抗体的制备及 TLR8 在双峰驼脾脏中的年龄相关性分布。

BACKGROUND: Toll-like receptor 8 (TLR8) can recognize specific pathogen-associated molecular patterns and exert multiple immunological functions through activation of signaling cascades. However, the precise distribution and age-related alterations of TLR8 in the spleens of Bactrian camels have not yet been investigated. This study aimed to prepare a rabbit anti-Bactrian camel TLR8 polyclonal antibody and elucidate the distribution of TLR8 in the spleens of Bactrian camels at different age groups. The methodology involved the construction of the pET-28a-TLR8 recombinant plasmid, followed by the expression of TLR8 recombinant protein via prokaryotic expression. Subsequently, rabbits were immunized with the purified protein to prepare the TLR8 polyclonal antibody. Finally, twelve Alashan Bactrian camels were categorized into four groups: young (1-2 years), pubertal (3-5 years), middle-aged (6-16 years) and old (17-20 years). These camels received intravenous sodium pentobarbital (20 mg/kg) anesthesia and were exsanguinated to collect spleen samples. Immunohistochemical techniques were employed to observe and analyze the distribution patterns and age-related changes of TLR8 in the spleen. RESULTS: The results showed that the TLR8 recombinant protein was expressed in the form of inclusion body with a molecular weight of 52 kDa, and the optimal induction condition involved 0.3 mmol/L IPTG induction for 8 h. The prepared antibody yielded a titer of 1:32 000, and the antibody demonstrated specific binding to TLR8 recombinant protein. TLR8 positive cells exhibited a consistent distribution pattern in the spleen across different age groups of Bactrian camels, primarily scattered within the periarterial lymphatic sheath of the white pulp, marginal zone, and red pulp. The predominant cell type expressing TLR8 was macrophages, with expression also observed in neutrophils and dendritic cells. Statistical analysis revealed that there were significant differences in the distribution density of TLR8 positive cells among different spleen regions at the same age, with the red pulp, marginal zone, and white pulp showing a descending order (P<0.05). Age-related changes indicated that the distribution density in the marginal zone and red pulp exhibited a similar trend of initially increasing and subsequently decreasing from young to old camels. As camels age, there was a significant decrease in the distribution density across all spleen regions (P<0.05). CONCLUSIONS: The results confirmed that this study successfully prepared a rabbit anti-Bactrian camel TLR8 polyclonal antibody with good specificity. TLR8 positive cells were predominantly located in the red pulp and marginal zone of the spleen, signifying their pivotal role in the innate immune response of the spleen. Aging was found to significantly reduce the density of TLR8 positive cells, while leaving their scattered distribution characteristics unaffected. These findings provide valuable support for further investigations into the immunomorphology and immunosenescence of the spleen in Bactrian camels.

背景：Toll 样受体 8（TLR8）可识别特定的病原体相关分子模式，并通过激活信号级联反应发挥多种免疫功能。然而，Bactrian 骆驼脾脏中 TLR8 的精确分布和与年龄相关的变化尚未得到研究。本研究旨在制备兔抗 Bactrian 骆驼 TLR8 多克隆抗体，并阐明不同年龄组 Bactrian 骆驼脾脏中 TLR8 的分布。该方法包括构建 pET-28a-TLR8 重组质粒，然后通过原核表达表达 TLR8 重组蛋白。随后，用纯化的蛋白免疫兔子制备 TLR8 多克隆抗体。最后，将 12 只阿拉善双峰驼分为 4 组：幼龄（1-2 岁）、青春期（3-5 岁）、中年（6-16 岁）和老年（17-20 岁）。这些骆驼接受静脉注射戊巴比妥钠（20 mg/kg）麻醉，并放血收集脾脏样本。采用免疫组织化学技术观察和分析 TLR8 在脾脏中的分布模式和年龄相关性变化。

结果：结果表明，TLR8 重组蛋白以 52 kDa 的分子量形成包涵体表达，最佳诱导条件为 0.3 mmol/L IPTG 诱导 8 h。制备的抗体效价为 1:32 000，抗体特异性结合 TLR8 重组蛋白。TLR8 阳性细胞在不同年龄组的 Bactrian 骆驼脾脏中表现出一致的分布模式，主要散在白髓的动脉周围淋巴鞘、边缘区和红髓内。表达 TLR8 的主要细胞类型是巨噬细胞，也观察到中性粒细胞和树突状细胞表达。统计分析表明，同一年龄不同脾脏区域 TLR8 阳性细胞的分布密度存在显著差异，红髓、边缘区和白髓呈降序排列（P<0.05）。年龄相关变化表明，从幼龄到老龄骆驼，边缘区和红髓的分布密度呈先增加后减少的相似趋势。随着骆驼年龄的增长，所有脾脏区域的分布密度均显著降低（P<0.05）。

结论：本研究成功制备了兔抗 Bactrian 骆驼 TLR8 多克隆抗体，具有良好的特异性。TLR8 阳性细胞主要位于脾脏的红髓和边缘区，表明其在脾脏固有免疫反应中发挥重要作用。研究发现，衰老显著降低了 TLR8 阳性细胞的密度，但不影响其分散分布的特征。这些发现为进一步研究 Bactrian 骆驼脾脏的免疫形态学和免疫衰老提供了有价值的支持。