Prokaryotic Expression, Antibody Preparation of TLR8 in Bactrian Camel (Camelus bactrianus) and Its Distribution with Age in the Spleen

Abstract

Abstract Background Toll-like receptor 8 (TLR8) can recognize special pathogen-associated molecular patterns and exert multiple immunological functions by activating signaling cascades. However, the precise characteristics and age-related changes of TLR8 in the spleen of Bactrian camels have not been reported. The aim of this study was to prepare rabbit anti-Bactrian camel TLR8 polyclonal antibody and elucidate distribution of TLR8 in the spleen of Bactrian camels of different ages. In this study, the pET-28a-TLR8 recombinant plasmid was firstly constructed, and the TLR8 recombinant protein was obtained by prokaryotic expression. Then, the TLR8 polyclonal antibody was prepared by immunizing rabbits with purified protein. Finally, Twelve Alashan Bactrian camels were divided into four groups: young (1–2 years), pubertal (3–5 years), middle-aged (6–16 years) and old (17–20 years). The distribution characteristics and age-related changes of TLR8 in the spleen were observed and analyzed using immunohistochemcal techniques. Results The results showed that the TLR8 recombinant protein was expressed in the form of inclusion body with a size of 52 kDa, and the optimal induction condition was 0.3 mmol·L-1 IPTG induced for 8 h. The titer of the prepared antibody was 1: 32 000, and the antibody could specifically bind to TLR8 recombinant protein. The distribution of TLR8 positive cells was similar in the spleen of Bactrian camels of different ages, all of which were mainly scattered in the periarterial lymphatic sheath of the white pulp, marginal zone and red pulp. TLR8 positive cell type was mainly macrophages, but also expressed in neutrophils and dendritic cells. Statistical results showed that the distribution density of TLR8 positive cells in different spleen regions at all age groups from high to low was the red pulp, marginal zone and white pulp, and there were significant differences between different regions (p < 0.05). The distribution density in the marginal zone and red pulp of the spleen showed similar changes with age, with a trend of first increasing and then decreasing from young to old. With the aging of the camels, the distribution density in each region of the spleen decreased significantly (p < 0.05). Conclusions The results confirmed that the rabbit anti-bactrian camel TLR8 polyclonal antibody with good specificity was successfully prepared, and TLR8 positive cells were mainly distributed in the red pulp and marginal zone of the spleen, suggesting that these two areas play an important role in innate immune response of the spleen. The aging could significantly decrease the density of positive cells, but had no impact on the scattered characteristics. These findings will provide support for the further studies on the immunomorphology and immunosenescence of the spleen of Bactrian camels.