Artificial antigen, anti-sheep scavenger receptor polyclonal antibody prepared from artificial antigen and application of anti-sheep scavenger receptor polyclonal antibody

Abstract

The invention discloses an artificial antigen, an anti-sheep scavenger receptor polyclonal antibody prepared from the artificial antigen and application of the anti-sheep scavenger receptor polyclonal antibody, and belongs to the technical field of immunity. Specifically, firstly, a sheep collagen-like structure macrophage receptor (MARCO) antigen is screened through antigen structure design, and the amino acid sequence of the sheep collagen-like structure macrophage receptor (MARCO) antigen is shown as SEQ ID NO.4. When the antigen is used for immunizing and preparing the antibody, the generated polyclonal antibody is high in titer and strong in specificity. Subsequently, an ELISA detection method for sheep MARCO is established based on the prepared polyclonal antibody, the polyclonal antibody is used as a coating antibody, an HRP-labeled donkey anti-goat antibody is used as a detection antibody, and the detection sensitivity of MARCO in a host is high. Therefore, a new original element is provided for MARCO detection, and the constructed MARCO detection method can effectively assist in screening and diagnosis of the mycoplasma ovipneumoniae.

Description

Technical Field

[0001] The present invention relates to an artificial antigen, an anti-sheep scavenger receptor polyclonal antibody prepared therefrom and application thereof, belonging to the field of immunological technology. Background Art

[0002] Scavenger receptors (SRs) are pattern recognition receptors that macrophages use to recognize and eliminate pathogens. They are divided into eight different classes (A–H) based on their functional and structural characteristics. Macrophage receptor with collagenous structure (MARCO) belongs to the A-class scavenger receptor class (SRAs). Elomaa O et al. first cloned and characterized the MARCO gene from mouse spleen and lymph nodes. Studies have shown that MARCO can bind to cell wall components of various Gram-positive and Gram-negative bacteria, such as bacterial lipopolysaccharide, teichoic acid, and acetylated low-density lipoprotein. By recognizing components of the bacterial cell wall, MARCO can mediate the phagocytosis of a wide range of pathogens and play a vital role in clearing pathogenic infections.

[0003] The expression distribution of MARCO in the body is strictly restricted and varies across species. For example, in pigs, MARCO is expressed in the lungs, spleen, and lymph nodes, but is essentially absent in pig alveolar macrophages. In sheep, MARCO is highly expressed in the lungs and low in alveolar macrophages. Whether this specificity of sheep MARCO expression is related to its ability to clear pathogenic infections remains to be determined.

[0004] However, the current lack of anti-sheep MARCO antibodies limits the research on sheep MARCO. Therefore, it is of great significance to find a method to stably obtain high-performance anti-sheep scavenger receptor polyclonal antibodies. Summary of the Invention

[0005] To address the above problems, the present invention attempts to prepare a polyclonal antibody against sheep MARCO based on the current lack of research on MARCO, especially the lack of research on MARCO in sheep and the specificity of sheep MARCO expression. The antibody has been tested to have good reactogenicity and immunogenicity, and can be used for subsequent research on the MARCO gene.

[0006] The first object of the present invention is to provide an artificial antigen, which comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO.4.

[0007] A second object of the present invention is to provide a method for preparing an artificial antigen, comprising the following steps: inserting a gene sequence encoding a polypeptide into an expression vector to obtain a recombinant plasmid, introducing the recombinant plasmid into a host cell to obtain a recombinant cell, and culturing the recombinant cell to prepare the artificial antigen;

[0008] The amino acid sequence of the polypeptide is shown in SEQ ID NO.4.

[0009] Furthermore, the gene sequence encoding the polypeptide is shown in SEQ ID NO.3.

[0010] Furthermore, the host cell includes a prokaryotic cell; preferably, the prokaryotic cell is Escherichia coli.

[0011] The third object of the present invention is to provide anti-sheep scavenger receptor polyclonal antibodies obtained by directly immunizing animals with the artificial antigen or the artificial antigen prepared by the preparation method.

[0012] Furthermore, the animals are non-human animals, including but not limited to mice, rabbits, etc.

[0013] Furthermore, the step of obtaining polyclonal antibodies includes the step of separating or purifying the serum of immune animals.

[0014] The fourth object of the present invention is to provide the use of the anti-sheep scavenger receptor polyclonal antibody in the preparation of MARCO protein detection products.

[0015] Furthermore, the MARCO protein detection products include but are not limited to enzyme-linked immunosorbent assay products (ELISA), immunoblotting detection products, immunofluorescence detection products, etc.; preferably, in the enzyme-linked immunosorbent assay products, the coating antibody is an anti-sheep scavenger receptor polyclonal antibody, and the detection antibody is a secondary antibody with an identification label.

[0016] The fifth object of the present invention is to provide the use of the anti-sheep scavenger receptor antibody in the preparation of a diagnostic product for a disease associated with MARCO protein abnormalities.

[0017] Furthermore, diseases associated with abnormal MARCO protein include but are not limited to lung diseases, liver diseases, infectious diseases, tumors, etc.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention obtains a MARCO antigen with good immunogenicity through design and screening. The antigen contains a region common to different MARCO spliceosomes and has excellent immunogenicity.

[0020] (2) The present invention expresses the above-mentioned MARCO antigen through prokaryotes, prepares polyclonal antibodies against MARCO by animal immunization, and proves its good immunogenicity and reactogenicity through Western blot and indirect ELISA tests. The expression distribution content of MARCO in various tissues of sheep is detected by the prepared polyclonal antibodies. Moreover, the present invention directly uses the recombinant protein inclusion bodies successfully expressed in prokaryotes to immunize mice. The prepared polyclonal antibodies have good effects, can induce a rapid immune response in the body, and can be widely used in the study of MARCO. Compared with conventional polyclonal antibody preparation, the operation is simple and the cost is low. Compared with monoclonal antibodies, the polyclonal antibodies prepared by the present invention have better reaction strength, simple preparation and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the prediction of the sheep MARCO functional region, where the area before the rectangle is the intracellular region, the area after the rectangle is the extracellular region, and the pentagon represents the scavenger receptor functional domain.

[0022] Figure 2 The structure prediction and antigenic region of sheep MARCO protein.

[0023] Figure 3 This is the sheep scoring curve for the Marco antigen region.

[0024] Figure 4 Shown are the Western blot results of different recombinant proteins.

[0025] Figure 5 Western blot results of polyclonal antibodies, where: M: Protein marker; 1: MARCO inclusion body protein; a: Serum from mice immunized with MARCO; b: His-tag antibody.

[0026] Figure 6 Western blot analysis of the MARCO distribution in various sheep tissues, including: M. Protein Marker; 1. Alveolar macrophages; 2. Lung; 3. Spleen; 4. Liver; 5. Inguinal lymph nodes; 6. Kidney; 7. Small intestine.

[0027] Figure 7 This is the standard curve for MARCO assay.

[0028] Figure 8 This is the ROC curve of the polyclonal antibody prepared in the present invention used for MARCO detection. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0030] Example 1 Screening and determination of antigens

[0031] MARCO is a pattern recognition receptor that is primarily expressed on the cell membrane ( Figure 1 ), the antibodies prepared from the intracellular region and transmembrane region are less effective, and the intracellular region and transmembrane region are shorter, so the prepared antibodies sometimes cannot be used for immunofluorescence detection; therefore, we choose the extracellular region to design antigens, and the extracellular region antigen structure is specific, which is a rational region for preparing antigens ( Figure 2 ).

[0032] Antigens: (1) Sheep MARCO isoform X1, see SEQ ID NO. 1; (2) Sheep MARCO isoform X2, see SEQ ID NO. 2; (3) Antigen design based on sheep MARCO variants isoform X1 and isoform X2:

[0033] Table 1 Antigen epitope prediction

[0034]

[0035]

[0036] The online tool was used to predict the antigen epitopes obtained from the above screening, and the results showed that they had good immunogenicity and high antigen scores ( Figure 3 ).

[0037] Based on the above results, we selected the antigen region with the best performance, SEQ ID NO.3 (amino acid sequence see SEQ ID NO.4), and conducted subsequent verification.

[0038] The sequence involved in the above steps is as follows:

[0039] SEQ ID NO. 1: sheep MARCO isoform X1, 1299 bp

[0040] ATGGAAAATGAGGAAATTCTCAAAGAGAAAGAGCTCTTGGCTAGC

[0041] ACTGAAGACGGAACGGCTCTCGACCAAACCATGTTGTCTAGGATGGAG

[0042] ACATTTGAAGTTAATGATCCAAAGCCCAAGAGGAGAAATGGAATGAACT

[0043] GCATCATGGTCCTAGTGGTCACGTACCTGGTTCTGCTCACTGTGGGCGCT

[0044] GGGTTATTGGTGATACAAGTTCTAAACCTGCAGGAGCGGCTCCGGATCC

[0045] TGGAGACACCCTACAACAATGAAACACAGGACCAGCTCACCCAAGTCC

[0046] TCACCAAGCAGGAGAGCCTACAGCAACGGATGGACAACTTTATTCAGAT

[0047] CCCAGGACCCCCAGGACCCCCAGGACCCCGAGGCCCACCAGGAATCAA

[0048] GGGAGAGGCAGGAAGAAAAGGAGACATGGGCATGAAAGGAAACATGG

[0049] GCATGAAAGGAGACGTGGGCATGAAAGGAGACGTGGGCATGAAAGGA

[0050] GACGTGGGCATGAAAGGAGACGTGGGCATGAAAGGAGACGTGGGCATG

[0051] AAAGGAGACATGGGGGTCATGGGACCCCCCGGAGCTCGGGGGGATAAA

[0052] GGTGACTCAGGAAAGCCAGGTTCCCCAGGATTGGCTGGAATTCCTGGAA

[0053] TCAAAGGTGATCAAGGACCACCTGGAGTGAAGGGTCTTCCAGGCTTTC

[0054] CAGGAGCTGCGGGATCCCCAGGTGCCAAGGGTGAGACTGGCAGCACTG

[0055] GCCCCACTGGGCCGATAGGACCACCAGGGATGCCCGGGAGACCAGGAG

[0056] TTGAAGGCGTAAAAGGAAGCAAGGGGGACACAGGACTTCAAGGACAG

[0057] AAAGGAACAAAAGGAGAATCAGGATTTCCAGGCCTTGCAGGCATGAAG

[0058] GGAGAGAAGGGAAGCCCGGGCCTGGGAGGCCTCAAGGGGGCACCTGG

[0059] ACCAAGTGGCCAAAAGGGAGAGCCCGGATCGAGAGGAGCTACTGGGC

[0060] CACAAGGATCCAAGGGAGAAAAAGGTCAAAAAGGTGACTCCTCGTTAA

[0061] CCGTCCGGATTATCGGCTCTAGGAACCGAGGCCGAGCTGAAGTTTTCTAT

[0062] AATGGTGCATGGGGGACAATCTGTGATGACAACTGGGACAATTCAGATG

[0063] CCACTGTCTTCTGCCGCATGCTGGGTTACTCCTCTGGAAGTGCTCTCTAC[[ID=:22]]

[0064] AATGTGGGAGCTGGCTCTGGAAACATCTGGCTGGATGATGTTGCATGTT

[0065] CAGGGTCAGAATGGACCCTGTGGGACTGCAACAAGAGCAGCTGGGGCT

[0066] CTCACAACTGCAACCACAATGAGGATGCGGGCGTGAACTGCAGCTGA

[0067] SEQ ID NO.2: Sheep MARCO isoform X2, 1092bp

[0068] ATGGAAAATGAGGAAATTCTCAAAGAGAAAGAGCTCTTGGCTAGC

[0069] ACTGAAGACGGAACGGCTCTCGACCAAACCATGTTGTCTAGGATGGAG

[0070] ACATTTGAAGTTAATGATCCAAAGCCCAAGAGGAGAAATGGAATGAACT

[0071] GCATCATGGTCCTAGTGGTCACGTACCTGGTTCTGCTCACTGTGGGCGCT

[0072] GGGTTATTGGTGATACAAGTTCTAAACCTGCAGGAGCGGCTCCGGATCC

[0073] TGGAGACACCCTACAACAATGAAACACAGGACCAGCTCACCCAAGTCC

[0074] TCACCAAGCAGGAGAGCCTACAGCAACGGATGGACAACTTTATTCAGAT

[0075] CCCAGGACCCCCAGGACCCCCAGGACCCCGAGGCCCACCAGGAATCAA

[0076] GGGAGAGGCAGGATTGGCTGGAATTCCTGGAATCAAAGGTGATCAAGG

[0077] ACCACCTGGAGTGAAGGGTCTTCCAGGCTTTCCAGGAGCTGCGGGATC

[0078] CCCAGGTGCCAAGGGTGAGACTGGCAGCACTGGCCCCACTGGGCCGAT

[0079] AGGACCACCAGGGATGCCCGGGAGACCAGGAGTTGAAGGCGTAAAAG

[0080] GAAGCAAGGGGGACACAGGACTTCAAGGACAGAAAGGAACAAAAGGA

[0081] GAATCAGGATTTCCAGGCCTTGCAGGCATGAAGGGAGAGAAGGGAAGC

[0082] CCGGGCCTGGGAGGCCTCAAGGGGGCACCTGGACCAAGTGGCCAAAA

[0083] GGGAGAGCCCGGATCGAGAGGAGCTACTGGGCCACAAGGATCCAAGGG

[0084] AGAAAAAGGTCAAAAAGGTGACTCCTCGTTAACCGTCCGGATTATCGGC

[0085] TCTAGGAACCGAGGCCGAGCTGAAGTTTTCTATAATGGTGCATGGGGGA

[0086] CAATCTGTGATGACAACTGGGACAATTCAGATGCCACTGTCTTCTGCCG

[0087] CATGCTGGGTTACTCCTCTGGAAGTGCTCTCTACAATGTGGGAGCTGGC

[0088] TCTGGAAACATCTGGCTGGATGATGTTGCATGTTCAGGGTCAGAATGGA

[0089] CCCTGTGGGACTGCAACAAGAGCAGCTGGGGGCTCTCACAACTGCAACC

[0090] ACAATGAGGATGCGGGCGTGAACTGCAGCTGA

[0091] SEQ ID NO.3: Prokaryotic expressed MACRO gene sequence (italics indicate enzyme cleavage sites, which are for illustration only and are not included in SEQ ID NO.3)

[0092] CATATG(MluI)

[0093] TTGGCTGGAATTCCTGGAATCAAAGGTGATCAAGGACCACCTGGAGTGAAGGGTCTTCCAGGCTTTCCAGGAGCTGCGGGATCCCCAGGTGCCAAGGGTGAGACTGGCAGCACTGGCCCCACTGGGCCGATAGGACCACCAGGGATGCCCGGGAGACCAGGAGTTGAAGGCGTAAAAGGAAGCAAGGGGGACACAGGACTTCAAGGACAGAAAGGAACAAAAGGAGAATCAGGATTTCCAGGCCTTGCAGGCATGAAGGGAGAGAAGGGAAGCCCGGGCCTGGGAGGCCTCAAGGGGGCACCTGGACCAAGTGGCCAAAAGGGAGAGCCCGGATCGAGAGGAGCTACTGGGCCACAAGGATCCAAGGGAGAAAAAGGTCAAAAAGGTGACTCCTCGTTAACCGTCCGGATTATCGGCTCTAGGAACCGAGGCCGAGCTGAAGTTTTCTATAATGGTGCATGGGGGACAATCTGTGATGACAACTGGGACAATTCAGATGCCACTGTCTTCTGCCGCATGCTGGGTTACTCCTCTGGAAGTGCTCTCTACAATGTGGGAGCTGGCTCTGGAAACATCTGGCTGGATGATGTTGCATGTTCAGGGTCAGAATGGACCCTGTGGGACTGCAACAAGAGCAGCTGGGGCTCTCACAACTGCAACCACAATGAGGATGCGGGCGTGAACTGCAGC CTCGAG(XhoI)

[0094] SEQ ID NO.4: Marco antigen sequence

[0095] LAGIPGIKGDQGPPGVKGLPGFPGAAGSPGAKGETGSTGPTGPIGPPGMPGRPGVEGVKGSKGDTGLQGQKGTKGESGFPGLAGMKGEKGSPGLGLKGAPGPSGQKGEPGSRGA TGPQGSKGEKGQKGDSSLTVRIIGSRNRGRAEVFYNGAWGTICDDNWDNSDATVFCRMLGYSSGSALYNVGAGSGNIWLDDVACSGSEWTLWDCNKSSWGSHNCNHNEDAGVNCS

[0096] Example 2 Preparation of polyclonal antibodies

[0097] 1. Inducible expression of recombinant protein

[0098] The antigen sequences prepared in Example 1 were inserted into the pET28a vector to prepare the recombinant plasmid pet-28a-MARCO. The recombinant plasmid pet-28a-MARCO was transfected into the competent cells BL21, and a single colony was picked and inoculated into 5 ml of LB liquid medium containing Kana, and cultured at 37°C with shaking overnight. The next day, the bacterial liquid was inoculated into LB liquid medium containing Kana at a ratio of 1:100, and cultured at 37°C with shaking until D 600 When the pH value reaches 0.6, IPTG is added to a final concentration of 1 mmol / L, and then the culture is cultured at 37°C for 4-6 hours, and the bacterial solution is centrifuged at 4°C and 10,000 rpm for 10 minutes. The precipitated bacteria after centrifugation are resuspended in PBS buffer and washed 3 times with PBS, and then placed in -80°C and repeatedly frozen and thawed 3 times; the frozen and thawed bacterial solution is ultrasonically disrupted on ice and then centrifuged at 4°C and 12,000 rpm for 5 minutes. The supernatant and precipitate are separated and stored separately, and polyacrylamide gel electrophoresis (SDS-PAGE) is used to analyze the induced expression of the target protein.

[0099] 2. SDS-PAGE analysis of expression products

[0100] Fix the glass plate in the electrophoresis tank as required, prepare the separation gel and stacking gel according to the kit instructions, and pour the prepared separation gel between the glass plates until it is about 3 cm from the edge of the glass plate. Cover the gel surface with a layer of distilled water to make the gel surface flat. After the separation gel is completely solidified, use filter paper to absorb the upper layer of water, then pour the stacking gel into the glass plate, insert a clean sample comb, and let it stand at room temperature for 20 minutes. Mix the sample with 4μL 5× SDS loading buffer and 12μL PBS, heat in a 100℃ water bath for 10 minutes to denature the protein, and then centrifuge at 4℃ 10000rpm for 5 minutes. After the stacking gel is solidified, add electrophoresis buffer to the electrophoresis tank and cover the glass plate. Carefully remove the sample comb and add the protein marker and sample in order. Initially, perform electrophoresis at a constant voltage of 80V. After all the samples have entered the separation gel, adjust the voltage to 120V. End the electrophoresis when the bromophenol blue indicator reaches the bottom. Remove the gel, stain it with Coomassie blue for 1 hour, rinse it with distilled water, and place it in a decolorizing solution for decolorization until the blue background of the gel is removed for observation.

[0101] See the results Figure 4 A, a target band of approximately 29 kDa can be found in the separated precipitate, demonstrating that MARCO represented by SEQ ID NO. 3 is mainly expressed in the form of inclusion bodies. Figure 4 B shows that SEQ ID NO. 1 and SEQ ID NO. 2 showed clear bands of 44 kDa and 52 kDa in the supernatant, and were mainly expressed in the form of inclusion bodies. Figure 4 C Coomassie Brilliant Blue staining showed that: 4 hours after induction (lane 1), the expression level of SEQ ID NO. 3 MARCO protein reached a peak, and the gray value increased by 7.8 times compared with the uninduced group (lane 2).

[0102] 3. Preparation of polyclonal antibodies

[0103] The resuspended recombinant protein inclusion body precipitate was mixed and emulsified with Freund's complete adjuvant, and mice were immunized with a protein content of 100 μg / mouse through multiple injections at the neck / back. Then, booster immunizations were performed every other week for a total of 5 immunizations. After the fifth immunization, blood was collected from the mice's eyeballs, and the collected blood was centrifuged and stored at -20°C.

[0104] 4. Antibody titer detection

[0105] The above-mentioned different recombinant proteins were used as antigens and coated on a 96-well ELISA plate at 4°C overnight, washed three times with PBST, and patted dry; blocked with 5% skim milk at 37°C for 1 hour, washed three times with PBST, and patted dry; immune mouse serum diluted at a ratio of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000 was added, incubated at 37°C for 1 hour, washed three times with PBST, and patted dry; diluted HRP-labeled sheep anti-mouse IgG was added, incubated at 37°C for 1 hour, washed three times with PBST, and patted dry; TMB color development solution was added to each well and color was developed in the dark for 10 minutes, then stop solution was added to terminate the reaction, and the OD was measured. 450 value.

[0106] The ELISA assay performed on the immunized mouse serum tested the titers of the polyclonal antibodies. The results showed that the titer of the polyclonal antibody against SEQ ID NO. 1 reached a P / N ratio of greater than 2.1 at a titer of 1:8000, the titer of the polyclonal antibody against SEQ ID NO. 2 reached a P / N ratio of greater than 2.1 at a titer of 1:4000, and the titer of the polyclonal antibody against SEQ ID NO. 3 reached a P / N ratio of greater than 2.1 at a titer of 1:16000. Therefore, the antibody against SEQ ID NO. 3 had the highest titer, with the titer of the prepared polyclonal antibody reaching 1:16000. This demonstrates that the prepared polyclonal antibody can stimulate an immune response in mice.

[0107] Table 1 Potency test

[0108]

[0109] +: effective dilution; -: ineffective dilution

[0110] 5. Polyclonal Antibody Western Blot Analysis

[0111] First, perform SDS-PAGE electrophoresis on the recombinant protein inclusion body precipitate. After electrophoresis, soak the cut PVDF membrane in methanol for 1-2 minutes. Then, soak the filter paper in transfer buffer. Place a layer of sponge on the black panel, followed by three layers of filter paper, gel, PVDF membrane, three layers of filter paper, and finally a layer of sponge. Align the filter paper, gel, and PVDF membrane, carefully removing any air bubbles. Place the clips in the transfer tank and carefully close the cover. Cycle at 300mA for 70 minutes. After the transfer is complete, remove the PVDF membrane and place it in 5% skim milk for blocking for 1 hour. Then add TBST and shake for 10 minutes, repeating three times. Incubate with 1:500 primary antibody (immune mouse serum) at 4°C overnight, discard the primary antibody solution, add TBST and shake for 10 minutes, repeating three times. Incubate with 1:5000 secondary antibody (HRP-labeled sheep anti-mouse IgG) at 37°C for 50 minutes, discard the secondary antibody solution, add TBST and shake for 10 minutes, repeating three times, and then add ECL color development solution for exposure and observation. Then, use His-tag-labeled sheep anti-mouse IgG as the primary antibody to detect and analyze the recombinant protein inclusion bodies by Western blot.

[0112] Western blot results showed that ( Figure 5 ), whether the immune mouse serum or His-tag labeled IgG was used as the primary antibody, an obvious target band could be found at 29 kDa, indicating that the prepared polyclonal antibody had good reactivity and was specific to the recombinant MARCO protein.

[0113] Western blot was used to detect the distribution of proteins in various tissues of sheep: the alveolar macrophages, liver, spleen, lung, inguinal lymph node, kidney and small intestine of healthy sheep were lysed and extracted, and the protein was detected by Western blot method, with the serum of immune mice as the primary antibody and sheep anti-mouse β-actin as internal reference. Protein lysis buffer was used to extract proteins from sheep alveolar macrophages, lung, spleen, liver, inguinal lymph node, kidney and small intestine, and after SDS-PAGE transfer to PVDF membrane, Western blot was performed, with β-actin as internal reference. The results are as follows Figure 6 As shown, MARCO is expressed in the lung, liver, spleen and inguinal lymph nodes, and a clear target band can be found at 58 kDa in the figure, while it is almost not expressed in alveolar macrophages, small intestine and kidney, and no target band can be observed.

[0114] Example 3 Detection of MARCO in Serum of Sheep Infected with Mycoplasma ovipneumoniae (MO)

[0115] The MARCO content in the serum of sheep in the MO infection group showed an increasing trend, with significant differences (Table 2). The increase in MARCO content can indirectly determine the MO infection status of sheep.

[0116] The polyclonal antibody prepared in Example 2 was used for MARCO detection. The specific steps are as follows: (1) The purified polyclonal antibody was diluted to 5 μg / ml with coating buffer (pH 9.6 0.05 M carbonate buffer), 200 μl / well, and incubated at 4°C overnight. (2) The antibody was discarded, patted dry, and 300 μl / well of washing solution (PBST) was added. The plate was shaken and patted dry. Repeat twice. (3) Blocking solution containing 2% BSA was added at 250 μl / well and incubated at 37°C for 2 h. (4) MARCO standards (0, 1, 2.5, 5, 10, 50, 100 ng / ml) or serum to be tested were dissolved in 1 ml of distilled water. 50 μl of standard and 150 μl of buffer (PBST containing 0.2% BSA) were added to each well and incubated at 37°C for 2 h. (5) The plate was washed three times, and HRP-labeled donkey anti-sheep antibody diluted in buffer was added at 200 μl / well and incubated at 37°C for 1 h. (6) Wash 5 times. Add TMB colorimetric solution, 200 μl / well, and develop at room temperature in the dark for 10 min. (7) Add 150 μl / well stop solution (1 M H2SO4), shake for 2 min, and measure the OD value of each well at a wavelength of 450 nm using a microplate reader.

[0117] Standard curve see Figure 7 The regression equation was y = 0.7997x + 0.0235, with a slope of 0.7997 (OD / μg / mL), an intercept of 0.0235 (close to the negative control OD value of 0.042), and an R² of 0.9994 (>0.99, indicating excellent linearity). The linear range was 0-2 μg / mL (covering the highest value of 0.728 μg / mL in the infection group). Sensitivity validation results (Table 2) showed that the OD value of the 0.1 μg / mL standard was 0.105, significantly higher than the blank (p < 0.001). The difference between the lowest concentration (0.1 μg / mL) and the blank was 0.063 OD.

[0118] The results in Table 3-4 show that the lower limit of detection is 0.044 μg / mL (negative control mean + 3SD), which can effectively distinguish the concentration difference between the normal group (0.208 μg / mL) and the infected group (0.641 μg / mL), showing a detection dynamic range of more than 3 times; the CV% of repeated detection within the group is controlled at 1.62-4.63%, with high accuracy; the 95% confidence interval shows that there is no overlap between the normal group [0.193-0.223] and the infected group [0.553-0.728], and the extremely significant p value (<0.0001) confirms the statistical reliability of the detection difference; the negative control value (0.044 μg / mL) is only 6.8% of the infected group, and the concentration distribution of the normal group and the infected group shows a clear bimodal feature, with good specificity; the standard deviation of the three repeated detections is <0.03 μg / mL, the CV% of the normal group is controlled between 1.7-3.7%, and the CV% of the infected group is maintained at 2.9-4.4%. ROC curve analysis is shown in Figure 8 .

[0119] Table 2 Standard curve

[0120]

[0121] Table 3 Detection of MARCO content in serum of MO infected sheep

[0122]

[0123]

[0124] Table 4 MO infection ELISA test results

[0125]

[0126] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An antigen, characterized in that The antigen includes a polypeptide having an amino acid sequence as shown in SEQ ID NO.

4.

2. A method for preparing an antigen, characterized in that: The following steps are involved: Inserting a gene sequence encoding the polypeptide into an expression vector to obtain a recombinant plasmid, introducing the recombinant plasmid into a host cell to obtain a recombinant cell, and culturing the recombinant cell to prepare the antigen; The amino acid sequence of the polypeptide is shown in SEQ ID NO.

4.

3. The preparation method according to claim 3, characterized in that The gene sequence encoding the polypeptide is shown in SEQ ID NO.

3.

4. The preparation method according to claim 3, characterized in that The host cell includes a prokaryotic cell; Preferably, the prokaryotic cell is Escherichia coli.

5. Anti-sheep scavenger receptor polyclonal antibodies obtained by immunizing an animal with the antigen according to claim 1 or the antigen prepared by the preparation method according to any one of claims 2 to 4 as an immunogen.

6. Use of the anti-sheep scavenger receptor polyclonal antibody according to claim 5 in the preparation of a MARCO protein detection product.

7. The use according to claim 6, characterized in that The MARCO protein detection products include enzyme-linked immunosorbent assay products, immunoblotting assay products or immunofluorescence assay products.

8. The use according to claim 7, characterized in that The MARCO protein detection product includes a coating antibody and a detection antibody. The coating antibody is the anti-sheep scavenger receptor polyclonal antibody, and the detection antibody is a secondary antibody with an identification label.

9. A MARCO protein detection product, characterized in that: The MARCO protein detection product contains the anti-sheep scavenger receptor polyclonal antibody according to claim 5.

10. Use of the anti-sheep scavenger receptor polyclonal antibody according to claim 5 in the preparation of a diagnostic product for diseases associated with MARCO protein abnormalities.

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