肽转化为商业抗蜱疫苗将取决于找到适当的、技术上和经济上可行的方法将其呈现给宿主免疫系统。两种缀合物（p64K-CyspP0 和 p64K-βAlapP0）使用来自大肠杆菌的脑膜炎奈瑟菌的 p64K 载体蛋白、相同的交联试剂和 pP0 的两种类似物合成。SDS-PAGE 分析表明，与 p64K-βAlapP0 相比，p64K-CyspP0 是一种异质缀合物，p64K-βAlapP0 被检测为 91kDa 的蛋白质带。通过 MALDI-MS 确定的 p64K-CyspP0 中的 pP0/p64K 比值范围为 1 至 8，3-5 是主要比值，而在 p64K-βAlapP0 的情况下，该比值为 5-7。CyspP0 部分连接到 39 个赖氨酸残基中的 35 个和 N 末端，而 βAlapP0 主要连接到 6 个游离半胱氨酸残基、N 末端以及较少程度的赖氨酸残基。缀合位点和副反应的分配基于 2 型肽的鉴定。当将 p64K-CyspP0 用作疫苗抗原时，兔免疫显示出针对 Rhipicephalus sanguineus 蜱的最佳抗 pP0 效价和最高功效。在 p64K-CyspP0 的 SDS-PAGE 分析中观察到的高分子质量聚集体的存在可能是对 pP0 产生更好免疫反应的原因，从而使其成为更好的抗蜱疫苗。
