A29-C71A-C72A-H74R-R107H recombinant protein of monkey pox virus as well as coding gene, kit and preparation method of A29-C71A-C72A-H74R-R107H recombinant protein

Abstract

The invention discloses a monkey pox virus A29-C71A-C72A-H74R-R107H (recombinant protein), and the recombinant protein has an amino acid sequence as shown in SEQ ID No.1. The invention also discloses a monkey pox virus A29-C71A-C72A-H74R-R107H recombinant protein. The monkey pox virus A29-C71A-C72A-H74R-R107H-recombinant protein disclosed by the invention can be expressed in quantity, and has better antigenicity and hydrophilicity. The invention also discloses a gene used for coding the recombinant protein of the monkey pox virus A29-C71A-C72A-H74R-R107H in the claim 1, wherein the gene is shown as SEQ ID No.2. The invention also discloses a recombinant protein of the monkey pox virus A29-C71A-C72A-H74R-R107H in the claim 1. The invention also discloses an indirect ELISA kit for the recombinant protein of the monkey pox virus A29-C71A-C72A-H74R-R107H, and the kit not only can be used for serological diagnosis after the monkey pox virus infection, but also can be used for evaluating the immune response of a human body to the monkey pox virus, and has a remarkable application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a monkeypox virus A29-C71A-C72A-H74R-R107H recombinant protein and its encoding gene, a kit and a preparation method. Background Art

[0002] Monkeypox (MPox) is a zoonotic disease caused by the monkeypox virus (MPox). The virus belongs to the genus Orthopoxvirus in the family Poxviridae. The reservoir of the virus remains unknown, but African rodents are currently considered the likely natural reservoir. The source of infection includes cases of monkeypox and infected rodents, monkeys, apes, and other primates.

[0003] Typical monkeypox virus infection in humans can be diagnosed based on clinical symptoms combined with pathological changes and epidemiological blood analysis. However, effective diagnostic methods are lacking for asymptomatic or atypical cases, requiring definitive laboratory diagnosis. Current diagnostic methods for monkeypox infection primarily include quantitative PCR, electron microscopy of viral particles, and virus isolation and culture. However, these methods suffer from drawbacks such as cumbersome procedures, high costs, and low sensitivity and specificity.

[0004] A29 is the major envelope protein of monkeypox virus (MPXV) and a hot topic in monkeypox virus serological diagnostic research. This protein consists of 110 amino acids and can be divided into four functional domains: an N-terminal signal peptide; a lysine / arginine-rich heparin-binding site (HBS) with the sequence "STKAAKNPETKR"; an α-helical coil domain; and a C-terminal leucine zipper motif. MPXV A29 is a homologous protein to vaccinia virus (VACV) A27, and its binding affinity for heparin is similar to that of VACV A27. The primary functions of this protein are related to viral replication, recognition, and regulation of cell entry and release.

[0005] Currently, the primary method for preventing monkeypox is the smallpox vaccine. According to the WHO, smallpox vaccination is approximately 85% effective in preventing monkeypox. While the smallpox vaccine provides some degree of cross-protection, its effectiveness may decrease over time, particularly for those who have already been vaccinated. Furthermore, the smallpox vaccine may not provide long-term and complete cross-protection because the monkeypox virus and smallpox virus, while belonging to the same family, are not identical. This may result in the smallpox vaccine being ineffective in preventing monkeypox in some cases. Furthermore, the storage and distribution of the smallpox vaccine require specialized conditions, which may complicate vaccination implementation in some areas.

[0006] Therefore, there is an urgent need for a serological diagnostic technology that can quickly and effectively detect and evaluate antibody levels after vaccine immunization. Summary of the Invention

[0007] In response to the above technical problems, the present invention provides a monkeypox virus A29-C71A-C72A-H74R-R107H (hereinafter referred to as A29) recombinant protein and its gene, kit and preparation method.

[0008] The present invention is achieved through the following technical solutions:

[0009] The first object of the present invention is to provide a monkeypox virus A29 recombinant protein, the amino acid sequence of the A29 recombinant protein is shown in SEQ ID No. 1.

[0010] SEQ ID No. 1:

[0011] MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKAAKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE

[0012] The present invention also provides a gene for encoding the above-mentioned monkeypox virus A29 recombinant protein, and the nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0013] SEQ ID No. 2:

[0014] ATGGACGGAACTCTTTTCCCCGGAGATGACGATCTTGCAATTCCAGCAACTGAATTTTTCTCTACAAAGGCTGCTAAAAATCCAGAGACTAAACGCGAAGCAATTGTTAAAGCCTATGGAGACGATAATGAGGAAACTCTCAAACAACGGCTAACTAATTTGGAA AAAAAGATTACTAATATAACAACAAAGTTTGAACAAATAGAAAAGGCTGCTAAACGCAACGATGAAGTTCTATTTAGGTTGGAAAATCACGCTGAAACTCTAAGAGCGGCTATGATATCTCTGGCTAAAAAGATTGAATGTACAGACTGGACGGCATCCATATGAG

[0015] The second object of the present invention is to provide an indirect ELISA detection kit for monkeypox virus A29 recombinant protein antibodies, the kit comprising a recombinant protein coating antigen, and the recombinant protein coating antigen is the above-mentioned monkeypox virus A29 recombinant protein.

[0016] Preferably, the kit further comprises a sample diluent, an enzyme-labeled antibody, a TMB substrate solution, a stop solution, a concentrated washing solution, a sealing film, a dilution plate, a negative control, and a positive control;

[0017] The positive control solution is prepared by diluting monkeypox positive serum with sample diluent; the negative control solution is prepared by diluting healthy person negative serum with sample diluent;

[0018] The enzyme-labeled antibody shown is a goat anti-human enzyme-labeled antibody.

[0019] The third object of the present invention is to provide a method for preparing the above-mentioned monkeypox virus A29 recombinant protein, comprising the following steps:

[0020] S1. Using monkeypox virus genomic DNA as a template, site-directed mutagenesis was performed by overlapping PCR using specific primers to obtain the A29-specific target gene.

[0021] The S2.A29-specific target gene was seamlessly cloned and assembled, and connected to the pET30a(+) expression vector to construct the recombinant vector pETA29;

[0022] S3. The recombinant vector pETA29 was transformed into BL21 Escherichia coli to obtain a recombinant expression strain;

[0023] S4. The recombinant expression strain was induced with IPTG, lysed, and centrifuged. The supernatant and precipitate were collected and purified using nickel-agarose gel affinity chromatography to obtain the monkeypox virus A29 recombinant protein.

[0024] A fourth object of the present invention is to provide an indirect ELISA method for detecting monkeypox virus, comprising the following steps:

[0025] S1. Coating: Dilute the recombinant protein coating antigen with carbonate buffer, wherein the amino acid sequence of the recombinant protein coating antigen is as shown in SEQ ID NO: 1;

[0026] S2. Blocking: Add blocking solution to each well, spin dry, and wash with PBST.

[0027] S3. Test serum conditions: The test serum was diluted with diluent, incubated, dried, and washed with PBST.

[0028] S4. Secondary antibody conditions: Dilute HRP-labeled goat anti-human IgG in PBST, incubate, spin dry, and wash with PBST.

[0029] S5. Color development: Add color development solution to each well and add stop solution after incubation to terminate the reaction.

[0030] S6. Reading: Read the OD data at 450 min using a microplate reader and calculate the results.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1. The A29 recombinant protein used in the present invention is easy to prepare and purify in large quantities. The A29 gene can be stably and efficiently expressed in the pET prokaryotic expression system, and the recombinant protein carrying the histidine tag is easy to purify.

[0033] 2. The kit of the present invention has strong specificity and can effectively eliminate interference from clinically similar diseases of monkeypox such as measles, varicella-zoster infection, and healthy people who have been vaccinated with smallpox vaccine, and specifically detect monkeypox antibodies.

[0034] 3. The diagnostic kit of the present invention has strong practicality and can be used for antibody detection of monkeypox virus infection in humans or diagnosis of monkeypox, and can also be used for antibody monitoring after monkeypox virus infection in humans.

[0035] 4. The kit of the present invention is convenient, sensitive, accurate and reliable. It uses TMB substrate for color development and uses a microplate reader to measure the OD value to determine the monitoring results of the sample to be tested. The difference between positive and negative results is obvious, and it is more sensitive, reliable and stable than OPD color development.

[0036] 5. The kit of the present invention is simple and quick to operate, and can complete sample detection within 1.5 hours, which is time-saving and low-cost, and is suitable for the detection of a large number of serum samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the nucleic acid electrophoresis identification diagram of the A29 gene.

[0038] Lanes 1 and 2 are PCR amplification products of A29 gene cloning;

[0039] M stands for DL2000 Marker.

[0040] Figure 2 To construct the prokaryotic expression vector pETA29 clone, the results were identified by PCR and double enzyme digestion.

[0041] Lane 1 is the identification of PCR amplification products of pETA29 bacterial solution;

[0042] Lane 2 is the identification of double enzyme digestion products of pETA29 plasmid;

[0043] Lane 3 is the pEIA29 plasmid control;

[0044] M stands for DL2000 Marker.

[0045] Figure 3 The figure is the SDS-PAGE electrophoresis identification diagram of the expression and purification products of A29 recombinant protein.

[0046] Lanes 1 and 4 are control samples without IPTG-induced expression;

[0047] Lane 2 is the A29 recombinant protein product before purification;

[0048] Lane 3 is the A29 recombinant protein purified by Ni column;

[0049] M stands for Protein Marker. DETAILED DESCRIPTION

[0050] In order to better understand the present invention, the present invention is further described in detail below with reference to the embodiments and drawings. However, those skilled in the art will understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] Example 1 Construction of Monkeypox Virus A29 Recombinant Protein

[0054] 1. Cloning of the monkeypox virus A29 gene and construction of a prokaryotic expression vector

[0055] Specific primers for the A29 gene segment were designed with reference to the monkeypox virus genome sequence (NC_003310.1) in GenBank. The primer sequences used in the present invention are:

[0056] Upstream primer 1:

[0057] CACATGGACAGCCCAGATCTGATGGACGGAACTCTTTTCCCCGGAGA TGACG (as shown in SEQ ID NO:3)

[0058] Upstream primer 2:

[0059] GAACAAATAGAAAAGGCTGCTAAACGCAACGATGAAGTTCTATTTAG (as shown in SEQ ID NO: 4)

[0060] Downstream primer 1:

[0061] TTAGTGGTGGTGGTGGTGGTGCTCATATGGATGCCGTCCAGTCTGTAC (as shown in SEQ ID NO:5)

[0062] Downstream primer 2:

[0063] CTTGTCGACGGAGCTCGAATTCTTAGTGGTGGTGGTGGTGGTGCTCA TATGGA (as shown in SEQ ID NO: 6)

[0064] The A29-specific target gene (nucleotide sequence shown in SEQ ID NO: 2) with a size of 330 bp was amplified by PCR, and its electrophoresis pattern was shown in FIG. Figure 1 As shown. By seamless cloning assembly technology and connecting with pET30a (+) expression vector, the prokaryotic expression vector pETA29 was constructed. PCR, double enzyme digestion and sequencing identification showed that the A29 gene prokaryotic expression vector was successfully constructed. The electrophoresis diagram is shown in Figure 2 .

[0065] SEQ ID NO:2

[0066] ATGGACGGAACTCTTTTCCCCGGAGATGACGATCTTGCAATTCCAGCAACTGAATTTTTCTCTACAAAGGCTGCTAAAAATCCAGAGACTAAACGCGAAGCAATTGTTAAAGCCTATGGAGACGATAATGAGGAAACTCTCAAACAACGGCTAACTAATTTGGAA AAAAAGATTACTAATATAACAACAAAGTTTGAACAAATAGAAAAGGCTGCTAAACGCAACGATGAAGTTCTATTTAGGTTGGAAAATCACGCTGAAACTCTAAGAGCGGCTATGATATCTCTGGCTAAAAAGATTGAATGTACAGACTGGACGGCATCCATATGAG

[0067] 2. High-efficiency expression and purification of A29 recombinant protein

[0068] The prokaryotic expression vector pETA29 was transformed into BL21 (DE3), and the positive recombinant bacteria were picked and inoculated into LB medium containing kanamycin and cultured at 37 ° C with shaking. 600When the pH reached 0.6, IPTG was added to a final concentration of 0.5 mM to induce expression. The host bacteria expressing in large quantities were collected by centrifugation and lysed by ultrasonication. The supernatant was obtained by centrifugation and filtered through a 0.45 filter membrane. The recombinant protein was purified by nickel agarose gel affinity chromatography. SDS-PAGE electrophoresis showed that the A29 recombinant protein was approximately 20 kD in size and had an amino acid sequence as shown in SEQ ID NO: 1. The electrophoresis diagram is shown in FIG. Figure 3 .

[0069] SEQ ID NO: 1

[0070] MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKAAKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE

[0071] Example 2 Establishment of an indirect ELISA method using purified recombinant protein A29 as a coating antigen

[0072] 1. Optimal antigen coating amount and coating method for ELISA detection method

[0073] A29 recombinant protein was subjected to an array test at coating concentrations of 0.125 μg, 0.25 μg, 0.5 μg, and 1 μg per well. The results are shown in Table 1. The array test showed that the optimal coating concentration of the antigen was 0.5 μg / well.

[0074] Table 1 Optimal antigen coating amount of A29 recombinant protein

[0075] Antigen coating amount per ELISA well 0.125 μg 0.25 μg 0.5 μg 1 μg P (monkeypox-positive serum) 1.014 1.586 2.404 2.784 N (monkeypox negative serum) 0.169 0.178 0.212 0.27 P / N 6.00 8.91 11.34 10.31

[0076] Using carbonate buffer (pH 9.6) as the coating solution, the purified A29 recombinant protein was diluted to 5 μg / mL and added to the ELISA plate at 100 μL / well for overnight coating at 4°C. The plate was washed five times with PBST, and 200 μL of blocking solution (NRA blocking solution from Wantai Company) was added to each well and blocked at 37°C for 2 hours. The plate was washed five times with PBST, air-dried at room temperature, placed in a desiccant, vacuum-packed, and stored at 4°C.

[0077] 2. Establishment of the operating procedures and judgment criteria for the monkeypox virus A29 antibody detection kit

[0078] Kit detection operating procedures

[0079] The testing procedure is:

[0080] 1) Dilute 10× concentrated washing solution 10 times to make washing solution;

[0081] 2) The serum to be tested, positive control serum and negative control serum were diluted 1:50 with sample diluent, 100 μL was added to each well of the ELISA detection plate, and incubated at 37°C for 30 min, and then centrifuged;

[0082] 3) 200 μL of washing solution was added to each well, and washed 5 times, each time for 1 min, and then centrifuged;

[0083] 4) 100 μL of enzyme-labeled secondary antibody working solution was added to each well, and incubated at 37°C for 30 min, and then centrifuged;

[0084] 5) 200 μL of washing solution was added to each well, and washed 5 times, each time for 1 min, and then centrifuged;

[0085] 6) 100 μL of TMB color developing solution was added to each well, and incubated at 37°C for 10 min in the dark;

[0086] 7) 50 μL of stop solution was added to each well, and the absorbance (OD value) was read at 450 nm by using an enzyme-labeled instrument. 450

[0087] Determination criteria of the detection results

[0088] The antibody detection was performed on 150 healthy human negative samples which were not infected with monkeypox, and the detection results are shown in Table 2. The cut-off value of the ELISA detection method was obtained by calculating the average value and standard deviation (X+3SD) of the negative samples. The judgment criteria of the sample to be tested were defined by the calculated cut-off value: when the OD 450 of the sample to be tested was greater than or equal to 0.35, it was determined to be positive; when the OD 450 was less than or equal to 0.29, the sample was determined to be negative; when 0.35>OD 450 >0.29, it was determined to be suspicious, and the sample needed to be retested once. If the OD 450 was greater than or equal to 0.35, it was determined to be positive; and if the OD 450 was less than or equal to 0.29, it was determined to be negative.

[0089] Table 2 Detection data of 147 healthy human negative serum samples which were not infected with monkeypox by the ELISA kit of the application

[0090] 0.056 0.023 0.074 0.065 0.102 0.079 0.119 0.128 0.087 0.093 0.076 0.135 0.179 0.128 0.069 0.078 0.054 0.092 0.087 0.138 0.195 0.093 0.065 0.099 0.156 0.068 0.055 0.074 0.098 0.061 0.135 0.143 0.121 0.082 0.132 0.129 0.055 0.131 0.068 0.726 0.053 0.128 0.189 0.058 0.064 0.103 0.154 0.089 0.077 0.065 0.138 0.110 0.157 0.093 0.172 0.086 0.059 0.091 0.154 0.128 0.133 0.147 0.098 0.069 0.172 0.083 0.079 0.169 0.138 0.048 0.082 0.159 0.172 0.051 0.112 0.090 0.141 0.088 0.126 0.060 0.134 0.053 0.122 0.068 0.098 0.119 0.101 0.059 0.137 0.103 0.084 0.058 0.069 0.073 0.199 0.082 0.097 0.136 0.108 0.091 0.058 0.081 0.049 0.069 0.103 0.119 0.142 0.093 0.072 0.139 0.110 0.086 0.058 0.103 0.125 0.087 0.095 0.581 0.116 0.130 0.069 0.134 0.078 0.059 0.123 0.110 0.135 0.083 0.090 0.054 0.195 0.110 0.139 0.127 0.083 0.054 0.190 0.152 0.084 0.091 0.103 0.112 0.073 0.051 0.084 0.192 0.058 / / / / / / / / /

[0091] Example 3 Indirect ELISA kit of monkeypox virus A29 recombinant protein and specificity test thereof

[0092] ​The kit consists of: a 96-well ELISA plate coated with 0.5 μg / mL of the A29 recombinant protein prepared in Example 1 as the coating antigen, 100 μL of positive control serum, 100 μL of negative control serum, 12 mL of enzyme-labeled secondary antibody, 6 mL of colorimetric solution A, 6 mL of colorimetric solution B, 6 mL of stop solution, 50 mL of washing solution, and 25 mL of sample diluent.

[0093] The A29 recombinant protein is a protein obtained by expressing the monkeypox virus A29 gene in an Escherichia coli expression system, and is purified by nickel-agarose gel affinity chromatography and identified by SDS-PAGE to obtain the purified target protein (see Example 1). The A29 recombinant protein contains the amino acid sequence shown in SEQ ID NO: 1. The A29 recombinant protein is diluted to 5 μg / mL using coating buffer.

[0094] The coating buffer solution is formulated as follows: 1.59 g of Na2CO3 and 2.93 g of NaHCO3 are dissolved in deionized water, the volume is fixed to 1000 mL, and the pH is adjusted to 9.5-9.8.

[0095] The positive control serum was monkeypox positive serum.

[0096] The negative control serum was negative serum from healthy individuals.

[0097] The sample diluent is PBST.

[0098] The blocking solution was purchased from Beijing Wantai.

[0099] The enzyme-labeled secondary antibody was an HRP-labeled goat anti-human enzyme-labeled antibody, and the concentration was 1:2000.

[0100] The color developing solution is TMB substrate solution, and the detection wavelength is 450 nm.

[0101] The stop solution is: 2 mol / L sulfuric acid.

[0102] The washing solution is PBST buffer, and the formula is: 8.0 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, 9 g of Na2HPO4·12H2O2, and 0.5 mL of Tween-20 are dissolved in 800 mL of distilled water, the pH is adjusted to 7.4, and the volume is made up to 1000 mL.

[0103] Specificity test

[0104] The specificity test of the diagnostic method was carried out using 30 sera from patients with varicella-zoster, 5 positive sera from patients with measles and 3 healthy people who had received smallpox vaccine. The results showed that the OD of the samples was 450 The values ​​are all less than 0.25.

[0105] Table 3 ELISA kit of the present invention for the detection of serum of patients with varicella-zoster, measles and healthy people who have been vaccinated with smallpox vaccine

[0106] Sample No. OD450 value Sample No. OD450 value Sample No. OD450 value VZV1 0.09 VZV16 0.21 Mea1 0.08 VZV2 0.13 VZV17 0.14 Mea2 0.05 VZV3 0.1 VZV18 0.11 Mea3 0.1 VZV4 0.07 VZV19 0.08 Mea4 0.07 VZV5 0.2 VZV20 0.08 Mea5 0.16 VZV6 0.11 VZV21 0.16 Vac1 0.08 VZV7 0.11 VZV22 0.13 Vac2 0.15 VZV8 0.08 VZV23 0.20 Vac3 0.11 VZV9 0.09 VZV24 0.09 VZV10 0.16 VZV25 0.12 VZV11 0.09 VZV26 0.10 VZV12 0.11 VZV27 0.05 VZV13 0.13 VZV28 0.09 VZV14 0.07 VZV29 0.17 VZV15 0.11 VZV30 0.09

[0107] Note: VZV is the serum of a patient infected with varicella-zoster; Meal is the serum of a patient with measles; Vac is the serum of a healthy person who has been vaccinated with smallpox vaccine.

[0108] Comparison of Acute and Convalescent Samples: Antibody testing was performed on sera from five monkeypox patients during the acute and convalescent phases. The test data are shown in Table 4. The results showed that IgG antibody readings in the convalescent phase were more than four times those in the acute phase.

[0109] Table 4 ELISA kit of the present invention detects the serum of 5 patients with acute and convalescent monkeypox

[0110]

[0111] (3) Comparison of clinical sample detection: The present invention conducted antibody detection on the sera of 54 monkeypox patients in the convalescent period. The detection data are shown in Table 5. The corresponding nucleic acid detection background of monkeypox patients is shown in Table 6.

[0112] Table 5 ELISA kit of the present invention detects 54 cases of monkeypox patients in the convalescent period.

[0113] A B C D E F G H I J K L 1 1.25 1.13 1.02 1.08 1.21 1.30 1.54 1.11 0.98 1.31 1.01 0.92 2 0.95 1.04 1.11 0.98 1.02 1.23 0.94 0.85 1.21 1.10 0.89 1.03 3 1.43 1.07 0.92 0.87 1.01 1.08 0.91 1.03 1.51 0.98 1.22 0.89 4 0.89 1.02 0.99 1.08 1.12 0.89 1.46 1.25 0.99 1.13 0.96 1.10 5 1.31 1.09 0.96 0.88 1.03 0.85 / / / / / /

[0114] Table 6 Ct values ​​of nucleic acid detection in 54 patients with convalescent monkeypox at the time of admission

[0115]

[0116]

[0117] (Note: The monkeypox nucleic acid test for the patient was performed using the Beijing Jinhao Monkeypox Nucleic Acid Detection Kit (fluorescence quantitative PCR method). According to the kit instructions, a Ct value ≤ 40 was considered positive.)

[0118] The above experiments show that the ELISA diagnostic method of the present invention has strong specificity, stable and reliable results, can specifically detect monkeypox IgG antibodies, and is suitable for diagnosis and antibody monitoring after monkeypox virus infection in humans.

[0119] In summary, the A29 recombinant protein used in the present invention is convenient for large-scale preparation and purification, the A29 gene can be stably and efficiently expressed in the pET prokaryotic expression system, and the recombinant protein carrying the histidine tag is easy to purify. The kit of the present invention has strong specificity, can effectively eliminate the interference of clinical similar diseases of monkeypox such as measles, varicella-zoster infection, and healthy people who have been vaccinated with smallpox vaccine, and specifically detect monkeypox antibodies. The diagnostic kit of the present invention has strong practicality and can be used for antibody detection of monkeypox virus human infection or diagnosis of monkeypox, and can also be used for antibody monitoring after monkeypox virus infection of the human body. The judgment is convenient, sensitive, accurate and reliable. TMB substrate is used for color development, and the OD value is measured with an enzyme marker to determine the monitoring result of the sample to be tested; the difference in positive and negative results is obvious, and it is more sensitive, reliable and stable than OPD color development. It is simple and quick to operate, and sample detection can be completed within 1.5 hours, which is time-consuming and low-cost, and is very suitable for the detection of a large number of animal serum samples.

[0120] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A monkeypox virus A29-C71A-C72A-H74R-R107H recombinant protein, characterized in that: The amino acid sequence of the A29-C71A-C72A-H74R-R107H recombinant protein is shown in SEQ ID No.

1.

2. A gene encoding the monkeypox virus A29-C71A-C72A-H74R-R107H recombinant protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.

2.

3. An indirect IgG ELISA detection kit for monkeypox virus A29-C71A-C72A-H74R-R107H recombinant protein antibody, characterized in that: It comprises a recombinant protein coating antigen, wherein the recombinant protein coating antigen is the monkeypox virus A29-C71A-C72A-H74R-R107H recombinant protein according to claim 1.

4. The detection kit according to claim 3, characterized in that It also includes sample diluent, enzyme-labeled antibody, TMB substrate solution, stop solution, concentrated washing solution, as well as sealing film, dilution plate, negative control, and positive control; The positive control solution is prepared by diluting monkeypox positive serum with sample diluent; the negative control solution is prepared by diluting healthy person negative serum with sample diluent; The enzyme-labeled antibody shown is a goat anti-human enzyme-labeled antibody.

5. A method for preparing the monkeypox virus A29-C71A-C72A-H74R-R107H recombinant protein according to claim 1, characterized in that: The following steps are included: S1. Using monkeypox virus genomic DNA as a template, gene amplification was performed using specific primers by PCR to obtain the A29-C71A-C72A-H74R-R107H specific target gene; The S2.A29-specific target gene was seamlessly cloned and assembled, and connected to the pET30a(+) expression vector to construct the recombinant vector pETA29; S3. The recombinant vector pETA29 was transformed into BL21 Escherichia coli to obtain a recombinant expression strain; S4. The recombinant expression strain was induced with IPTG, lysed, and centrifuged. The supernatant and precipitate were collected and purified using nickel-agarose gel affinity chromatography to obtain the monkeypox virus A29-C71A-C72A-H74R-R107H recombinant protein.

6. An indirect ELISA method for detecting monkeypox virus, characterized in that: The following steps are involved: S1. Coating: Dilute the recombinant protein coating antigen with carbonate buffer, wherein the amino acid sequence of the recombinant protein coating antigen is shown in SEQ ID NO: 1; S2. Blocking: Add blocking solution to each well, spin dry, and wash with PBST. S3. Test serum conditions: The test serum was diluted with diluent, incubated, dried, and washed with PBST. S4. Secondary antibody conditions: Dilute HRP-labeled goat anti-human IgG in PBST, incubate, spin dry, and wash with PBST. S5. Color development: Add color development solution to each well and add stop solution after incubation to terminate the reaction. S6. Reading: Read the OD data at 450 min using a microplate reader and calculate the results.

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