A recombinant protein of monkey serum amyloid A, a monoclonal antibody thereof, and a preparation method and application thereof

Abstract

The present invention provides a recombinant protein of monkey serum amyloid A, a monoclonal antibody thereof, and a preparation method and application thereof. The amino acid sequence of the recombinant protein is as shown in SEQ ID No.1, and the nucleotide sequence encoding the recombinant protein is as shown in SEQ ID No.2. By optimizing the antigen peptide sequence and expression vector, the efficient and soluble expression of monkey serum amyloid A in Escherichia coli is achieved. A murine monoclonal antibody specifically binding to monkey serum amyloid A is screened based on the recombinant protein of monkey serum amyloid A, and a pair of monoclonal antibodies that can be paired is screened using the above murine monoclonal antibody. Further, a double-antibody sandwich immuno-ELISA detection method or kit for the recombinant protein of monkey serum amyloid A is constructed based on the combination of the pair of monoclonal antibodies, which has high detection sensitivity and can be well applied to the detection of monkey serum.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a recombinant protein of monkey serum amyloid A, a monoclonal antibody, and a preparation method and application thereof. Background Art

[0002] Infectious diseases are extremely common clinically, and laboratory tests are crucial for the diagnosis and treatment of infectious diseases. The four most commonly used indicators are C-reactive protein (CRP), procalcitonin (PCT), serum amyloid A (SAA), and interleukin-6 (IL-6). The details of the four indicator detections are as follows in the table:

[0003]

[0004] Serum amyloid A (SAA) is an acute-phase protein mainly derived from liver cells. When healthy, the blood concentration of serum amyloid A is extremely low. When the human body has an inflammation, its blood concentration will increase rapidly, especially in the early stage after the inflammatory reaction. And because of its relatively fast metabolism, the blood concentration of SAA will rapidly decline during the recovery period of inflammation. Therefore, the determination of SAA level is often used clinically for the diagnosis and condition monitoring of infectious diseases.

[0005] Laboratory animals are the main experimental subjects for preclinical evaluation of drugs, and monkeys are an important species for preclinical research. The application of laboratory animals in new drug research and development covers the preclinical research and clinical research stages, especially more in preclinical research. The pharmacological effects of drugs are evaluated through different experimental methods, their action mechanisms are studied, and their toxic effects are observed to prove the effectiveness and safety of the drugs. Monkeys are the closest to humans in terms of genetic relationship, with 75% - 98.5% homology with human genetic material, which is the animal with the highest homology with humans. Therefore, monkeys are also the most ideal animals closest to humans in experiments.

[0006] The available amino acid sequence of serum amyloid A (shown as SEQ ID No.11) and the corresponding nucleotide sequence (shown as SEQ ID No.12) can be queried now. However, the antibody prepared from the protein expressed using this sequence has relatively low sensitivity and cannot meet the application of the monkey serum amyloid A ELISA kit. Currently, there is no ELISA detection kit with high sensitivity for monkey serum amyloid A applied to the detection of monkey serum amyloid A, which is one of the four major indicators of monkey inflammation. Summary of the Invention

[0007] In view of this, the present invention provides a recombinant protein of monkey serum amyloid A, a monoclonal antibody thereof, and a preparation method and application thereof. The present invention utilizes bioinformatics software to optimize and select a polypeptide of monkey serum amyloid A, which is predicted to be expressed in Escherichia coli. Through immunization and screening, a specific paired monoclonal antibody against monkey serum amyloid A is obtained, and a double-antibody sandwich ELISA detection system is established, providing a new approach for preclinical research on monkey inflammation models.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] One object of the present invention is to provide: a recombinant protein of monkey serum amyloid A, and the amino acid sequence of the recombinant protein is as shown in SEQ ID No.1.

[0010] Another object of the present invention is to provide: a nucleotide sequence encoding the above-mentioned recombinant protein of monkey serum amyloid A, and the nucleotide sequence is as shown in SEQ ID No.2.

[0011] A third object of the present invention is to provide: a preparation method of the above-mentioned recombinant protein of monkey serum amyloid A, comprising the following steps: first, connecting the gene encoding the recombinant protein to an expression vector to construct a recombinant plasmid; then, transforming the recombinant plasmid into competent cells, inducing expression and purifying, to obtain the recombinant protein of monkey serum amyloid A.

[0012] Furthermore, the gene encoding the recombinant protein is codon-optimized by the E.coli expression system;

[0013] The expression vector is any one of pET28a, pET32a, pET40b, pSmart1, and pCold-TF;

[0014] The competent cells are any one of BL21, Rosetta, and OrigamiB.

[0015] A fourth object of the present invention is to provide: a monoclonal antibody that specifically reacts with the above-mentioned recombinant protein of monkey serum amyloid A, and the monoclonal antibody includes SAA-3# and SAA-8#;

[0016] Among them, the amino acid sequence of the heavy chain variable region of the monoclonal antibody SAA-3# is as shown in SEQ ID No.3, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No.4, the amino acid sequence of the light chain variable region is as shown in SEQ ID No.5, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID No.6;

[0017] The amino acid sequence of the heavy chain variable region of the monoclonal antibody SAA-8# is shown in SEQ ID No. 7, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No. 8, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 9, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No. 10.

[0018] The fifth object of the present invention is to provide: A method for preparing the above monoclonal antibody, comprising the following steps:

[0019] S1. Add an adjuvant to the above-mentioned recombinant simian serum amyloid A protein, mix, and immunize female BALB / c mice.

[0020] S2. Take the spleen cells of the immunized mice and fuse them with SP2 / 0 myeloma cells in the logarithmic growth phase, screen and culture to obtain hybridoma cells.

[0021] S3. Expand and culture the screened hybridoma cells, intraperitoneally inject female BALB / c mice, collect ascites, purify and screen after centrifugation to obtain the monoclonal antibody.

[0022] The sixth object of the present invention is to provide: An ELISA detection method based on the above monoclonal antibody.

[0023] The seventh object of the present invention is to provide: The application of the above ELISA detection method in detecting simian serum amyloid A.

[0024] The eighth object of the present invention is to provide: A simian serum detection kit, which contains the above monoclonal antibody.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] (1) The present invention provides a recombinant simian serum amyloid A protein, and through the optimization of the antigen peptide sequence and expression vector, the efficient, soluble and active expression of simian serum amyloid A in Escherichia coli is achieved.

[0027] (2) The present invention screens a murine monoclonal antibody that specifically binds to simian serum amyloid A based on the recombinant simian serum amyloid A protein, and uses the above murine monoclonal antibody to screen a pair of paired monoclonal antibodies. Further, a double-antibody sandwich ELISA detection method or kit for simian serum amyloid A is constructed based on this pair of monoclonal antibody combinations, which can specifically detect simian serum amyloid A protein without cross-reaction, has a high detection sensitivity (reaching 0.08 ng / mL), and provides a new approach for preclinical simian inflammation model research. Description of the Drawings

[0028] Figure 1 Electrophoresis diagram of the recombinant protein of monkey serum amyloid A described in Example 1 of the present invention.

[0029] Figure 2 ELISA detection standard curve diagram of monkey serum amyloid A described in Example 3 of the present invention. Detailed implementation manners

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the scope protected by the present invention.

[0031] Example 1

[0032] This example provides a recombinant protein of monkey serum amyloid A, the amino acid sequence of which is shown in SEQ ID No.1, and the gene sequence encoding the recombinant protein is shown in SEQ ID No.2. The specific sequences are as follows:

[0033] SEQ ID No.1: MKLLTGLVQAGANLGVNSRSWFSFLGEAYDGARDMWRAYSKEANYINADKYFHARGNSFAAQRGPGGAWAAEQKSDARENIQKLLGDTLADQAANEWGRSDMWRNHFRPAGLPEKY.

[0034] SEQ ID No.2: atgaaactgctgaccggcctggtgcaggcgggcgcgaacctgggcgtgaacagccgcagctggtttagctttctgggcgaagcgtatgatggcgcgcgcgatatgtggcgcgcgtatagcaaagaagcgaactatattaacgcggataaatattttcatgcgcgcggcaacagctttgcggcgcagcgcggcccgggcggcgcgtgggcggcggaacagaaaagcgatgcgcgcgaaaacattcagaaactgctgggcgataccctggcggatcaggcggcgaacgaatggggccgcagcgatatgtggcgcaaccattttcgcccggcgggcctgccggaaaaatat.

[0035] The preparation method of the above-mentioned recombinant monkey serum amyloid A protein comprises the following steps:

[0036] (1) Plasmid construction: Use computational software to predict and select the one containing the main antigenic epitopes of monkey serum amyloid A protein for expression, as shown in SEQ ID No.2. This polypeptide segment can be expressed in the E. coli prokaryotic expression system. The gene is cloned into the pET28a vector through the E. coli expression system to obtain the recombinant plasmid pET28a-SAA expressing monkey serum amyloid A protein.

[0037] (2) Induced expression: Transform the recombinant plasmid pET28a-SAA into BL21(DE3) competent cells. Spread the transformed bacteria on an LB agar plate containing 50 μg / mL kanamycin and culture overnight at 37°C. Pick a single colony and inoculate it into 4 mL of LB medium containing 50 μg / mL kanamycin, and culture overnight with shaking at 37°C and 230 rpm. Inoculate according to 1% of the total volume of the medium into an LB medium containing 50 μg / mL kanamycin, and culture with shaking at 37°C and 220 rpm for about 3 hours until the OD 600 reaches 0.6. Add IPTG with a final concentration of 0.1 mM, and induce for 15 hours at 16°C and 180 rpm, then collect the bacterial cells.

[0038] (3) Purification of the recombinant protein: Since the expressed recombinant protein carries a histidine tag, use a protein purifier and a chromatography column for preliminary crude purification, and then obtain the purified recombinant protein, which is the recombinant monkey serum amyloid A protein, through ion exchange chromatography. The amino acid sequence is as shown in SEQ ID No.1.

[0039] (4) Identify the purity of the recombinant protein by SDS-PAGE electrophoresis: Protein sample pretreatment: Take samples respectively and add the same volume of 2×SDS loading buffer, place in a boiling water bath for 10 min, centrifuge at 12000 rpm for 3 min, and take the supernatant and add it to the sample well. After detection, the purified protein is the recombinant monkey serum amyloid A protein, and the electrophoresis pattern is as Figure 1 shown.

[0040] Example 2

[0041] This example provides a method for screening monoclonal antibodies based on the recombinant protein monkey serum amyloid A. The specific operation is as follows:

[0042] S1. Mouse immunization: Mix 50 μg of recombinant simian serum amyloid A protein with an equal volume of Freund's complete adjuvant until completely emulsified to obtain a mixture. Subcutaneously inject the mixture at multiple points to immunize 6-week-old female BALB / c mice. At week 0 and week 2, emulsify the Freund's incomplete adjuvant and recombinant protein in equal proportions according to the above method and immunize again. At week 6, collect mouse serum to detect the antibody titer, and select the mouse with the highest titer to boost immunize 20 μg of recombinant simian serum amyloid A protein through the tail vein. Three days later, take the mouse spleen for the preparation of hybridoma cells.

[0043] S2. Screening of hybridoma cell lines: After fusing all the spleen cells of the immunized mice with SP2 / 0 myeloma cells in the logarithmic growth phase, place them in HAT medium for screening culture. When the fused cells grow to 1 / 2 of the well bottom, screen for positive clones by indirect ELISA with simian serum amyloid A protein. And achieve a 100% positive rate through 2 - 3 consecutive limited dilution cloning methods. Expand the culture and cryopreserve the cell lines; among them, the method for screening positive clones by indirect ELISA is as follows: First, coat the recombinant protein in a microplate: The coating buffer is carbonate buffer (prepared by dissolving 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate in 1 L of pure water), the coating concentration is 1 μg / mL, 50 μl / well, overnight at 4°C; Block with 3% sucrose and 1% BSA, 150 μL per well, 2 hours at 37°C, wash the plate once with washing solution PBST (PBS containing 0.05% Tween-20), pat dry; Add 50 μL of cell culture supernatant, react at 37°C for 30 min. Discard the liquid in the well, wash the plate 4 times with PBST washing solution, pat dry, then add 50 μL of HRP-labeled goat anti-mouse (diluted 1:5000 with PBS) per well, react at 37°C for 30 min, wash the plate 4 times again, pat dry, add 50 μL of TMB chromogenic solution per well and develop color at room temperature for 10 min, and finally add 0.5 M sulfuric acid as a terminator to terminate the reaction, and measure the OD 450nm value.

[0044] S3. Preparation of monoclonal antibody ascites: After expanding the culture of the screened monoclonal cell line, intraperitoneally inject 0.2 mL (containing 2.5×10 6 cells) of female BALB / c mice pretreated with Freund's incomplete adjuvant. Approximately 10 days later, when the mouse abdomen is significantly swollen, collect ascites using a sterile syringe needle. Centrifuge the collected ascites at 3000 r / min for 10 minutes and collect the middle layer.

[0045] S4. Purification of monoclonal antibody: Centrifuge the ascites at 12,000 r / min for 5 minutes, take 5 mL of the supernatant, add 20 mL of acetate buffer (68 mmol / L, pH 4.5), mix well, then slowly add 50 μL of n-octanoic acid while stirring, continue to stir for 30 minutes after addition, centrifuge at 12,000 r / min for 30 minutes at 2 - 8 °C, and take the supernatant. Filter the supernatant through absorbent cotton, add saturated ammonium sulfate at a ratio of 50% (V / V) of the final volume while stirring, continue to stir for 30 minutes after addition, precipitate at 2 - 8 °C for 4 - 5 hours, centrifuge at 12,000 r / min for 30 minutes at 2 - 8 °C, and take the precipitate. Resuspend the precipitate with 3 mL of Tris-HCl (10 mmol / L, pH 9.0), load it into a dialysis bag (MW: 8000 - 14000), and dialyze in 2 L of Tris-HCl (10 mmol / L, pH 9.0) solution at 2 - 8 °C for 14 hours. Transfer the liquid in the dialysis bag to a centrifuge tube, centrifuge at 12,000 r / min for 5 minutes, and the supernatant is the purified monoclonal antibody. Determine the concentration of the purified monoclonal antibody using an ultra-micro spectrophotometer and store it at -20 °C, avoiding repeated freezing and thawing.

[0046] S5. Identification of the binding activity of monoclonal antibody: Use the indirect ELISA method to coat the recombinant protein of simian serum amyloid A and the control vector protein (simian PCT recombinant protein, manufacturer: Abbexa, catalog number: abx262430) respectively, and perform ELISA detection on the purified antibody to identify the affinity of the monoclonal antibody. Dilute the antibody to 1 μg / ml, and the results are shown in Table 1. In Table 1, "Ctrl" represents the negative control simian PCT murine monoclonal antibody.

[0047] Table 1 Results of the identification of the binding activity of monoclonal antibody

[0048]

[0049] As can be seen from Table 1, for the 15 selected monoclonal antibodies against simian serum amyloid A, their binding activities with simian serum amyloid A protein and the control vector protein were detected by indirect ELISA. The data showed that the monoclonal antibodies could specifically bind to simian serum amyloid A protein.

[0050] Example 3

[0051] This example provides the screening and application of paired monoclonal antibodies, and the specific steps are as follows:

[0052] 1. HRP labeling of the antibody

[0053] The selected monoclonal antibodies were labeled with HRP according to the classical sodium periodate method. Finally, the labeled antibodies were dialyzed overnight in a 0.01M PBS buffer at pH 7.4, and glycerol was added in a 1:1 volume ratio, and then stored at -20°C in aliquots.

[0054] 2. Establishment of a double-antibody sandwich immunoassay method

[0055] The purified monoclonal antibodies were diluted to concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6), and 50 μL per well was added to the enzyme-linked immunosorbent assay (ELISA) plate and coated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 3% sucrose and 1% BSA, 150 μL per well, and incubated at 37°C for 2 h. The blocking solution was discarded and the plate was patted dry. The sample to be tested, recombinant monkey serum amyloid A protein, and the control sample, recombinant monkey PCT protein (manufacturer: Abbexa, catalog number: abx262430), were diluted with PBS to 2 μg / mL, 1 μg / mL, and 0.5 μg / mL and then added to the ELISA plate, 50 μL per well, and incubated at 37°C for 35 min. The plate was washed 4 times with PBST washing solution, patted dry, and then enzyme-labeled monoclonal antibodies diluted 500-, 1000-, and 2000-fold with PBS were added, 50 μL per well, and incubated at 37°C for 35 min. The plate was washed 4 times again, patted dry, and then 50 μL of TMB chromogenic solution was added per well and developed at room temperature for 10 min. Finally, 50 μL of 0.5M sulfuric acid was added per well to terminate the reaction, and the optical density (OD) 450nm value was measured using an enzyme-linked immunosorbent assay reader.

[0056] Then, by adjusting the dilution ratios of the coated antibody and the HRP antibody, the most suitable reaction conditions were determined. Specifically: monoclonal antibody was coated in a microplate (coating buffer: carbonate buffer, 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate were dissolved in 1 L of pure water), and the coating concentration gradients were 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL per well, overnight at 4°C; the plate was washed once with the washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose and 1% BSA, 150 μL per well, at 37°C for 2 hours, patted dry and stored dry for later use; the recombinant protein of monkey serum amyloid A was diluted to a concentration of 10 ng / ml with PBS, 50 μL was added to the microplate coated with monoclonal antibody, and at the same time, PCT protein was diluted to 10 ng / ml as a negative control, reacted at 37°C for 30 min. The liquid in the wells was flicked out, and the plate was washed 4 times with PBST washing solution, patted dry; the HRP-labeled monoclonal antibody was diluted to concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL with PBS, 50 μL per well was added, reacted at 37°C for 30 min, washed 4 times again, patted dry, and 50 μL of TMB chromogenic solution was added per well for color development at room temperature for 10 min, and finally the reaction was terminated by adding the termination solution, and the OD 450nm value was measured with an enzyme-linked immunosorbent assay reader. The coating conditions with the most obvious difference between the negative and positive results and the optimal reaction conditions for the HRP-labeled antibody were selected.

[0057] By separately coating the monoclonal antibody and labeling it with HRP as described above, and based on the detection of negative and positive samples, the combination with the most obvious difference in the detection values between negative and positive was selected as the optimal pairing combination. Finally, it was determined that the coated monoclonal antibody was SAA-3#, the concentration was 1 μg / mL, and the concentration of the HRP-labeled monoclonal antibody SAA-8# was 4 μg / mL.

[0058] The full-length amino acid sequence of the variable region of the heavy chain (H chain) of the monoclonal antibody SAA-3# is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.4; the full-length amino acid sequence of the variable region of the light chain (L chain) is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.6. The specific sequences are as follows:

[0059] SEQ ID No.3: EVQLVESGGGLVPPGGSLKLSCAASGNSGRFSSMSWVRQTPGKSLELVATISYNDRNDYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMFYCARDFISRSTWYTYWGQGTLVTVSA.

[0060] SEQ ID No.4: gaagtgcagctggtggaaagcggcggcggcctggtgccgccgggcggcagcctgaaactgagctgcgcggcgagcggcaacagcggccgctttagcagcatgagctgggtgcgccagaccccgggcaaaagcctggaactggtggcgaccattagctataacgatcgcaacgattattatccggatagcgtgaaaggccgctttaccattagccgcgataacgcgaaaaacaccctgtatctgcagatgagcagcctgaaaagcgaagataccgcgatgttttattgcgcgcgcgattttattagccgcagcacctggtatacctattggggccagggcaccctggtgaccgtgagcgcg。

[0061] SEQ ID No.5: DIVMTQSQKFMSTSVGDRVSVTCKASQNGVRTNVAWYQQKPGQSPKALIYSSSYRYSGVPDRFTGSGSGTDFTLTISHVQSEDLADYFCQGFTISNTFGPGTKLELK。

[0062] SEQ ID No.6: gatattgtgatgacccagagccagaaatttatgagcaccagcgtgggcgatcgcgtgagcgtgacctgcaaagcgagccagaacggcgtgcgcaccaacgtggcgtggtatcagcagaaaccgggccagagcccgaaagcgctgatttatagcagcagctatcgctatagcggcgtgccggatcgctttaccggcagcggcagcggcaccgattttaccctgaccattagccatgtgcagagcgaagatctggcggattatttttgccagggctttaccattagcaacacctttggcccgggcaccaaactggaactgaaa。

[0063] The full-length amino acid sequence of the heavy chain (H chain) variable region of the monoclonal antibody SAA-8# is shown in SEQ ID No.7, and the nucleotide sequence is shown in SEQ ID No.8; the full-length amino acid sequence of the light chain (L chain) variable region is shown in SEQ ID No.9, and the nucleotide sequence is shown in SEQ ID No.10. The specific sequences are as follows:

[0064] SEQ ID No.7: QVQLVESGGGVVQPGRSLRLSCTASGGFLNLYSMYWVRQAPGKGLEWVAVINTGDYRTYNEDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCADLDVYWYFYWGQGTLVTVSS.

[0065] SEQ ID No.8: caggtgcagctggtggaaagcggcggcggcgtggtgcagccgggccgcagcctgcgcctgagctgcaccgcgagcggcggctttctgaacctgtatagcatgtattgggtgcgccaggcgccgggcaaaggcctggaatgggtggcggtgattaacaccggcgattatcgcacctataacgaagatagcgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcggatctggatgtgtattggtatttttattggggccagggcaccctggtgaccgtgagcagc.

[0066] SEQ ID No.9: EIVLTQSPGTLSLSPGERATLSCRASQYFSRSYLAWYQQKPGQAPRLLIYGPSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQDYQTSNTFGPGTKVDIK.

[0067] SEQ ID No.10: gaaattgtgctgacccagagcccgggcaccctgagcctgagcccgggcgaacgcgcgaccctgagctgccgcgcgagccagtattttagccgcagctatctggcgtggtatcagcagaaaccgggccaggcgccgcgcctgctgatttatggcccgagcagccgcgcgaccggcattccggatcgctttagcggcagcggcagcggcaccgattttaccctgaccattagccgcctggaaccggaagattttgcggtgtattattgccaggattatcagaccagcaacacctttggcccgggcaccaaagtggatattaaa。

[0068] 3. Linear detection based on the double antibody sandwich ELISA detection method

[0069] Referring to the above detection steps, first, the standard protein of simian serum amyloid A was serially diluted with PBS buffer solution, and the diluted concentrations were 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.63 ng / mL, 0.31 ng / mL, 0.16 ng / mL, and 0 ng / mL respectively. 50 μL of the diluted standard protein with different concentrations was taken for detection, and three parallel experiments were performed for each concentration to prepare a standard curve. The standard curve is as Figure 2 shown. According to the calculation of the four-parameter fitting results, the linearity reached 0.999.

[0070] 4. Sensitivity detection based on the double antibody sandwich ELISA detection method

[0071] Use the amino acid sequence of monkey serum amyloid A retrieved: MKLLTGLVFCSLVLGVNSRSWFSFLGEAYDGARDMWRAYSDMKEANYINADKYFHARGNYDAAQRGPGGAWAAEVISDARENIQKLLGRGAEDTLADQAANEWGRSGKDPNHFRPAGLPEKY (SEQ ID No.11), and the corresponding nucleotide sequence: atgaaactgctgaccggcctggtgttttgcagcctggtgctgggcgtgaacagccgcagctggtttagctttctgggcgaagcgtatgatggcgcgcgcgatatgtggcgcgcgtatagcgatatgaaagaagcgaactatattaacgcggataaatattttcatgcgcgcggcaactatgatgcggcgcagcgcggcccgggcggcgcgtgggcggcggaagtgattagcgatgcgcgcgaaaacattcagaaactgctgggccgcggcgcggaagataccctggcggatcaggcggcgaacgaatggggccgcagcggcaaagatccgaaccattttcgcccggcgggcctgccggaaaaatat (SEQ ID No.12). Use this sequence to express the protein, prepare monoclonal antibodies, and finally prepare a sandwich ELISA. First, serially dilute the monkey serum amyloid A calibration standard protein with PBS buffer solution. The concentrations of the diluted standards are 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.3 ng / mL, 3.1 ng / mL, 1.6 ng / mL, and 0 ng / mL respectively. Take 50 μL of the diluted standard proteins at different concentrations and 20 PBS buffer solutions for detection and perform a sensitivity test. The measured values of the standard curve and PBS buffer solution are shown in Table 2. Calculate the sensitivity of the double antibody sandwich ELISA detection method as 1.53 ng / mL according to the formula "mean + 2SD".

[0072] Table 2 Sensitivity test data

[0073]

[0074] Referring to the above detection steps, using the method provided by the present invention, first, the monkey serum amyloid A calibration standard protein is serially diluted with PBS buffer solution, and the diluted concentrations are 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.63 ng / mL, 0.32 ng / mL, 0.16 ng / mL, and 0 ng / mL respectively. Take 50 μL of the diluted standard protein with different concentrations and 20 PBS buffer solutions for detection to perform a sensitivity test. The standard curve and the measured values of the PBS buffer solution are shown in Table 3. According to the formula "average value + 2SD", the sensitivity of the double antibody sandwich ELISA detection method is calculated to be 0.08 ng / mL.

[0075] Table 3 Sensitivity test data

[0076]

[0077] According to the comparison results, the sensitivity of the sandwich ELISA detection method of the present invention after optimization is significantly higher than that before optimization.

[0078] Example 4

[0079] Furthermore, in order to verify the accuracy of the technical solution provided by the present invention, the following verification examples were also carried out.

[0080] Referring to the aforementioned detection steps, based on the double antibody sandwich ELISA detection method, the OD values of 16 non-infected monkey sera and 16 bacterially infected monkey sera were measured and identified, and the measured OD values are shown in Table 4.

[0081] Table 4 Identification results of non-infected monkey sera and infected monkey sera

[0082]

[0083] It can be seen from the detection results that there are significant differences between the infected and non-infected. This shows that the double antibody sandwich immuno-ELISA detection method provided by this application not only has high sensitivity but also can accurately detect whether there is an infection in monkey sera.

[0084] In the present invention, the specific raw materials not described are all existing substances and can be directly purchased from the market.

[0085] The above are only the preferred implementation schemes of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant monkey serum amyloid A protein, characterized in that The amino acid sequence of the recombinant protein is shown in SEQ ID No.

1.

2. The nucleotide sequence encoding the monkey serum amyloid A recombinant protein according to claim 1, characterized in that: The nucleotide sequence is shown as SEQ ID No.

2.

3. A method for preparing the monkey serum amyloid A recombinant protein according to claim 1, characterized in that: The method comprises the following steps: firstly connecting the gene encoding the recombinant protein to an expression vector to construct a recombinant plasmid; then transforming the recombinant plasmid into competent cells, inducing expression and then purifying to obtain the monkey serum amyloid A recombinant protein.

4. The preparation method according to claim 3, characterized in that: The gene encoding the recombinant protein is codon-optimized in the E. coli expression system; The expression vector is any one of pET28a, pET32a, pET40b, pSmart1, and pCold-TF; The competent cells are any one of BL21, Rosetta, and OrigamiB.

5. A monoclonal antibody that specifically reacts with the monkey serum amyloid A recombinant protein according to claim 1, characterized in that: The monoclonal antibody includes SAA-3# or SAA-8#; Wherein, the monoclonal antibody SAA-3# comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.5; The monoclonal antibody SAA-8# includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

9.

6. The monoclonal antibody according to claim 5, characterized in that The nucleotide sequence of the heavy chain variable region of the monoclonal antibody SAA-3# is shown in SEQ ID No.4, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.6; The nucleotide sequence of the heavy chain variable region of the monoclonal antibody SAA-8# is shown in SEQ ID No.8, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.

10.

7. An ELISA detection method constructed based on the monoclonal antibodies SAA-3# and SAA-8# described in claim 5.

8. A monkey inflammation detection kit, characterized in that: The kit contains the monoclonal antibodies SAA-3# and SAA-8# described in claim 5.

Images