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

Abstract

The present invention provides a monkey serum amyloid A recombinant protein, a monoclonal antibody and a preparation method and application thereof, wherein the amino acid sequence of the recombinant protein is shown in SEQ ID No. 1, and the nucleotide sequence encoding the recombinant protein is shown in SEQ ID No. 2. By optimizing the antigen peptide sequence and the expression vector, the efficient and soluble expression of monkey serum amyloid A in Escherichia coli is achieved, and a mouse monoclonal antibody specifically binding to monkey serum amyloid A is screened based on the monkey serum amyloid A recombinant protein, and the above mouse monoclonal antibody is used to screen a pairable monoclonal antibody, and further based on the monoclonal antibody combination, a double antibody sandwich immune ELISA detection method or kit applied to monkey serum amyloid A recombinant protein is obtained, which has high detection sensitivity and can be well applied to the detection of monkey serum.

Description

Technical Field

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

[0002] Infectious diseases are extremely common in clinical practice, and laboratory auxiliary examinations 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). Details of the four indicators are as follows:

[0003] Serum amyloid A (SAA) is an acute phase protein mainly derived from liver cells. The blood concentration of serum amyloid A is extremely low when healthy. When inflammation occurs in the human body, its concentration in the blood will increase rapidly, especially in the early stages after the inflammation occurs. Because of its rapid metabolism, the blood concentration of SAA will drop rapidly during the recovery period of inflammation. Therefore, SAA level determination is often used clinically for the diagnosis and monitoring of infectious diseases.

[0004] Laboratory animals are the main experimental subjects for preclinical evaluation of drugs, and monkeys are an important species in preclinical research. The application of laboratory animals in the development of new drugs covers both preclinical and clinical research stages, especially in preclinical research. Different experimental methods are used to evaluate the pharmacological effects of drugs, study their mechanisms of action, and observe their toxic effects to prove the effectiveness and safety of drugs. Monkeys are the closest to humans in terms of kinship, with 75% to 98.5% homology with human genetic material. They are the animals with the highest homology with humans, and therefore are the ideal animals closest to humans in experiments.

[0005] The applicable amino acid sequence of serum amyloid protein A that can be queried now (shown in SEQ ID No.11) and the corresponding nucleotide sequence (shown in SEQ ID No.12) are relatively low in sensitivity for the antibody prepared by the protein expressed using this sequence, and cannot meet the application of the monkey serum amyloid protein A ELISA kit. Currently, there is no monkey serum amyloid protein A high-sensitivity ELISA detection kit for the detection of monkey serum amyloid protein A, one of the four major indicators of monkey inflammation. Summary of the invention

[0006] In view of this, the present invention provides a monkey serum amyloid A recombinant protein, a monoclonal antibody, and a preparation method and application thereof. The present invention uses bioinformatics software to select and optimize a segment of monkey serum amyloid A polypeptide, which can be expressed in Escherichia coli after prediction and analysis, obtains a specific paired monoclonal antibody of monkey serum amyloid A through immunization and screening, establishes a double antibody sandwich ELISA detection system, and provides a new approach to preclinical monkey inflammation model research.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide: a monkey serum amyloid A recombinant protein, the amino acid sequence of the recombinant protein is shown in SEQ ID No.1.

[0008] The second object of the present invention is to provide: a nucleotide sequence encoding the above-mentioned monkey serum amyloid A recombinant protein, and the nucleotide sequence is shown as SEQ ID No.2.

[0009] The third object of the present invention is to provide: a method for preparing the above-mentioned monkey serum amyloid A recombinant protein, comprising 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.

[0010] Furthermore, the gene encoding the recombinant protein is codon-optimized in an 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.

[0011] The fourth object of the present invention is to provide: a monoclonal antibody that specifically reacts with the above-mentioned monkey serum amyloid A recombinant protein, wherein the monoclonal antibody includes SAA-3# and SAA-8#; The amino acid sequence of the heavy chain variable region of the monoclonal antibody SAA-3# is shown in SEQ ID No.3, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No.4, the amino acid sequence of the light chain variable region is shown in SEQ ID No.5, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.6; 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.

[0012] A fifth object of the present invention is to provide: a method for preparing the above-mentioned monoclonal antibody, comprising the following steps: S1, adding an adjuvant to the above-mentioned monkey serum amyloid A recombinant protein, mixing, and immunizing female BALB / c mice; S2, taking spleen cells from immunized mice and fusing them with SP2 / 0 myeloma cells in the logarithmic growth phase, screening and culturing them to obtain hybridoma cells; S3. The hybridoma cells obtained by screening are expanded and cultured, and injected intraperitoneally into female BALB / c mice. The ascites is collected, and the ascites is purified and screened after centrifugation to obtain monoclonal antibodies.

[0013] The sixth object of the present invention is to provide: an ELISA detection method constructed based on the above monoclonal antibody.

[0014] The seventh object of the present invention is to provide: application of the above-mentioned ELISA detection method in detecting monkey serum amyloid A.

[0015] The eighth object of the present invention is to provide: a monkey serum detection kit, which contains the above-mentioned monoclonal antibody.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a recombinant monkey serum amyloid A protein, which achieves efficient, soluble and active expression of monkey serum amyloid A in Escherichia coli by optimizing the antigen peptide sequence and expression vector.

[0017] (2) The present invention screened out a mouse monoclonal antibody that specifically binds to monkey serum amyloid A based on the monkey serum amyloid A recombinant protein, and used the above mouse monoclonal antibody to screen a pairing monoclonal antibody, and further constructed a double antibody sandwich ELISA detection method or kit for monkey serum amyloid A based on the monoclonal antibody combination, which can specifically detect monkey serum amyloid A protein without cross-reaction and has a high detection sensitivity (reaching 0.08 ng / mL), providing a new approach for preclinical monkey inflammation model research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the electrophoresis diagram of the monkey serum amyloid A recombinant protein described in Example 1 of the present invention.

[0019] Figure 2 This is a graph showing the monkey serum amyloid A ELISA test described in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0021] Example 1 This embodiment provides a monkey serum amyloid A recombinant protein, whose amino acid sequence is shown in SEQ ID No. 1, and the gene sequence encoding the recombinant protein is shown in SEQ ID No. 2. The specific sequence is as follows: SEQ ID No. 1: MKLLTGLVQAGANLGVNSRSWFSFLGEAYDGARDMWRAYSKEANYINADKYFHARGNSFAAQRGPGGAWAAEQKSDARENIQKLLGDTLADQAANEWGRSDMWRNHFRPAGLPEKY.

[0022] SEQ ID No.2: atgaaactgctgaccggcctggtgcaggcgggcgcgaacctgggcgtgaacagccgcagctggtttagctttctgggcgaagcgtatgatggcgcgcgcgatatgtggcgcgcgtatagcaaagaagcgaactatattaacgcggataaatattttcatgcgcgcggcaaca gctttgcggcgcagcgcggcccgggcggcgcgtgggcggcggaacagaaaagcgatgcgcgcgaaaacattcagaaactgctgggcga taccctggcggatcaggcggcgaacgaatggggccgcagcgatatgtggcgcaaccattttcgcccggcgggcctgccggaaaaatat.

[0023] The method for preparing the above-mentioned monkey serum amyloid A recombinant protein comprises the following steps: (1) Plasmid construction: Computational software was used to predict and select the polypeptide containing the major antigenic epitope of monkey serum amyloid A protein for expression, as shown in SEQ ID No. 2. This polypeptide can be expressed in the Escherichia coli prokaryotic expression system. The gene was cloned into the pET28a vector via the E. coli expression system to obtain the recombinant plasmid pET28a-SAA expressing monkey serum amyloid A protein.

[0024] (2) Induced expression: The recombinant plasmid pET28a-SAA was transformed into BL21 (DE3) competent cells, and the transformed bacteria were spread on LB agar plates containing 50 μg / mL kanamycin and cultured at 37°C overnight. A single colony was picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 230 rpm overnight. 1% of the total volume of the culture medium was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220 rpm for about 3 hours until the OD 600 The concentration of IPTG was 0.6, and the final concentration was 0.1 mM IPTG. After induction at 16°C and 180 rpm for 15 hours, the bacteria were collected.

[0025] (3) Purification of recombinant protein: Since the expressed recombinant protein carries a histidine tag, it is initially crudely purified using a protein purifier and a chromatography column, and then purified using ion exchange chromatography to obtain the recombinant protein, which is the monkey serum amyloid A recombinant protein, and the amino acid sequence is shown in SEQ ID No. 1.

[0026] (4) Use SDS-PAGE electrophoresis to identify the purity of the recombinant protein: Protein sample pretreatment: add an equal volume of 2×SDS loading buffer to each sample, place in a boiling water bath for 10 minutes, centrifuge at 12000rpm for 3 minutes, and add the supernatant to the sample well. The purified protein was found to be monkey serum amyloid A recombinant protein. The electrophoresis diagram is shown in the figure below. Figure 1 shown.

[0027] Example 2 This embodiment provides a method for obtaining monoclonal antibodies based on the recombinant protein monkey serum amyloid A, and the specific operations are as follows: S1. Mouse immunization: 50 μg of monkey serum amyloid A recombinant protein was mixed with an equal volume of Freund's complete adjuvant to obtain a mixture, which was then injected subcutaneously at multiple points to immunize 6-week-old female BALB / c mice. At week 0 and week 2, Freund's incomplete adjuvant was mixed with the recombinant protein in equal proportions and emulsified according to the above method, and then immunized again. At week 6, mouse serum was taken to test the antibody titer, and the mouse with the highest titer was selected to be boosted with 20 μg of monkey serum amyloid A recombinant protein through the tail vein. After 3 days, the spleen of the mouse was taken for the preparation of hybridoma cells.

[0028] S2. Screening of hybridoma cell lines: All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then placed in HAT medium for screening and culture. When the fused cells grew to 1 / 2 of the bottom of the well, positive clones were screened by indirect ELISA of monkey serum amyloid A protein, and the positive rate was 100% through 2-3 consecutive limiting dilution cloning methods, and the cell lines were expanded and frozen; among them, the method of screening positive clones by indirect ELISA is as follows: first, the recombinant protein was coated in the microplate: the coating buffer was carbonate buffer (1.59g sodium carbonate, 2.93g sodium bicarbonate, fixed to 1L pure water), the coating concentration was 1μg / mL, 50μl / well, 4°C overnight; 3% sucrose, 1% BSA blocking, 150μL per well, 37°C for 2 hours, and the plate was washed once with washing buffer PBST (PBS containing 0.05% Tween-20), and patted dry; 50μL cell culture supernatant was added, and the reaction was carried out at 37°C for 30min. The liquid in the wells was shaken off, and the plate was washed 4 times with PBST solution. After patting dry, 50 μL / well of HRP-labeled goat anti-mouse (diluted 1:5000 in PBS) was added. The plate was reacted at 37°C for 30 min. The plate was washed 4 times again, and after patting dry, 50 μL / well of TMB colorimetric solution was added to develop the color at room temperature for 10 min. Finally, 0.5 M sulfuric acid was added as a terminator to terminate the reaction, and the OD was measured with an enzyme reader. 450nm value.

[0029] S3. Preparation of monoclonal antibody ascites: After the selected monoclonal cell line was expanded and cultured, 0.2 mL (containing 2.5×10 6 Cells) were pretreated with Freund's incomplete adjuvant in female BALB / c mice. About 10 days later, when the abdomen of the mice was obviously swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000r / min for 10 minutes to collect the middle layer.

[0030] S4. Purification of monoclonal antibodies: Centrifuge the ascites at 12000r / min for 5 minutes, take 5mL of the supernatant, add 20ml of acetate buffer (68mmol / L, pH 4.5), mix well, slowly add 50μL of octanoic acid, stirring while adding, continue stirring for 30 minutes after adding, centrifuge at 12000r / min for 30 minutes at 2-8℃, and take the supernatant. Filter the supernatant with absorbent cotton, add saturated ammonium sulfate at a ratio of 50% (V / V) of the final volume, stirring while adding, continue stirring for 30 minutes after adding, precipitate at 2-8℃ for 4-5 hours, centrifuge at 12000r / min for 30 minutes at 2-8℃, and take the precipitate. The precipitate was resuspended with 3 mL Tris-HCl (10 mmol / L, pH 9.0), placed in a dialysis bag (MW: 8000-14000), and dialyzed for 14 hours in 2 L Tris-HCl (10 mmol / L, pH 9.0) solution at 2-8°C. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The concentration of the purified monoclonal antibody was measured with an ultra-micro spectrophotometer and stored at -20°C to avoid repeated freezing and thawing.

[0031] S5. Identification of binding activity of monoclonal antibodies: Use the indirect ELISA method to coat monkey serum amyloid A recombinant protein and control carrier protein (monkey PCT recombinant protein, manufacturer: Abbexa, product number: abx262430), respectively. Perform ELISA on the purified antibodies to identify the affinity of the monoclonal antibodies. The antibodies were diluted to 1 μg / ml. The results are shown in Table 1. "Ctrl" in Table 1 represents the negative control monkey PCT mouse monoclonal antibody.

[0032] Table 1 Binding activity identification results of monoclonal antibodies

[0033] As can be seen from Table 1, the binding activity of the 15 selected monkey serum amyloid A monoclonal antibodies to monkey serum amyloid A protein and the control carrier protein was detected by indirect ELISA. The data showed that the monoclonal antibodies could specifically bind to monkey serum amyloid A protein.

[0034] Example 3 This embodiment provides the screening and application of paired monoclonal antibodies, and the specific steps are as follows: 1. HRP labeling of antibodies The screened monoclonal antibodies were labeled with HRP according to the classic sodium periodate method. Finally, the labeled antibodies were dialyzed overnight in 0.01M PBS, pH 7.4 buffer, glycerol was added at a volume of 1:1, and the antibodies were stored in separate devices at -20°C.

[0035] 2. Establishment of double antibody sandwich immunoassay The purified monoclonal antibody was diluted to a concentration 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, fixed to 1 L of pure water, pH 9.6), and added to the ELISA plate at 50 μL / well for overnight coating at 4°C. The coating solution was discarded the next day, 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 at 150 μL / well. Incubated at 37°C for 2 h, the blocking solution was discarded, and patted dry. The monkey serum amyloid A recombinant protein and the control monkey PCT recombinant protein (manufacturer: Abbexa, catalog number: abx262430) were diluted with PBS at 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, and then added to the ELISA plate at 50 μL / well, incubated at 37°C for 35 min, washed the plate 4 times with PBST washing solution, patted dry, and added with 50 μL / well of enzyme-labeled monoclonal antibody diluted to 500, 1000, and 2000 times with PBS, incubated at 37°C for 35 min, washed the plate 4 times, patted dry, and added with 50 μL / well of TMB colorimetric solution, developed at room temperature for 10 min, and finally added 0.5 M sulfuric acid at 50 μL / well to terminate the reaction, and the OD was measured with an ELISA reader. 450nm value.

[0036] Then, the most suitable reaction conditions were determined by adjusting the dilution of the coated antibody and HRP antibody. Specifically, the monoclonal antibody was coated in the microplate (coating buffer carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate fixed to 1L pure water), the coating concentration gradient was 0.5μg / mL, 1μg / mL, 2μg / mL, 50μL / well, overnight at 4℃; the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose, 1% BSA, 150μL per well, 37℃ for 2 hours, patted dry and stored for later use; the monkey serum amyloid A recombinant protein was diluted to a concentration of 10ng / ml with PBS, and 50μL was added to the microplate coated with the monoclonal antibody, and PCT protein was diluted to 10ng / ml as a negative control, and reacted at 37℃ for 30min. The liquid in the wells was shaken off, the plate was washed 4 times with PBST, and patted dry; HRP-labeled monoclonal antibody was diluted with PBS to a concentration of 1 μg / mL, 2 μg / mL, and 4 μg / mL, and 50 μL / well was added, reacted at 37°C for 30 min, and the plate was washed 4 times, patted dry, and TMB color development solution was added at 50 μL / well for color development at room temperature for 10 min, and finally the stop solution was added to terminate the reaction, and the OD was measured with an enzyme reader. 450nm Select the coating conditions with the most obvious positive and negative differences and the optimal reaction conditions for the HRP-labeled antibody.

[0037] Through the above-mentioned coating and HRP labeling of monoclonal antibodies, the best pairing combination was selected based on the detection of positive and negative samples, and the difference between positive and negative detection values ​​was the most obvious. Finally, it was determined that the concentration of coated monoclonal antibody SAA-3# was 1μg / mL, and the concentration of HRP-labeled monoclonal antibody SAA-8# was 4μg / mL.

[0038] The full-length amino acid sequence of the heavy chain (H chain) variable region 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 light chain (L chain) variable region is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.6. The specific sequences are as follows: SEQ ID No. 3: EVQLVESGGGLVPPGGSLKLSCAASGNSGRFSSMSWVRQTPGKSLELVATISYNDRNDYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMFYCARDFISRSTWYTYWGQGTLVTVSA.

[0039] SEQ ID No.4: gaagtgcagctggtggaaagcggcggcggcctggtgccgccgggcggcagcctgaaactgagctgcgcggcgagcggcaacagcggccgctttagcagcatgagctgggtgcgccagaccccgggcaaaagcctggaactggtggcgaccattagctataacgatcgcaacgattatt atccggatagcgtgaaaggccgctttaccattagccgcgataacgcgaaaaacaccctgtatctgcagatgagcagcctgaaaagcgaagataccgcgatgtttattgcgcgcgcgattttattagccgcagcacctggtatacctattggggccagggcaccctggtgaccgtgagcgcg.

[0040] SEQ ID No. 5: DIVMTQSQKFMSTSVGDRVSVTCKASQNGVRTNVAWYQQKPGQSPKALIYSSSYRYSGVPDRFTGSGSGTDFTLTISHVQSEDLADYFCQGFTISNTFGPGTKLELK.

[0041] SEQ ID No.6:gatattgtgatgacccagccagaaatttatgagcaccagcgtgggcgatcgcgtgagcgtgacctgcaaagcgagccagaacggcgtgcgcaccaacgtggcgtggtatcagcagaaaccgggccagagcccgaaagcgctgatttatagcagcag ctatcgctatagcggcgtgccggatcgctttaccggcagcggcagcggcaccgattttaccctgaccattagccatgtgcagagcgaagatctggcggattatttttgccagggctttaccattagcaacacctttggcccgggcaccaaactggaactgaaa.

[0042] 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: SEQ ID No. 7: QVQLVESGGGVVQPGRSLRLSCTASGGFLNLYSMYWVRQAPGKGLEWVAVINTGDYRTYNEDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCADLDVYWYFYWGQGTLVTVSS.

[0043] SEQ ID No.8: caggtgcagctggtggaaagcggcggcggcgtggtgcagccgggccgcagcctgcgcctgagctgcaccgcgagcggcggctttctgaacctgtatagcatgtattgggtgcgccaggcgccgggcaaaggcctggaatgggtggcggtgattaacaccggcgattatcgcacctataacgaagatagcgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcggatctggatgtgtattggtatttttattggggccagggcaccctggtgaccgtgagcagc。

[0044] SEQ ID No.9: EIVLTQSPGTLSLSPGERATLSCRASQYFSRSYLAWYQQKPGQAPRLLIYGPSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQDYQTSNTFGPGTKVDIK。

[0045] SEQ ID No.10: gaaattgtgctgacccagagcccgggcaccctgagcctgagcccgggcgaacgcgcgaccctgagctgccgcgcgagccagtattttagccgcagctatctggcgtggtatcagcagaaaccgggccaggcgccgcgcctgctgatttatggcccgagcagccgcgcgaccggcattccggatcgctttagcggcagcggcagcggcaccgattttaccctgaccattagccgcctggaaccggaagattttgcggtgtattattgccaggattatcagaccagcaacacctttggcccgggcaccaaagtggatattaaa。

[0046] 3. Linear detection based on the double antibody sandwich ELISA detection method Referring to the above detection steps, the monkey serum amyloid A fixed value standard protein was first diluted with PBS buffer solution. The concentrations after dilution were 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.63ng / mL, 0.31ng / mL, 0.16ng / mL, and 0ng / mL, respectively. 50μL of the diluted standard protein of different concentrations was taken for detection. Three parallel experiments were performed for each concentration to prepare a standard curve. The standard curve is as follows: Figure 2 As shown, according to the four-parameter fitting results, the linearity reaches 0.999.

[0047] 4. Sensitivity test based on double antibody sandwich ELISA detection method The amino acid sequence of monkey serum amyloid A was used: MKLLTGLVFCSLVLGVNSRSWFSFLGEAYDGARDMWRAYSDMKEANYINADKYFHARGNYDAAQRGPGGAWAAEVISDARENIQKLLGRGAEDTLADQAANEWGRSGKDPNHFRPAGLPEKY (SEQ ID No.11), corresponding nucleotide sequence: atgaaactgctgaccggcctggtgttttgcagcctggtgctgggcgtgaacagccgcagctggtttagctttctgggcga agcgtatgatggcgcgcgcgatatgtggcgcgcgtatagcgatatgaaagaagcgaactatattaacgcggataaatattttcatgcgcgcggcaac tatgatgcggcgcagcgcggcccgggcggcgcgtgggcggcggaagtgattagcgatgcgcgcgaaaacattcagaaactgctgggccgcggcgcg gaagataccctggcggatcaggcggcgaacgaatggggccgcagcggcaaagatccgaaccattttcgcccggcgggcctgccggaaaaatat (seq ID No.12). This sequence was used to express protein, prepare monoclonal antibodies, and finally prepare sandwich ELISA. First, the monkey serum amyloid A fixed value standard protein was gradiently diluted with PBS buffer solution. The concentrations of the diluted standard were 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.3ng / mL, 3.1ng / mL, 1.6ng / mL, and 0ng / mL, respectively. 50μL of the diluted standard protein of different concentrations and 20 PBS buffers were taken for detection and sensitivity test. The standard curve and PBS buffer values ​​are shown in Table 2. According to the formula "mean + 2SD", the sensitivity of the double antibody sandwich ELISA detection method was calculated to be 1.53ng / mL.

[0048] Table 2 Sensitivity test data

[0049] Referring to the above detection steps, using the method provided by the present invention, the monkey serum amyloid A fixed value standard protein was first diluted with a PBS buffer solution, and the concentrations after dilution were 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. 50 μL of the diluted standard protein of different concentrations and 20 PBS buffers were taken for detection, and a sensitivity test was performed. The standard curve and PBS buffer measured values ​​are shown in Table 3. According to the formula "average value + 2SD", the sensitivity of the double antibody sandwich ELISA detection method was calculated to be 0.08 ng / mL.

[0050] Table 3 Sensitivity test data

[0051] 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.

[0052] Example 4 Furthermore, in order to verify the accuracy of the technical solution provided by the present invention, the following verification examples were also carried out.

[0053] Referring to the aforementioned detection steps, 16 uninfected monkey sera and 16 bacteria-infected monkey sera were measured and identified based on the double antibody sandwich ELISA detection method, and the measured OD values ​​are shown in Table 4.

[0054] Table 4 Identification results of uninfected monkey sera and infected monkey sera

[0055] From the test results, it can be seen that there is a significant difference between infected and uninfected monkeys, which shows that the double antibody sandwich immune ELISA detection method provided by the present application is not only highly sensitive, but also can accurately detect whether monkey serum is infected.

[0056] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0057] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in 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. A method for preparing a monoclonal antibody, characterized in that: The following steps are involved: S1. adding an adjuvant to the monkey serum amyloid A recombinant protein according to claim 1, mixing, and immunizing female BALB / c mice; S2, taking spleen cells from immunized mice and fusing them with SP2 / 0 myeloma cells in the logarithmic growth phase, screening and culturing them to obtain hybridoma cells; S3. The hybridoma cells obtained by screening are expanded and cultured, and injected intraperitoneally into female BALB / c mice. The ascites is collected, and the ascites is purified and screened after centrifugation to obtain monoclonal antibodies.

8. An ELISA detection method constructed based on the monoclonal antibody according to claim 5.

9. Use of the ELISA detection method according to claim 8 in detecting monkey serum amyloid A.

10. A monkey serum detection kit, characterized in that: The kit contains the monoclonal antibody according to claim 5.

Images