A monkeypox virus IgM antibody detection kit and a method for preparing monkeypox virus specific fusion protein antigen

Abstract

The invention discloses a monkeypox virus IgM antibody detection kit, comprising a monkeypox virus specific fusion protein antigen enzyme conjugate, an anti-human IgM monoclonal antibody strip, a sample diluent, a concentrated washing solution, a negative control, a positive control, a substrate solution A, a substrate solution B and a stop solution. The antigen used is a monkeypox virus specific fusion protein antigen: the amino acid sequence is shown in SEQ ID NO.3. The coding gene of the monkeypox virus specific fusion protein antigen: the nucleotide sequence is shown in SEQ ID NO.4. The kit of the invention can specifically distinguish between people immunized with smallpox virus vaccine and people newly infected with monkeypox virus, and can judge whether a human body is infected with monkeypox virus for the first time, with good specificity.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering technology and in vitro diagnostic technology, and in particular to a monkeypox virus IgM antibody detection kit and a method for preparing a monkeypox virus specific fusion protein antigen. Background Art

[0002] Monkeypox virus belongs to orthopoxvirus, which is the first subgenus of poxvirus. The size of poxvirus is about 200-400mm, brick-shaped or oval with rounded corners. The spherical nucleus is surrounded by an outer membrane, and the nucleus contains a large double-stranded DNA gene. There are two branches of monkeypox virus in genetic evolution. The mortality rate of the West African branch is about 1% to 5%, and the mortality rate of the Congo Basin (Central Africa) branch can be as high as 11%. Orthopoxviruses differ in host tropism and virulence, but orthopoxviruses show a high degree of similarity in virus morphology, life cycle and structure, and the homology between protein groups reaches 92-95%. Therefore, it is more important to find an antigen that can effectively distinguish between antibodies against smallpox virus vaccine and monkeypox virus infection, which is an important factor affecting the accuracy of monkeypox detection kits.

[0003] The monkeypox A44R protein is homologous to the vaccinia 169 protein and is localized in the cytoplasm, which is different from the virus. It plays a role in inhibiting host protein synthesis. Specifically, it inhibits the initiation of cap-dependent and cap-independent translation. In turn, it affects the outcome of infection by reducing the recruitment of inflammatory leukocytes and reducing memory CD8+T cell responses. Studies have shown that after infection with monkeypox virus, IgM antibodies can specifically bind to the A44R protein 6 days later, and become stronger and stronger; 28 days after infection, there are almost no IgM and IgA in the recovery period, and only IgG can be detected. However, the binding force of A44R to IgG is weakened compared to IgM, and it is even undetectable. The mechanism is still unknown. Therefore, A44R can specifically identify the characteristics of IgM antibodies, which can effectively distinguish between antibodies against smallpox virus vaccine and monkeypox virus infection.

[0004] Currently, there is no specific detection method for monkeypox virus IgM antibodies on the market. It is urgent to develop a detection kit that can effectively distinguish between antibodies against smallpox virus vaccine and monkeypox virus infection. Summary of the invention

[0005] In order to solve the above-mentioned problems existing in the prior art, the present invention provides a monkeypox virus IgM antibody detection kit and a method for preparing a monkeypox virus-specific fusion protein antigen; and achieves the following invention objectives: the antibody detection kit can effectively distinguish between smallpox virus vaccine immunity and recent monkeypox virus infection; the prepared monkeypox virus-specific fusion protein antigen has good antigenicity and specificity.

[0006] The technical solution adopted by the present invention is:

[0007] A monkeypox virus IgM antibody detection kit comprises a monkeypox virus specific fusion protein antigen enzyme conjugate, an anti-human IgM monoclonal antibody strip, a sample diluent, a concentrated washing solution, a negative control, a positive control, a substrate solution A, a substrate solution B and a stop solution.

[0008] The antigen used is a monkeypox virus-specific fusion protein antigen: the amino acid sequence is shown in SEQ ID NO.3.

[0009] The gene encoding the monkeypox virus-specific fusion protein antigen: the nucleotide sequence is shown in SEQ ID NO.4.

[0010] The antigen used is a monkeypox virus-specific fusion protein antigen: it includes a specific amino acid sequence, the sequence of which is SEQ ID NO.1; and the nucleotide sequence expressing the specific amino acid sequence is SEQ ID NO.2.

[0011] The present invention provides a method for preparing a monkeypox virus-specific fusion protein antigen, the specific steps of which are as follows:

[0012] (1) Based on the amino acid sequence of GenBank: AAL40612.1, a specific amino acid sequence was screened, and the corresponding sequence was SEQ ID NO. 1; the nucleotide sequence A44R was obtained by codon optimization, and the corresponding sequence was SEQ ID NO. 2.

[0013] (2) The first nucleotide sequence A44R is connected to the second nucleotide sequence A44R by a linker, and the second nucleotide sequence A44R is connected to the third nucleotide sequence A44R by a linker. In this case, the corresponding amino acid sequence is SEQ ID NO. 3; the nucleotide sequence is SEQ ID NO. 4.

[0014] (3) Add restriction endonucleases BamHI and NotI to both ends of the concatenated monkeypox virus-specific fusion protein antigen nucleotide sequence and clone them into the expression vector PET28a to obtain a recombinant expression vector; then transfer the recombinant expression vector into the host bacteria BL21 (DE3) to obtain a recombinant engineered bacterium;

[0015] (4) Using recombinant engineered bacteria to induce the expression of monkeypox virus-specific fusion protein antigen;

[0016] (5) Extract and purify the monkeypox virus-specific fusion protein antigen.

[0017] The beneficial effects of the present invention are:

[0018] 1. The present invention has developed a monkeypox virus IgM antibody detection kit, which can effectively distinguish between smallpox virus vaccine immunity and recent monkeypox virus infection.

[0019] 2. The present invention has developed a monkeypox virus-specific fusion protein antigen and expressed it through recombinant engineering bacteria. The monkeypox virus-specific fusion protein antigen prepared by expression has good antigenicity and specificity;

[0020] 3. The present invention is based on the A44R protein, selects specific fragments to avoid crossover, constructs a triploid amino acid sequence, and performs prokaryotic expression, so that the repeated specific sequence is effectively expressed, avoiding the omission of specific sites due to steric hindrance, resulting in missed detection. The protein is used as a coating antigen in an enzyme-immunoassay platform to prepare a kit for detecting monkeypox IgM antibodies, and the specificity of the detection is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The analysis results of Bepipred Linear Epitope Prediction 2.0 in Example 1;

[0022] Figure 2 This is the SDS-PAGE result of the positive expression result of the recombinant engineering bacteria in Example 1;

[0023] Among them, serial number 1 is the control group, and serial numbers 2-3 are the positive expression groups.

[0024] Figure 3 This is a diagram showing the experimental results of the clinical experiment in Example 4. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0027] Example 1 A method for preparing a monkeypox virus-specific fusion protein antigen

[0028] Step 1: Screening of antigenic epitopes

[0029] According to the amino acid sequence of the A44R protein of the monkeypox virus published on NCBI, the secondary and tertiary structures of the protein were obtained using online analysis tools such as ExPasy and UniProt, and the dominant linear sites of the A44R protein of the monkeypox virus were screened out in combination with the analysis results of Bepipred Linear Epitope Prediction2.0. The specific linear sites of the A44R protein of the monkeypox virus were screened out and located from the 25th amino acid to the 70th amino acid; the specific amino acid sequence obtained was SEQ ID NO.1; the nucleotide sequence A44R was obtained by codon optimization, and the corresponding sequence was SEQ ID NO.2.

[0030] The specific Bepipred Linear Epitope Prediction 2.0 analysis results are as follows Figure 1 shown.

[0031] SEQ ID NO.1: ITIDVTPKKKKEKDVLLAQSVAVEEAKDVKVEEKNIIDIEDDDDMD;

[0032] SEQ ID NO.2: attaccatgatgtgaccccgaaaaaaaaaaagagaaagatgtgctgctggcccagagcgtggcggtggaagaagcgaaagatgtgaaagtggaagaaaaaaacatcatcgatattgaagatgatgatgatgatggat.

[0033] Step 2: Obtaining the antigen epitope gene of monkeypox virus A44R protein-specific antigen

[0034] The genes encoding the dominant antigen epitopes are concatenated in the following order: A44R 25-70 , linker (GGGGS) 4, A44R 25-70 , linker (GGGGS) 4, A44R 25-70 , a fusion antigen nucleotide sequence containing a trivalent epitope was constructed, and finally the monkeypox virus-specific fusion protein antigen nucleotide sequence was synthesized, and BamH and NotⅠ restriction endonucleases were added to both ends of the nucleotide. The corresponding amino acid sequence is SEQ ID NO.3; the nucleotide sequence is SEQ ID NO.4, and the antigen epitope gene of the monkeypox virus-specific fusion protein antigen is obtained.

[0035] SEQ ID NO. 3:

[0036] ITIDVTPKKKKEKDVLLAQSVAVEEAKDVKVEEKNIIDIEDDDDMD GGGGSGGGGSGGGGSGGGGSITIDVTPKKKKEKDVLLAQSVAVEEAKDVKVEEKNIIDIEDDDDMD GGGGSGGGGSGGGGSGGGGS ITIDVTPKKKKEKDVLLAQSVAVEEAKDVKVEEKNIIDIEDDDDMD;

[0037] SEQ ID NO.4:

[0038] atcaccattgatgtgacgccgaaaaaaaaaaaagaaaaagacgtgctgctggcgcagagcgtggcggttgaagaagcaaaagatgtgaaagtggaagaaaaaaacatcattgatattgaagatgatgatgatatggatggcggcggcggtagcggcggtggcggcagcggcggcggtggcagcggcggcggcggcagcattaccattgatgtgaccccgaaaaaaaaaaaagaaaaagatgtgctgctggcacagagcgtggccgtggaagaagcgaaagacgtgaaagtggaagaaaaaaatatcatcgacatcgaagatgatgatgatatggatggcggtggcggctcgggcggcggcggcagcggcggcggcggcagcggcggcggtggctcgattaccattgatgtgaccccgaagaaaaaaaaagaaaaagacgtgctgctggcccagagcgtggccgtggaagaagcgaaagatgttaaagttgaagaaaaaaacattattgatattgaagatgatgatgacatggat。

[0039] Step 3, construct an expression vector for the specific antigen of the monkeypox virus A44R protein

[0040] Extract the PET28a plasmid, double-digest the plasmid with BamHⅠ and NotⅠ, and recover the double-digested large fragment after electrophoresis, that is, the double-digested plasmid. Double-digest the monkeypox virus specific fusion protein antigen with BamHⅠ and NotⅠ, recover the gel after electrophoresis, and keep it at -20℃ for standby use to obtain the double-digested target fragment. The double-digested plasmid and the double-digested target fragment are connected at a ratio of 1:5 using T4 ligase at 16℃ overnight. After connection, the expression vector of the monkeypox virus A44R protein specific antigen is obtained, which is a recombinant expression vector named PET28a-A44R.

[0041] Step 4: Screening and identification of recombinant expression vectors

[0042] The recombinant expression vector was transferred into E. coli BL21 (DE3). The recombinant expression vector was transformed into E. coli BL21 (DE3), coated on LB plates containing 50ug / ml kanamycin sulfate, and incubated overnight at 37°C. The E. coli BL21 (DE3) containing plasmid PET28a was used as the control colony, and coated and cultured under the same conditions.

[0043] The next day, 5 transformed colonies and 2 control colonies (plasmid PET28a) were randomly picked and plasmids were extracted from each.

[0044] The plasmid PET28a-A44R extracted from the transformed colony was double-digested with BamHⅠ and NotⅠ. After digestion, electrophoresis was performed and the corresponding target fragments and vector fragments were visible. The results showed that the recombinant expression vector was successfully constructed, which was a positive expression bacterium.

[0045] The plasmid extracted from the control bacteria was double-digested with BamHI and NotⅠ and then subjected to electrophoresis, but no target fragment was observed.

[0046] Step 5: Efficient expression after positive expression bacteria identification

[0047] The positive expression bacteria and control bacteria were inoculated into 2 ml LB medium test tubes, shaken at 37°C for 2 hours, and IPTG was added. The final concentration of IPTG was 1 mmol / l. The induction was continued at 30°C for 6 hours. The bacteria were collected by centrifugation and tested by SDS-PAGE. The LB medium contained 50 ug / ml kanamycin sulfate.

[0048] The test found that after the positive expression bacteria were cultured, the target protein expressed by plasmid PET28a-A44R was about 21KD in size, which was consistent with the prediction result of ExPSY ProtParam online software. There was no target protein band in the control bacteria. This indicates that the recombinant engineering bacteria expressing monkeypox virus-specific fusion protein antigen were obtained (the SDS-PAGE result of the positive expression result of the recombinant engineering bacteria is shown in the attached figure). Figure 2 as shown).

[0049] Step 6: Purification of expressed protein

[0050] (1) The recombinant engineered bacteria expressing the monkeypox virus-specific fusion protein antigen and its culture medium (i.e., the original bacterial solution) were centrifuged at 12,000 rpm and 4°C for 15 min to separate and retain the precipitate.

[0051] (2) First resuspension: Resuspend the retained precipitate in PBS buffer with a volume of 1 / 10 of the original bacterial solution, wash thoroughly once, and centrifuge at 12,000 rpm and 4°C for 15 min to separate and retain the precipitate.

[0052] (3) Second resuspension: The precipitate obtained after the first resuspension was resuspended in PBS buffer with a volume of 1 / 10 of the original bacterial solution, ultrasonicated on ice for 30 min, centrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was separated and retained. The PET28a-A44R expression protein was present in the supernatant.

[0053] (4) Chromatography: Use an IMAC Bestarose FF column (purchased from Bogelon (Shanghai) Biotechnology Co., Ltd.) for affinity chromatography. Equilibrate the column with 10 column volumes of ultrapure water. Equilibrate the column with 10 column volumes of equilibration buffer and then add the protein sample. After loading, wash away unbound proteins with 10 column volumes of equilibration buffer. Finally, elute the target protein with elution buffer to obtain a purified PET28a-A44R expression protein solution, i.e., the monkeypox virus-specific fusion protein antigen.

[0054] The PBS buffer has a concentration of 0.01 M and a pH value of 7.4.

[0055] The equilibration buffer is: 10 mM PBS buffer, pH 7.4;

[0056] The elution buffer is 10 mM PBS buffer containing 500 mM imidazole and a pH value of 7.4.

[0057] Example 2 Monkeypox virus IgM antibody detection kit

[0058] The monkeypox virus IgM antibody detection kit includes a monkeypox virus-specific fusion protein antigen-enzyme conjugate, an anti-human IgM monoclonal antibody strip, a sample diluent, a concentrated washing solution, a negative control, a positive control, a substrate solution A, a substrate solution B and a stop solution.

[0059] Preparation method of monkeypox virus IgM antibody detection kit:

[0060] Step 1: Preparation of monkeypox virus specific fusion protein antigen enzyme conjugate

[0061] (1) Dialysis

[0062] 1 mL of horseradish peroxidase solution was added to 2 L of pH 4.4 acetate buffer and dialyzed at 4° C. for 24 hours. The horseradish peroxidase solution had a concentration of 5 mg / mL and contained 3.5 mg / mL sodium periodate.

[0063] 1 mL of the purified PET28a-A44R expression protein solution prepared in Example 1 was taken, and 2 L of carbonate buffer solution with a concentration of 1.0 mol / L and a pH value of 9.6 was added, and dialyzed at 4° C. for 24 hours.

[0064] (2) Labeling and purification of PET28a-A44R expressed protein

[0065] The dialyzed horseradish peroxidase solution of step (1) and the dialyzed purified PET28a-A44R expression protein solution of step (1) are mixed at a volume ratio of 1:1 to obtain a mixture A.

[0066] 2 mL of mixture A was added to 2 L of carbonate buffer solution with a concentration of 1.0 mol / L and a pH value of 9.6, and dialyzed for 6 hours under stirring. Then 0.1 mL of 6 mg / mL sodium borohydride solution was added to the dialyzed mixture A to obtain a mixture B.

[0067] The mixed solution B was passed through a Sephadex G-200 column and eluted with a pH 7.2 phosphate buffer. The effluent was collected in 1 mL sections. The effluent was detected with a detection strip, and the effluent that was collected and displayed blue with the detection strip was the labeled purified PET28a-A44R expression protein.

[0068] The detection strip is prepared by using anti-monkeypox virus IgM antibody to obtain an anti-monkeypox virus IgM antibody strip, which is the detection strip. The anti-monkeypox virus IgM antibody is commercially available.

[0069] (3) Preparation of monkeypox virus specific fusion protein antigen enzyme conjugate

[0070] The labeled purified PET28a-A44R expression protein prepared in step (2) is added to a phosphate buffer; the volume ratio of the protein to the phosphate buffer is 1:5000; the phosphate buffer has a concentration of 60 mM and a pH value of 7.2; and contains 10% calf serum and 0.5% PC300, wherein the percentages are by volume.

[0071] The labeled purified PET28a-A44R expression protein and the buffer solution were evenly mixed to prepare a monkeypox virus specific fusion protein antigen enzyme conjugate.

[0072] Step 2: Preparation of anti-human IgM monoclonal antibody strips

[0073] Anti-human IgM monoclonal antibody was diluted to 1µg / mL with carbonate buffer at a concentration of 50mM and pH 9.6 to obtain antibody diluent. Then, the ELISA plate was coated with the antibody diluent at a protein amount of 100ng / well and washed overnight at 4°C.

[0074] To the washed ELISA plate, add PBS containing 10% calf serum by volume at a volume of 200 µl / well, block at 37°C for 2 hours, dry and store at 4°C for later use, to prepare an anti-human IgM monoclonal antibody strip.

[0075] Step 3: Prepare sample dilution solution

[0076] 70 g of BSA was added to 1 L of 60 mM phosphate buffer with a pH of 7.2, and the mixture was mixed well to obtain a sample dilution solution.

[0077] Step 4: Prepare concentrated washing solution

[0078] 0.3 mL of Tween 20 was added to 1 L of 60 mM phosphate buffer with a pH of 7.2, and the mixture was mixed well to obtain a concentrated washing solution.

[0079] Step 5: Prepare negative control

[0080] 9 g of sodium chloride, 20 g of sucrose, and 5 mL of PC300 were added to 1 L of 60 mM phosphate buffer with a pH of 7.4, and the mixture was mixed well to obtain a negative control.

[0081] Step 6. Prepare positive control

[0082] 0.1 mL of anti-monkeypox virus IgM antibody was added to 1 L of negative control and mixed evenly to obtain a positive control. The anti-monkeypox virus IgM antibody was commercially available.

[0083] Step 7: Prepare substrate solution A

[0084] Add 5 g of trisodium citrate, 9 g of citric acid, and 0.6 mL of hydrogen peroxide to 1 L of 50 mM acetic acid-sodium acetate buffer at pH 5.5, mix well, and obtain substrate solution A.

[0085] Step 8: Prepare substrate solution B

[0086] Add 1.5 g TMB·HCl and 1 g EDTA to 1 L pure water and mix well to obtain substrate solution B.

[0087] Step 9. Prepare stop solution

[0088] Add 110 mL of sulfuric acid to 890 mL of pure water and mix well to obtain a stop solution.

[0089] Example 3 Application method of monkeypox virus IgM antibody detection kit

[0090] 1. Sample collection

[0091] Take venous blood, let it stand for 30 minutes to allow it to shrink naturally, then centrifuge it for 10 minutes to obtain serum, which is the sample to be tested.

[0092] 2. Sample addition test

[0093] a. Take out the monkeypox virus IgM antibody test kit stored in a refrigerated environment and equilibrate it to room temperature.

[0094] b. Dilute the concentrated washing solution with purified water at a volume ratio of 1:40 and shake well; the diluted washing solution is ready for use.

[0095] c. Take the anti-human IgM monoclonal antibody strip and add 100 µL of sample diluent into the reaction wells. Reserve one well for blank control, negative control, and positive control respectively. Do not add sample diluent to the blank control well, negative control well, and positive control well.

[0096] d. Take 10µL of the sample to be tested and add it to the reaction wells of the strip. Add 100µL of negative control to the negative control wells. Add 100µL of positive control to each positive control well. Leave the blank control wells empty.

[0097] e. Oscillate the reaction plate for 20 seconds to mix thoroughly and incubate at 37°C for 20 minutes.

[0098] f. Take out and wash the plate 5 times. When washing the plate, fill each well with diluted washing solution, leave it for 10 seconds, then suck it out and pat it dry.

[0099] g. Add 50 µL of monkeypox virus-specific fusion protein antigen-enzyme conjugate to each well, and do not add any to the blank control well. Vortex the reaction plate for 20 seconds to mix thoroughly, and incubate at 37°C for 20 minutes.

[0100] h. Take out and wash the plate five times.

[0101] i. Add 50µL of substrate solution A and substrate solution B to each well. Add nothing to the blank control well. Vortex the reaction plate for 20 seconds to mix thoroughly. Apply sealing film and incubate at 37℃ for 10 minutes.

[0102] j. After taking out the reaction strips, add 50µL of stop solution to each well, and do not add any to the blank control wells. Vortex the reaction strips for 20 seconds to mix thoroughly, and read the results using an ELISA reader.

[0103] 3. Results Analysis

[0104] At a wavelength of 450 nm, the blank control well was used to adjust the concentration to zero and the OD value of each well was read.

[0105] Critical value = 2.1 × average OD value of negative control. When the average OD value of negative control is less than 0.05, it is calculated as 0.05; when the average OD value of negative control is greater than 0.05, it is calculated as the actual value.

[0106] The specimen OD value ≥ the critical value was positive;

[0107] The sample OD value < the critical value is negative.

[0108] Validity judgment: The positive control OD value should be ≥0.8, and the negative control OD value should be ≤0.1, otherwise the test results are invalid and the test should be repeated.

[0109] Example 4 Performance index test of the monkeypox virus IgM antibody detection kit of the present invention

[0110] (1) Negative compliance rate:

[0111] 10 negative sera were taken as samples to be tested, and the test was performed using the kit of Example 2 and the method of Example 3. The test results are shown in Table 1:

[0112] Table 1

[0113]

[0114] Note: OD value ≥ 0.105 is positive; OD value < 0.105 is negative.

[0115] As can be seen from Table 1, all the test results were negative, and the negative compliance rate was 10 / 10 -.

[0116] (2) Positive compliance rate:

[0117] 0.006%, 0.001%, 0.003%, 0.015% and 0.012% of anti-monkeypox virus IgM antibody were added to the 5 negative controls respectively to prepare samples to be tested, which were numbered P1, P2, P3, P4 and P5 respectively. The test was performed using the kit of Example 2 and the method of Example 3. The test results are shown in Table 2:

[0118] Table 2

[0119]

[0120] Note: OD value ≥ 0.105 is positive; OD value < 0.105 is negative.

[0121] As can be seen from Table 2, all the test results were positive, with a positive compliance rate of 5 / 5+.

[0122] (3) Minimum detection limit:

[0123] 0.003% of anti-monkeypox virus IgM antibody was added to the negative control to prepare the internal minimum detection limit reference product. The internal minimum detection limit reference product was serially diluted for detection, and the dilution ratios were 1:2, 1:4, 1:8, 1:16, and 1:32, respectively. The kit of Example 2 was used to detect according to the method of Example 3. The detection results are shown in Table 3:

[0124] Table 3

[0125]

[0126] Note: OD value ≥ 0.105 is positive; OD value < 0.105 is negative.

[0127] It can be seen from Table 3 that the titration endpoint is not less than 1:8; therefore, the minimum detection limit of the detection kit of the present invention is 1:8; that is, the anti-monkeypox virus IgM antibody content is 0.000375%.

[0128] (4) Repeatability:

[0129] 0.0025% of anti-monkeypox virus IgM antibody was added to the negative control to prepare an internal repeatability reference. The internal repeatability reference was used as the sample to be tested, and the kit of Example 2 was used. According to the method of Example 3, the test was repeated 10 times. The test results are shown in Table 4:

[0130] Table 4

[0131]

[0132] As shown in Table 4, the coefficient of variation is 3.18%, and its coefficient of variation (CV) is no higher than 15%, indicating good repeatability.

[0133] (5) Stability:

[0134] The monkeypox virus IgM antibody detection kit of the present invention was used to perform negative coincidence rate, positive coincidence rate, minimum detection limit, and repeatability CV detection experiments after the preparation was completed, placed at normal storage conditions of 2-8°C for 6 months, and placed at 37°C for 8 days. The experimental method was the same as the above methods (1)-(4). The detection results are shown in Table 5:

[0135] Table 5

[0136]

[0137] As can be seen from Table 5, the negative compliance rate of the monkeypox virus IgM antibody detection kit placed at 2-8°C for 6 months was 10 / 10-, the positive compliance rate was 5 / 5+, the minimum detection limit was 1:8, and the coefficient of variation (CV) was 7.00%; the negative compliance rate of the monkeypox virus IgM antibody detection kit placed at 37°C for 8 days was 10 / 10-, the positive compliance rate was 5 / 5+, the minimum detection limit was 1:8, and the coefficient of variation (CV) was 7.21%, indicating that the monkeypox virus IgM antibody detection kit of the present invention has high stability.

[0138] (6) Clinical trials:

[0139] The capture enzyme-linked immunosorbent assay was used to detect 45 sera of people over 60 years old who had been immunized with vaccinia virus and 45 sera of people under 30 years old who had not been immunized with vaccinia virus. The monkeypox virus IgM antibody detection kit of the present invention was used to detect anti-monkeypox virus IgM antibodies. The OD value was measured at a wavelength of 450nm. The results are shown in Table 6.

[0140] Table 6

[0141]

[0142] Note: OD value ≥ 0.105 is positive; OD value < 0.105 is negative.

[0143] This kit did not react with 45 sera from people over 60 years old who had been immunized with vaccinia virus and 45 sera from people under 30 years old who had not been immunized with vaccinia virus, with an OD value of <0.105; however, it produced a strong color development with anti-monkeypox virus IgM antibodies, with an OD value of 1.476, which was greater than 0.105; this indicated that this kit had good specificity.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A monkeypox virus IgM antibody detection kit, characterized in that: It includes a monkeypox virus-specific fusion protein antigen-enzyme conjugate, an anti-human IgM monoclonal antibody strip, a sample diluent, a concentrated washing solution, a negative control, a positive control, a substrate solution A, a substrate solution B and a stop solution. The amino acid sequence of the monkeypox virus-specific fusion protein antigen is shown in SEQ ID NO.

3.

2. A monkeypox virus IgM antibody detection kit according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the monkeypox virus-specific fusion protein antigen is shown in SEQ ID NO.

4.

3. A method for preparing a monkeypox virus-specific fusion protein antigen, characterized in that: The preparation method includes the steps of screening antigenic epitopes, obtaining antigenic epitope genes of monkeypox virus A44R protein-specific antigens, constructing expression vectors of monkeypox virus A44R protein-specific antigens, screening and identifying recombinant expression vectors, efficiently expressing positive expression bacteria after identification, and purifying the expressed proteins; the amino acid sequence of the monkeypox virus-specific fusion protein antigen is shown in SEQ ID NO.

3.

4. The method for preparing a monkeypox virus-specific fusion protein antigen according to claim 3, characterized in that: Screening of the antigenic epitope: The specific linear site of the monkeypox virus A44R protein is screened out, which is located between the 25th amino acid and the 70th amino acid; the amino acid sequence is shown in SEQ ID NO.1; the nucleotide sequence A44R is obtained by codon optimization, and the nucleotide sequence is shown in SEQ ID NO.

2.

5. The method for preparing a monkeypox virus-specific fusion protein antigen according to claim 4, characterized in that: The nucleotide sequence of the antigen epitope gene is SEQ ID NO.

4.

6. The method for preparing a monkeypox virus-specific fusion protein antigen according to claim 5, characterized in that: The expression vector for constructing the monkeypox virus A44R protein-specific antigen: The PET28a plasmid is used as the expression vector, and restriction endonucleases BamHI and NotI are added to both ends of the serially connected monkeypox virus-specific fusion protein antigen nucleotide sequence and cloned into the expression vector PET28a to obtain a recombinant expression vector.

7. The method for preparing a monkeypox virus-specific fusion protein antigen according to claim 6, characterized in that: Screening and identification of the recombinant expression vector: The recombinant expression vector is transferred into Escherichia coli BL21 DE3 to obtain positive expression bacteria.

8. The method for preparing a monkeypox virus-specific fusion protein antigen according to claim 7, characterized in that: The purification of the expressed protein includes centrifugation, first resuspension, second resuspension and chromatography steps.

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