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

doi:10.21203/rs.3.rs-2957164/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-2957164/v1

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published 16 Dec, 2023

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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.

Bactrian camels

Toll-like receptor 8

antibody preparation

spleen

aging

distribution

Innate immunity serves as the first line of the body defense against invading pathogenic microorganisms, and the innate immune system of mammals detects pathogenic microorganisms invading the body through pattern recognition receptors encoded by germline genes, of which Toll-like receptors (TLRs) play an important role in initiating innate immune responses by recognizing pathogen-associated molecular patterns (the conserved molecular structures of pathogens) and damage-associated molecular patterns (components released by damaged own cells and tissues) [1–3]. Activation of TLRs can mediate inflammatory response, modulation of cell cycle, apoptosis and other biological effects [4]. TLRs, as a “bridge” connecting innate immunity and adaptive immunity, according to sub-cellular localization, are classified into two types. One type is located at the plasma membrane, such as TLR1, 2, 4, 5, 6 and TLR10, and mainly recognizes bacterial cell wall components and related molecules. The other type is expressed on the endosomes and phagosome membranes, including TLR3, 7, 8 and TLR9, which mainly recognizes bacterial and viral nuclear components [5, 6].

TLR8, as one of the main members of the TLR7/8/9 subfamily, is a type I membrane-spanning proteins and consists of an extracellular leucine-rich repeat (LRR) domain which is responsible for ligand recognition, a transmembrane domain and an intracellular Toll/IL-1 receptor (TIR) domain responsible for signal transduction [7, 8]. TLR8 is mainly expressed in monocytes/ macrophages, neutrophils, dendritic cells, natural killer cells [9, 10], and can recognize single-stranded RNA [9, 11], synthetic oligonucleotides [10], guanosine analogs with antiviral activities [11], synthetic imidazoquinoline compounds [12], and activate specific signaling pathways to exert multiple immunological functions. TLR8 recognizes the corresponding pathogen-associated molecular patterns (PAMPs), and brings the intracellular TIR domains closer to each other by binding with PAMPs, thereby initiating signal transduction. The TIR domains can bind to the adapter protein myeloid differentiation factor 88 (MyD88) through homotypic interaction, which can recruit IL-1R-associated kinase 1 (IRAK1) and 4, and activate ubiquitin ligase TNF receptor-associated factor 6 (TRAF6). TRAF6 can activate the downstream transcription factors NF-κB and interferon regulatory factor 7 (IRF7) by initiating the corresponding signal transduction pathways. Ultimately, pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-12, IL-27, and type I interferon are produced to participate in the body's innate and adaptive immune response [7, 13, 14]. Activation of TLR8 signaling pathway can produce IFN-α and pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-12, etc., suggesting that TLR8 plays an important role in cellular immunity, anti-tumor, antiviral and anti-infection of the body. In addition, Th2-type cytokines, such as IL-4, IL-5 and IL-13, are associated with the occurrence and development of some allergic diseases. TLR8 can exert anti-allergic effects by inhibiting the abnormal secretion of Th2-type cytokines [15]. Crucially, CD4⁺ regulatory T (Treg) cells can suppress immune responses of body, while TLR8 can activate the TLR8-MyD88-IRAK4 signaling pathway through recognition of ligands to inhibit the immunosuppressive function of Treg cells, thereby enhancing the body's anti-tumour immunity [16]. Therefore, basic studies on the distribution, expression and signaling mechanism of TLR8 are of great significance for revealing the relevant mechanisms of the body's anti-infection, anti-tumor and anti-allergy.

As of now, studies on TLR8 have been reported in human [17, 18] and many animals, mainly including bovine [19], goat [20], porcine [21], equine [22], nyctereutes procyonoides [23], mouse [24, 25] and other animals [26–28]. These studies are mainly concerned with the molecular cloning, characterization and expression patterns of TLR8 [22–28], the development and clinical application of TLR8 agonists [17, 29], especially in application of tumor immunotherapy [17], the mechanism of TLR8 signaling pathway and anti-infection [19, 21, 26], and the relationship between TLR8 and the occurrence and development of some clinical diseases has also been reported, such as tumors [17], tuberculosis [18] and asthma [29]. Bactrian camel, as an important special economic animal in China, is a particularly special species because of its ability to survive in arid and semi-arid desert environments [30]. Compared with other ruminants, Bactrian camels have unique biological characteristics, such as greater resistance to rough-feeding, hunger, thirst, heat, cold and so on [31, 32]. Meanwhile, studies on the mucosal immunity of Bactrian camels have also revealed that they have unique mucosal immunological characteristics [33, 34]. The spleen, as the largest immune organ of Bactrian camels, is a key site for the migration and settlement of lymphocytes and immune response, and has biological functions such as filtering and storing blood, regulating blood volume and participating in the body's immune response [35]. As a highly expressed site of TLR8, the spleen can generate corresponding immune responses when the body is stimulated by antigens. The multiple biological effects mediated by TLR8 may be an essential part of maintaining the strong immune function of Bactrian camels. Although the distribution and expression patterns of TLR8 have been elucidated in many animals, studies on Bactrian camel TLR8 have not been reported, and the distribution characteristics and age-related changes of TLR8 in the spleen of Bactrian camel remains unclear. As an important reagent necessary for basic research, polyclonal antibody is widely used due to its short preparation cycle and high cost-effectiveness. However, there are still no commercial Bactrian camel TLR8 antibodies. To clarify these questions, in this study, the TLR8 recombinant protein of Bactrian camel was successfully obtained through the prokaryotic expression, and rabbit anti-Bactrian camel TLR8 polyclonal antibody was prepared. The distribution characteristics and age-related changes of TLR8 in the spleen of Bactrian camel were carefully observed, analyzed and compared. These data will provide support for further studies on the immunomorphology and immunosenescence of Bactrian camel spleen and mechanism of TLR8 signaling pathway.

TLR8 gene synthesis and recombinant plasmid construction of Bactrian camel

The prediction of transmembrane structure (Fig. 1A) showed that TLR8 protein of Bactrian camel contained a total of 1078 amino acids, of which 1-862 amino acids were the extra-membrane region. The extra-membrane region was cut by Editseq program of DNAStar7.0, and the prediction of signal peptide (Fig. 1B) showed that TLR8 protein had no signal peptide. According to the prediction results of protein hydrophilicity (Fig. 2A) and antigenic epitope (Fig. 2B), TLR8 protein of Bactrian camel was a hydrophobic protein with a high antigenic index. The extra-membrane amino acid sequence (70–518 amino acids) with high antigenic index of TLR8 protein was intercepted by Editseq program of DNAStar7.0, and then the restriction site BamH Ⅰ and the start codon were added upstream of the corresponding base sequence, and the restriction site Xho Ⅰ and the stop codon were added downstream. The length of optimized sequence of Bactrian camel TLR8 gene was 1365 bp, encoding a total of 450 amino acids, with a size of 52 kDa. The double digestion identification of the recombinant plasmid showed that there was obvious band between 1 000 and 1 500 bp of Marker, and the size was consistent with expectations, suggesting that the recombinant plasmid pET-28a-TLR8 was successfully constructed (Fig. 3A).

Prokaryotic expression results of pET-28a-TLR8 recombinant plasmid

The recombinant bacteria before and after induction by 1.0 mmol·L^-1 IPTG were detected by 12% SDS-PAGE, and the results showed that compared with the pre-induction product of recombinant bacteria, after the recombinant bacteria were induced by IPTG, an obvious band appeared between 45 and 60 kDa (Fig. 3B), and the size was 52 kDa, indicating that the recombinant plasmid pET-28a-TLR8 had been successfully transferred into competent cells BL21 and could be normally expressed in the competent cells.

Optimization results of expression conditions of TLR8 recombinant protein in Bactrian camel

The recombinant bacteria were induced to express TLR8 by IPTG with different concentration gradients (0.1-1.0 mmol·L^-1), and the results of 12% SDS-PAGE showed (Fig. 3C) that when the IPTG concentration was 0.3 mmol·L^།1, the expression level of the target protein was highest, so the optimal IPTG induction concentration for TLR8 recombinant protein expression was 0.3 mmol·L^།1.

Under the optimal IPTG concentration, the recombinant bacteria were induced to express TLR8 at different time gradients (2–10 h). The results of 12% SDS-PAGE showed (Fig. 3D) that the target protein was expressed when IPTG was added for 2 h. As the induction time passed, the expression level of the target protein gradually increased, reached the highest level at 8 h, and then gradually decreased. Therefore, the optimal induction time for TLR8 recombinant protein expression was at 8 h.

Purification results of TLR8 recombinant protein in Bactrian camel

Under the optimal induction conditions, after the recombinant bacteria were induced to express the target protein, the recombinant bacteria were disrupted by ultrasound, and the lysed supernatant and precipitate of recombinant bacteria were collected respectively after centrifugation. The results of 12% SDS-PAGE showed (Fig. 3E) that compared with the lysed supernatant, a clear target band only appeared in the precipitate of recombinant bacteria with a size of 52 kDa, indicating that the TLR8 recombinant protein was successfully expressed in the form of inclusion body in competent cells BL21.

The recombinant bacteria were induced and cultivated, then disrupted by ultrasound and centrifuged to collect the lysed precipitate for protein purification. The eluate was collected during protein purification, and the highest protein content determined by Ultraviolet spectrophotometer was 2.009 mg⋅mL^− 1.The results of 12% SDS-PAGE showed (Fig. 3F) that the purified protein had a single band without other heteroproteins, the size was 52 kDa, indicating that the target protein was purified well and could be used for immunization of experiment animals.

Serum titer and specificity detection of rabbit Anti-Bactrian camel TLR8 polyclonal antibody

The results of serum titer detection by indirect ELISA showed (Fig. 4) that when the rabbit antiserum and pre-immune serum were diluted at 1:32 000 and 1:2000, respectively. The value of OD₄₅₀ (positive) /OD₄₅₀ (negative) was greater than 2.1, indicating that the titer of TLR8 polyclonal antibody was 1:32 000, and TLR8 recombinant protein had good immunogenicity and could induce rabbits to produce specific antibody.

The specificity of TLR8 polyclonal antibody was detected by Western blotting, and the results showed (Fig. 5) that there was an obvious blot between 45 and 60 kDa on the PVDF membrane, the size was 52kDa, and it was a single blot, indicating that the polyclonal antibody prepared in this study had good reactogenicity and could specifically bind to TLR8 recombinant protein.

Distribution characteristics of TLR8 in the spleen of Bactrian camels of different ages

According to histological characteristics of the spleen of Bactrian camel (Fig. 6), the spleen was divided into three regions: the white pulp, marginal zone and red pulp. The results of immunohistochemical staining showed (Fig. 7, Fig. 8) that TLR8 positive cells were round or oval in shape, with abundant cytoplasm, large in size and relatively small in nucleus, which were typical macrophages. In addition, the nuclei of some positive cells were rod-shaped or obviously lobulated, and a small number of positive cells were dendritic with multiple pseudopodial protrusions, indicating that TLR8 was also expressed in neutrophils and dendritic cells.

In pubertal camels, TLR8 positive cells were scattered in the periarterial lymphatic sheath of the white pulp (Fig. 7B1), mainly macrophages, followed by neutrophils, and relatively few dendritic cells. The positive cells were diffusedly distributed in the marginal zone (Fig. 7B2), mainly macrophages, followed by neutrophils. The positive cells were also diffusedly distributed in the splenic sinus and splenic cord of the red pulp (Fig. 7B3), mainly macrophages, followed by neutrophils. The distribution pattern of the positive cells in the spleen of young (Fig. 7B1-B3), middle-aged (Fig. 8B1-B3) and old (Fig. 8B1-B3) camels was essentially similar to that of pubertal camels, and the positive cells were mainly distributed in the red pulp and marginal zone, while the white pulp was relatively less.

Changes in the distribution density of TLR8 positive cells with age

The distribution density of TLR8 positive cells in different regions of the spleen of Bactrian camels (young, pubertal, middle-aged and old) was statistically analyzed, and the results showed (Fig. 9A) that the distribution density of positive cells in different regions of the spleen in each age group from high to low was the red pulp, marginal zone and white pulp, and there were significant differences between different regions (P < 0.05). That is, the distribution density of positive cells in the red pulp was significantly higher than that in the marginal zone and white pulp (P < 0.05), and the distribution density of positive cells in the marginal zone was significantly higher than that in the white pulp (P < 0.05). Meanwhile, the results of comparing the distribution density of positive cells in the same region of the spleen in different age groups showed that the distribution density of positive cells in the red pulp from high to low was pubertal, middle-aged, young and old camels, and there were significant differences among different age groups (P < 0.05). The overall trend of change was first increased and then decreased (Fig. 9B, Fig. 10C), and the change trend of distribution density in the marginal zone (Fig. 9B, Fig. 10B) was similar to that in the red pulp. Interestingly, the distribution density of positive cells in the white pulp was not significantly different among young, pubertal and middle-aged camels (P>0.05), but was significantly reduced in old camels (P < 0.05) (Fig. 9B, Fig. 10A).

Prokaryotic expression is the most widely used method to obtain protein antigens, which has many advantages, such as clear genetic background, low cost, rapidity, simplicity of operation, and can meet the requirements of rapid antigen preparation for antibody production [36, 37]. In the process of prokaryotic expression, the expression level of target protein is not only affected by the factors such as the bacterial strain type and the expression plasmid, but also related to other factors, including inducer concentration, induction time and induction temperature [38]. In this study, under the optimal IPTG concentration, as the induction time passed, the expression level of the TLR8 recombinant protein showed a trend of first increasing and then gradually decreasing, and the recombinant protein was mainly expressed in the form of inclusion bodies. These results were basically consistent with the previous studies, in which polyclonal antibodies were prepared by prokaryotic expression [30, 39]. The reason for this might be that the growth and reproduction of the bacterial population need to go through the lag phase, logarithmic phase, stationary phase and decline phase [40]. The expression level of the recombinant protein increased first, which may be due to the rapid growth of the bacterial population in a stable geometric progression after entering the logarithmic phase from the lag phase, and as the number of living bacteria linearly increased [41], the expression level of target protein also increased. However, as the induction time passed, the bacteria population entered the decline phase from the stationary phase, and the number of dead bacteria increased significantly, the bacteria aged, the cell wall thickened [41], and the incomplete release of protein during ultrasonic fragmentation resulted in the decrease of recombinant protein content. The recombinant protein appears in the form of inclusion bodies in the precipitate, which is related to the high expression of the protein in Escherichia coli [42], and the codon optimization of the truncated fragment may lead to the rapid expression of the recombinant protein and the formation of inclusion bodies. In addition, the low hydrophilicity of TLR8 recombinant protein may also be one of important reasons for its expression in the form of inclusion bodies. TLR8 mainly recognizes PAMPs through extracellular domain, so the extracellular domain of TLR8 was intercepted for prokaryotic expression, and the recombinant protein of expected size was obtained. After immunizing animals, the TLR8 polyclonal antibody was successfully prepared, and the titer of the antibody detected by indirect ELISA was 1:32000. Western blotting detection showed that the target band only appeared at 52 kDa, which was consistent with the expected size, indicating that this study successfully prepared a highly efficient and specific rabbit anti-bactrian camel TLR8 polyclonal antibody.

Innate immunity is a series of defense mechanisms formed during the germline evolution of the body, and pattern recognition receptors(PRRs)play an important role in innate immune responses [43]. TLR8, as an important PRR, can recognize specific PAMPs to active the body's innate immune response, and subsequently induce the adaptive immune responses for the clearance of infected pathogen [44]. As the largest peripheral immune organ of the body, the spleen contains multiple immune active cells and immune effect molecules, and is an important response site for innate and adaptive immunity, with a wide range of immunological functions [45]. The spleen is a highly expressed site of TLR8 [46], and the multiple immunological effects mediated by TLR8 may be an essential part of splenic immune function. Therefore, the distribution characteristics and age-related changes of TLR8 in the spleen were assessed in this study. The results of immunohistochemical staining showed that the distribution of TLR8 positive cells was similar in the spleen of Bactrian camels of different age groups, all of which were mainly scattered in the periarterial lymphatic sheath (PALS) of the white pulp, the marginal zone and the splenic cord and splenic sinus of the red pulp. Positive cell type was mainly macrophage, but also expressed in neutrophils and dendritic cells. These results were basically consistent with the previous studies, in which the distribution of TLR1 and TLR2 positive cells in the spleen and lymph nodes has been reported [47, 48], and also consistent with the cellular expression profile of TLR8 [9, 10], suggesting that macrophages may be the main cell type for TLR8 to exert immune function in the spleen. The white pulp of the spleen is the main immune region, and its PALS containing dense T lymphocytes is the main site of cellular immune response [49, 50]. TLR8 positive cells scattered in the PALS may effectively capture antigens and present them to T lymphocytes. The marginal zone, as the region where arterial blood first flows through, is the site where the spleen first contacts antigens and induces immune response [49, 50]. TLR8 positive cells scattered in the marginal zone may provide effective clearance of foreign bodies and antigens from the blood. The red pulp is the main part of the spleen to filter blood [49], and TLR8 positive cells scattered in the splenic cord and splenic sinus may be conducive to the phagocytosis and clearance of blood-borne antigens, as well as aging, deformed, and dysfunctional red blood cells, thereby maintaining iron homeostasis in the body. Macrophages, neutrophils, and dendritic cells in the spleen may recognize specific PAMPs through TLR8 homodimers to mediate innate immune responses. The scattered distribution of TLR8 positive cells in each region of the spleen may contribute to antigen capture, uptake, presentation, and clearance, and this distribution does not change with age.

Statistical results showed that the distribution density of TLR8 positive cells in different spleen regions at all ages from high to low was the red pulp, marginal zone and white pulp, and there were significant differences between different regions (p < 0.05), suggesting that the red pulp and marginal zone might play an important role in the innate immune response of the spleen. The reason for this might be that the majority of the splenic tissue (approximately 2/3) is red pulp, and the splenic sinuses and splenic cords are the main parts of the distribution of innate immune cells [51]. TLR8 positive cells are mainly macrophages, neutrophils and dendritic cells, which may be the reason for the high distribution density of TLR8 positive cells in the red pulp. In addition, the red pulp mainly serves as a blood filter, performing the functions of hematopoiesis, blood storage and removal of foreign materials, and the innate immune cells are mainly distributed in the red pulp [51], which may be conducive to phagocytize aging and dead red blood cells from the circulation, and detect pathogens and tissue damage. The marginal zone is the anatomical site where blood-borne pathogens enter the white pulp from the red pulp bloodstream [49]. TLR8 positive cells are more distributed in the marginal zone, which may be conducive to capture blood-borne antigens and apoptotic cells, and play an important role in immune surveillance and adaptive immune response. The white pulp is the main region of cellular and humoral immune responses in the spleen, accounting for 1/5 of the splenic tissue, mainly composed of T and B lymphocytes, and relatively few innate immune cells [51]. Scattered TLR8 positive cells can effectively mediate antigen presentation and regulate T cell responses. The distribution density of TLR8 positive cells 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. These results were consistent with the previous studies, in which the distribution of SIgA and IgG antibody-secreting cells has been reported in the small intestine and aggregated lymphoid nodules area of Bactrian camels of different ages [52, 53]. As the Bactrian camel grows older, its various growth and development indicators and organ functions gradually improve, and the immune function of the spleen also reaches the strongest. In this process, the increase of TLR8 positive cells such as macrophages, neutrophils and dendritic cells may be conducive to resist the invasion of pathogenic microorganisms for the body. With the aging of the camels, the density of TLR8 positive cells in each region of the spleen decreased significantly (p < 0.05). Several studies have reported that aging can lead to reduced expression and dysfunction of TLRs in macrophages and neutrophils [54–56], a decrease in the number of dendritic cells [57], and a decrease in the ability of macrophages to phagocytize apoptotic cells with age [56], which may reduce the distribution density of TLR8 positive cells in the spleen. These results provide support for the further study of immunomorphology and immunosenescence in the spleen of Bactrian camels.

In this study, the pET-28a-TLR8 recombinant plasmid was successfully constructed, the rabbit anti-Bactrian camel TLR8 polyclonal antibody with good specificity was prepared, and the distribution characteristics and age-related change patterns of TLR8 in the spleen of Bactrian camel were elucidated. TLR8 positive cells were mainly distributed in the red pulp and marginal region of the spleen, suggesting that these two areas play an important role in the 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 and mechanism of TLR8 signaling pathway.

Experimental animals and tissues collection

Twelve healthy Alashan Bactrian camels were divided into four groups according to age: young (1–2 years, n = 3), pubertal (3–5 years, n = 3), middle-aged (6–16 years, n = 3) and old (17–20 years, n = 3). These camels were obtained from the slaughterhouse (Minqin county, Gansu province of China), not starved before slaughter, anaesthetised intravenously with sodium pentobarbital (20 mg/kg) and exsanguinated to death. The spleen was separated from each Bactrian camel, and then gently washed with PBS to remove surface blood. Collected tissues were stored in labeled sampling bottles containing 4% paraformaldehyde (Solarbio, Beijing, China) for making paraffin sections.

Synthesis of TLR8 gene and construction of recombinant plasmid in Bactrian camel

The TLR8 mRNA sequence of Bactrian camel (GenBank: XM_010966216.2) has 4729 bp, and its coding sequence (247–3483) has 3237 bp, encoding a total of 1078 amino acids. The transmembrane structure of TLR8 protein was predicted by TMHMM Server 2.0 software (https://services.health-tech.dtu.

dk/services/TMHMM-2.0/), and Editseq program of DNAStar7.0 was used to intercept the extra-membrane region. SignalP-5.0 Server software (https://services.health-tech.-dtu.dk/services/SignalP-

5.0/) was used to predict and cut off the signal peptide of TLR8 protein, and then the hydrophobicity and antigenic epitope of TLR8 protein were predicted using Protean program of DNASTAR7.0, and Editseq program of DNAStar7.0 was used to intercept the extra-membrane amino acid sequence with higher antigen index as target fragment. The restriction sites of the base sequence corresponding to the amino acid were predicted using Primer Premier 5.0. The restriction site BamH Ⅰ and the start codon were added upstream of the base sequence, and the restriction site Xho Ⅰ and the stop codon were added downstream. Finally, the intercepted TLR8 base sequence was optimized and synthesized in Jinweizhi Biotechnology Co., Ltd., connected with the pET-28a (+) vector (Solarbio, Beijing, China) and transformed into competent cells DH5α (Solarbio, Beijing, China) for culture. The plasmid was extracted for sequencing and the positive recombinant plasmid with correct sequencing was named pET-28a-TLR8.

Prokaryotic expression of pET-28a-TLR8 recombinant plasmid

The pET-28a-TLR8 recombinant plasmid was transformed into competent cells BL21 (C1400, Solarbio, Beijing, China), and 700 µL LB liquid medium of uncontaining Kan⁺ was added for culture. Then, 100 µL of the bacterial liquid was spread on the plates (LB solid medium of containing Kan⁺) and cultured overnight at 37 ℃. A single colony was picked and inoculated in 5 mL LB liquid medium of containing Kan⁺, cultured at 37 ℃ until the OD₆₀₀ value reached 0.6–0.8, aspirated 1mL of the bacterial liquid as the control before induction and induced the remaining bacterial liquid using 1.0 mmol·L^-1 IPTG for 6 h. Then, all samples were detected by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Optimization of expression conditions of TLR8 recombinant protein in Bactrian camel

To improve the expression level of TLR8 recombinant protein, the concentration of IPTG and induction time were optimized. Different IPTG concentrations (0.1 mmol·L^-1, 0.3 mmol·L^།1, 0.5 mmol·L^།1, 0.7 mmol·L^།1, 0.9 mmol·L^།1, 1.0 mmol·L^།1 ) were used to induce the expression of TLR8 recombinant protein, and all samples were detected by 12% SDS-PAGE. The effect of different IPTG concentrations on recombinant protein expression was observed to determine the optimal inducer concentration. To determine the optimal induction time, under the optimal IPTG concentration, the expression level of TLR8 recombinant protein was detected by 12% SDS-PAGE at different induction times (2 h, 4 h, 6 h, 8 h, 10 h).

Purification of TLR8 recombinant protein in Bactrian camel

The expression of TLR8 recombinant protein was induced at the optimal IPTG concentration and optimal induction time. 1 mL of the bacterial liquid was aspirated as the control before ultrasonication, and the remaining portion was disrupted by ultrasound until the liquid became clear. After centrifugation, the lysed supernatant and precipitate were collected respectively for 12% SDS-PAGE detection to determine the expression form of the recombinant protein.

Based on the results of 12% SDS-PAGE detection, the lysed precipitates of the recombinant bacteria were collected for protein purification. The target protein was purified from lysed precipitates using His Tag Protein Purification Kit (Inclusion Body Protein) (CW0893S, CWBIO, Jiangsu, China) following the manufacturer’s instructions, and detected by 12% SDS-PAGE to observe the effect of protein purification. The content of target protein was detected using Ultraviolet spectrophotometer (GeneQuant 1300, General Electric Co., USA).

Preparation of rabbit anti-Bactrian camel TLR8 polyclonal antibody

To prepare rabbit anti-Bactrian camel TLR8 polyclonal antibody with good specificity and high titer, a reasonable immunization procedure was determined. Three healthy adult New Zealand white rabbits (male, 2.7±0.3 kg) were purchased from the experimental animal center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. After all rabbits were fed adaptively for 7 days, a small amount of blood was collected from the ear veins before immunization, and the serum was separated as negative control and stored in an ultra-low temperature refrigerator at -80 ℃ for later use. For the first immunization, the purified TLR8 recombinant protein was mixed with an equal volume of Freund’s complete adjuvant (F5881, Sigma, Missouri, USA) and fully emulsified, and then the rabbits were immunized at a dose of 800 µg per rabbit by subcutaneous multi-point injection at back, scapular and popliteal lymph nodes. One week later, the second immunization was carried out. The purified protein was fully emulsified with an equal volume of Freund’s incomplete adjuvant (F5506, Sigma, Missouri, USA), and then the rabbits were immunized by subcutaneous multi-point injection at back and popliteal lymph nodes at a dose of 400 µg per rabbit. After that, immunization was performed every other week (immunization method and dose were the same as the second). One week after the fourth immunization, the blood was collected from the heart, centrifuged to obtain rabbit anti-Bactrian camel TLR8 poly-antiserum and stored in an ultra-low temperature refrigerator at -80°C.

Detection of serum titer of Bactrian camel TLR8 polyclonal antibody

The titer of the prepared antibody was detected by indirect ELISA method. The purified TLR8 recombinant protein was used as the antigen, added to the enzyme linked immunosorbent assay plate at 5µg/well and kept at 4 ℃ overnight. After TBST washing for 5 min × 3 times, 5% skim milk powder (100 µL/well) was added to block at 37 ℃ for 1 h. After TBST washing for 5 min × 3 times, the rabbit antiserum diluted in gradient (dilution concentration from 1:2000 to 1:128000) was added at 100 µL/well, and incubated at 37 ℃ for 1 h. After TBST washing for 5 min × 3 times, HRP-labeled goat anti-rabbit IgG (BA1054, BOSTER, Wuhan, China) was diluted at 1:8000, added at 100 µL/well, and incubated at 37 ℃ for 1 h. Then, after washing with TBST again, TMB substrate chromogenic solution (PR1200, Solarbio, Beijing, China) was added at 100 µL/well in a dark environment. After color development at room temperature for 15 min, 2 mol·L^-1 H₂SO₄ was added to stop the reaction at 50 µL/well. Finally, the absorbance value of the sample at 450 nm was measured with ReadMax 1200 microplate reader (Shanghai Shanpu Biotechnology Co., Ltd., Shanghai, China). The highest dilution ratio of OD _positive /OD _negative ≥2.1 was used as the titer of the multi-antibody serum. The antiserum of each gradient was added in parallel three times, and the pre-immune rabbit serum diluted 1:2000 was added to the negative control group. Other operations were consistent with the experimental group, and blank control was also set.

Western blotting identification of Bactrian camel TLR8 polyclonal antibody

After the purified TLR8 recombinant protein was electrophoresed by 12% SDS-PAGE, the gel strip of appropriate size was cut at the 52 kDa, and electrotransferred to polyvinylidene fluoride (PVDF) membrane. TBST containing 5% skim milk powder was added to block at 37 ℃ for 2 h. Then, the rabbit antiserum (diluted 1: 800) as primary antibody was added, and incubated at 4 ℃ overnight. After TBST washing for 15 min × 3 times, HRP-labeled goat anti-rabbit IgG (diluted 1: 8000) was added, and incubated at room temperature for 2 h. After TBST washing for 15 min × 3 times, ECL luminescent solution (PE0010, Solarbio, Beijing, China) was added for color development, exposed, photographed and saved the picture.

Hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining

Fixed spleen tissues were respectively embedded in paraffin, and then cut into 5-µm-thick sections using a microtome (HistoCore MULTICUT, Leica, Germany) for routine hematoxylin and eosin (H&E) staining. The histological structure of the spleen of Bactrian camels was carefully observed by a light microscope and captured images using Olympus DP-71 microscopy system (Olympus, Hamburg, Germany).

The prepared paraffin sections were performed to indirect SABC immunohistochemical staining, and main staining steps were as follows: 1. The sections were deparaffinated, dehydrated and washed. 2. 3% H₂O₂ was added and incubated for 15 min at room temperature to eliminate endogenous peroxidase activity, and then washed with distilled water for 5 min × 3 times. 3. 0.1% trypsin was added and incubated for 40 min at 37 ˚C to repair the antigen, and then washed with distilled water for 5 min × 3 times; 4. To avoid non-specific binding, 5% BSA was added on the sections for blocking, and incubated for 45 min at 37 ˚C without washing. 5. The rabbit antiserum (diluted 1: 900), as primary antibody, was added to the positive group and incubated at 4 ˚C overnight to allow the antigen and antibody to react fully. PBS solution was added instead of the primary antibody in the negative control group. In subsequent experiment, both were washed separately with PBS for 5 min × 5 times. 6. HRP conjugated goat anti-rabbit IgG from immunohistochemical kit(SA1022, BOSTER, Wuhan, China), as secondary antibody, was added and incubated for 1 h at 37˚C, and then washed with PBS for 5 min × 5 times. 7. The SABC was added and incubated for 30 min at 37˚C, and then washed with PBS for 5 min × 5 times. 8. DAB colour rendering was performed at room temperature in a dark environment, and the sections were counterstained with hematoxylin dyeing solution (Solarbio, Beijing, China) and sealed with neutral balsam (Solarbio, Beijing, China).

Statistical analysis

Five sections were randomly selected from each sample, and ten microscopic fields for each of the white pulp, marginal zone and red pulp were randomly selected form each section, observed and photomicrographed. The number of TLR8 positive cells was counted in each microscopic field, and the density of positive cells was calculated by Image-Pro Plus 6.0, respectively. Statistically significant differences in the distribution density of TLR8 positive cells in different regions of the spleen of the same age group and in the same region of the spleen of different ages were analyzed by one-way analysis of variance followed by Duncan’s multiple range test. Data analyses were performed using IBM SPSS 23.0 (SPSS Inc., Chicago, USA). Graphs were constructed using Origin 2020 (OriginLab Inc., Northampton, USA). Differences were considered significant at P < 0.05.

TLRs: Toll-like receptors; TLR8: Toll-like receptor 8; PAMPs: Pathogen-associated molecular patterns; LRR: Leucine-rich repeat; TIR: Toll/IL-1 receptor; MyD88: Myeloid differentiation factor 88; IRAK1: IL-1R-associated kinase 1; TRAF6: TNF receptor-associated factor 6; IRF7: Interferon regulatory factor 7.

Acknowledgments

We would like to thank Wei-Wei Zeng, from Veterinary Pathology Laboratory of College of Veterinary Medicine, Gansu Agricultural University, China, for his help in the collected sample.

Authors contributions

Designed the experiments: Y-DF, W-HW. Collected sample: Y-DF, W-WZ, FX. Performed the experiments: Y-DF, J-YL. Analyzed and processed the data: Y-DF, L-PL, W-WZ. Contributed reagents/materials/analysis tools: Y-DF, L-PL, FX. Completed the writing of the manuscript: Y-DF. All authors read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (Grant number: 31960693 and 31760723).

Availability of data and materials

The data that support the findings of this study are available from the article or supplementary material, further inquiries can be directed to the corresponding authors.

Ethics approval and consent to participate

The authors confirm that all methods were performed in accordance with relevant guidelines and regulations. This study was approved by the Animal Care and Use Committee (IACUC) of College of Veterinary Medicine of Gansu Agricultural University (Approval No: GSAU-AEW-2021-0016). All experiments were performed in accordance with the IACUC guidelines, and all efforts were made to minimize animal suffering.

Consent for publication

Not applicable.

Competing interests

The authors declare that there is no financial or commercial conflict of interest.

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

Status:

Journal Publication

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Abstract

Background

Results

Conclusions

Figures

Background

Results

TLR8 gene synthesis and recombinant plasmid construction of Bactrian camel

Prokaryotic expression results of pET-28a-TLR8 recombinant plasmid

Optimization results of expression conditions of TLR8 recombinant protein in Bactrian camel

Purification results of TLR8 recombinant protein in Bactrian camel

Serum titer and specificity detection of rabbit Anti-Bactrian camel TLR8 polyclonal antibody

Distribution characteristics of TLR8 in the spleen of Bactrian camels of different ages

Changes in the distribution density of TLR8 positive cells with age

Discussion

Conclusions

Materials and methods

Experimental animals and tissues collection

Synthesis of TLR8 gene and construction of recombinant plasmid in Bactrian camel

Prokaryotic expression of pET-28a-TLR8 recombinant plasmid

Optimization of expression conditions of TLR8 recombinant protein in Bactrian camel

Purification of TLR8 recombinant protein in Bactrian camel

Preparation of rabbit anti-Bactrian camel TLR8 polyclonal antibody

Detection of serum titer of Bactrian camel TLR8 polyclonal antibody

Western blotting identification of Bactrian camel TLR8 polyclonal antibody

Hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining

Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1