Dengue type 2 virus envelope protein EDIII detection kit as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of immunology, and particularly discloses a detection kit for dengue type 2 virus envelope protein EDIII as well as a preparation method and application of the detection kit. The invention provides a recombinant dengue type 2 virus envelope protein EDIII antigen. A nucleotide sequence of a coding gene of the recombinant dengue type 2 virus envelope protein EDIII antigen is shown as SEQ ID NO. 1. The target protein immunogen with higher purity is prepared by means of prokaryotic expression, denaturation dissolution, nickel column purification, dialysis renaturation and the like. A New Zealand white rabbit is immunized by the dengue type 2 virus envelope protein EDIII polyclonal antibody with strong specificity and high titer is prepared. According to the present invention, the dengue type 2 virus envelope protein EDIII enzyme-linked immunosorbent assay method is established based on the polyclonal antibody, the polyclonal antibody and other types of flaviviruses have no serum cross reaction, and the established immunosorbent assay method can be used for detection of the dengue type 2 virus, has advantages of simple operation, high sensitivity, strong specificity and the like, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and particularly relates to a dengue type 2 virus envelope protein EDⅢ detection kit, a preparation method thereof, and an application thereof. Background Art

[0002] Dengue fever, as an acute infectious disease caused by dengue virus, poses a serious threat to global public health. Dengue virus (DENV) belongs to the genus Flavivirus of the family Flaviviridae. The main vectors and transmission media are Aedes albopictus and Aedes aegypti. It is mainly divided into four serotypes, among which serotype 2 has the widest spread. The disease course of dengue fever develops very rapidly, and it can progress from mild to severe within just a few days. The best window period for diagnosing dengue fever is usually from the onset of fever to ten days after infection. Therefore, it is crucial to develop rapid and accurate diagnostic tools for early disease diagnosis and management.

[0003] Currently, the research targets of detection kits mainly focus on IgG, IgM antibodies, and NS1 antigen. However, IgG and IgM antibody detections are commonly associated with cross-reactions with other flaviviruses, which are prone to producing false positive results; NS1 antigen is prone to forming NS1-IgG antigen-antibody immune complexes in the late stage of secondary infection, making it difficult to detect NS1, seriously affecting the accuracy and sensitivity of detection. In the sera of dengue fever patients, in addition to NS1 antigen, there is also a high concentration of envelope E protein antigen. The E protein is the most important surface-exposed structural protein of DENV, existing in the form of a homotrimer on the surface of mature virus particles, and can be divided into three envelope domains. Among them, the third domain (EDⅢ) has an IgG-like structure similar to immunoglobulin, independent of other domains of the E protein, and protrudes vertically from the virus surface to form a protrusion. Currently, EDⅢ has been proven to be the receptor recognition and binding domain of DENV, and can be used as a target for serological diagnosis for the detection of dengue virus. Therefore, a dengue virus detection method and product with high specificity, high sensitivity, and few cross-reactions based on EDⅢ can be developed. Summary of the Invention

[0004] Aiming at the deficiencies existing in the prior art, the present invention proposes an ELISA detection kit for dengue type 2 virus envelope protein EDⅢ, which can be used for early diagnosis and disease condition management of dengue fever patients by detecting the content of envelope protein EDⅢ of dengue type 2 virus in sera, and has the advantages of simple operation, strong specificity, high sensitivity, and few cross-reactions.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides a recombinant dengue type 2 virus envelope protein EDⅢ antigen, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO.1.

[0007] Second aspect, the present invention provides a method for preparing the above-mentioned dengue type 2 virus envelope protein EDⅢ antigen, comprising the following steps:

[0008] S1. Construct a vector: According to the optimized DNA fragment of the dengue type 2 virus envelope protein EDⅢ, select a restriction enzyme cleavage site in the reading frame of the pET series expression vector DNA to complete the construction of the recombinant expression vector;

[0009] S2. Express the protein: Transform the protein recombinant expression vector obtained in step S1 into Escherichia coli competent cells to form an engineered bacterium, culture the engineered bacterium to produce recombinant dengue type 2 virus envelope protein EDⅢ, and centrifuge to obtain the bacterial precipitate;

[0010] S3. Ultrasonic disruption: Use ultrasonic disruption on the bacterial precipitate obtained in step S2, and centrifuge to obtain the inclusion body precipitate of dengue type 2 virus envelope protein EDⅢ;

[0011] S4. Denaturation and purification: Wash the inclusion body precipitate obtained in step S3 2 - 5 times, suspend the precipitate in a denaturing reagent and dissolve it overnight at 4°C, centrifuge, and take the supernatant to filter through a membrane; Load Ni-NTA onto a column, balance the purification column with a denaturing reagent 2 - 3 times, load the solution after filtering through the membrane onto the column, and repeat 3 - 5 times; Wash the column with 1 - 2 column volumes of washing solution 8 - 10 times; Wash the column with 1 - 2 column volumes of elution solution 5 - 10 times;

[0012] S5. Renaturation dialysis: Dialyze the denatured protein obtained in step S4 successively in gradient renaturation solutions for 16 - 48 hours;

[0013] S6. Ultrafiltration concentration: Centrifuge the protein obtained in step S5 using an ultrafiltration tube for 5 - 10 hours.

[0014] Preferably, the engineered bacterium in step S2 is BL 21 or Rosetta series engineered bacterium.

[0015] Preferably, the parameters of the ultrasonic in step S3 are: the power is set to 10% - 40%, the total ultrasonic duration is 10 - 30 minutes, the time interval between on and off is 2 - 5 seconds, and the time when the ultrasonic is off needs to be greater than the time when the ultrasonic is on.

[0016] Preferably, the denaturing reagent in step S4 contains tris(hydroxymethyl)aminomethane hydrochloride, urea, sodium chloride, and sodium dihydrogen phosphate; both the washing solution and the elution solution contain tris(hydroxymethyl)aminomethane hydrochloride, urea, sodium chloride, and imidazole.

[0017] Further preferably, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 10 mM - 100 mM; the concentration of urea is 0.5 M - 8 M; the concentration of sodium chloride is 100 mM - 300 mM; the concentration of sodium dihydrogen phosphate in the denaturing reagent is 100 mM - 300 mM; the concentration of imidazole in the washing solution is 5 mM - 30 mM; the concentration of imidazole in the elution solution is 200 mM - 300 mM.

[0018] Preferably, the refolding solution in step S4 contains tris(hydroxymethyl)aminomethane hydrochloride, urea, sodium chloride, sodium dihydrogen phosphate, glycerol, ethylenediaminetetraacetic acid, polyethylene glycol octylphenyl ether, and dithiothreitol.

[0019] Further preferably, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 10 mM - 100 mM; the concentration of urea is 0.5 M - 8 M; the concentration of sodium chloride is 100 mM - 300 mM; the concentration of sodium dihydrogen phosphate is 100 mM - 300 mM; the mass concentration of glycerol is 10% - 20%; the concentration of ethylenediaminetetraacetic acid is 0.1 mM - 1 mM; the mass concentration of polyethylene glycol octylphenyl ether is 0.1% - 2%; the concentration of dithiothreitol is 0.1 mM - 1 mM.

[0020] In a third aspect, the present invention provides the use of the above-mentioned dengue type 2 virus envelope protein EDⅢ antigen in the preparation of dengue type 2 virus envelope protein EDⅢ antibody.

[0021] Preferably, the dengue type 2 virus envelope protein EDⅢ antibody is a polyclonal antibody.

[0022] In a fourth aspect, the present invention provides a method for preparing an anti-dengue type 2 virus envelope protein EDⅢ polyclonal antibody, comprising the following steps: emulsifying the above-mentioned dengue type 2 virus envelope protein EDⅢ antigen with Freund's adjuvant, immunizing a non-human animal by subcutaneous multi-point injection, using Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the booster immunization. After the immunization is completed, blood is taken, allowed to stand at 37°C for 1 - 2 hours, centrifuged to obtain the supernatant, and the high-titer serum is purified using the caprylic acid-ammonium sulfate method.

[0023] Preferably, the non-human animal used is a New Zealand white rabbit, the immunization dose is 0.2 mg - 1 mg / animal, and the animal is immunized 3 - 6 times at an interval of 14 - 15 days.

[0024] In a fifth aspect, the present invention provides an anti-dengue type 2 virus envelope protein EDⅢ polyclonal antibody prepared by the above method.

[0025] Sixthly, the present invention provides the use of the above-mentioned anti-Dengue virus type 2 envelope protein EDⅢ polyclonal antibody in the preparation of a reagent for detecting Dengue virus type 2. This polyclonal antibody exhibits high sensitivity and high specificity for Dengue virus type 2, and has no serum cross-reaction with flaviviruses of other types (Zika virus, Japanese encephalitis virus, Chikungunya virus, Yellow fever virus).

[0026] Seventhly, the present invention provides a method for detecting Dengue virus type 2 envelope protein EDⅢ, which is used for non-disease diagnosis purposes and includes the step of using the above-mentioned anti-Dengue virus type 2 envelope protein EDⅢ polyclonal antibody.

[0027] Eighthly, the present invention provides an ELISA detection kit for Dengue virus type 2 envelope protein EDⅢ, which includes a standard product of Dengue virus type 2 envelope protein EDⅢ, the above-mentioned anti-Dengue virus type 2 envelope protein EDⅢ specific polyclonal antibody, a Dengue virus type 2 envelope protein EDⅢ specific monoclonal antibody, a coating solution, a washing solution, a blocking solution, an HRP-labeled goat anti-mouse antibody, a substrate chromogenic solution, and a termination solution.

[0028] Finally, the present invention also provides a preparation method of an ELISA detection kit for Dengue virus type 2 based on the above-mentioned anti-Dengue virus type 2 envelope protein EDⅢ antibody, including the following steps:

[0029] (1) Coating: Dilute the above-mentioned anti-Dengue virus type 2 envelope protein EDⅢ specific polyclonal antibody 500 - 16,000 times with a coating buffer solution, 100 μL per well, use PBS solution as a blank control group, coat at 4°C for 12 - 16 hours or coat at 37°C for 2 hours;

[0030] (2) Washing: Wash the plate 3 - 5 times with PBST solution, soak with PBST solution for 1 - 2 minutes each time when washing the plate, and finally shake off the water;

[0031] (3) Blocking: Add PBS solution containing 2% - 5% skim milk powder, 350 μL per well, incubate at 37°C for 1 - 2 hours;

[0032] (4) Test sample: Repeat step (2), add the test sample, 100 μL per well, incubate at 37°C for 1 hour;

[0033] (5) Incubate the monoclonal antibody: Repeat step (2), dilute the Dengue virus type 2 envelope protein EDⅢ specific monoclonal antibody 200 - 12,800 times with PBS solution, 100 μL per well, incubate at 37°C for 1 hour;

[0034] (6) Incubate with secondary antibody: Repeat step (2), dilute the HRP-labeled goat anti-mouse antibody with PBS solution at a ratio of 1:8,000 - 10,000, 100 μL per well, and incubate at 37 °C for 1 hour;

[0035] (7) Color development: Repeat step (2), add freshly prepared substrate chromogenic solution, 100 μL per well, and incubate at 37 °C for 20 minutes;

[0036] (8) Termination: Add 2 M sulfuric acid termination solution, 50 μL per well, and observe the solution change from blue to yellow;

[0037] (9) Detection: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of each well of the ELISA plate at 450 nm, plot the standard curve and obtain the standard curve formula, substitute the absorbance value of the sample to be tested into the standard curve formula, and obtain the dengue virus type 2 content of the sample to be tested.

[0038] Compared with the prior art, the beneficial effects of this application are as follows:

[0039] The present invention uses means such as prokaryotic expression in Escherichia coli, denaturation and dissolution, nickel column purification, and dialysis renaturation to prepare an immunogen of the target protein with a purity of over 90%. By exploring the purification and renaturation conditions, a method for preparing an immunogen with high success rate and strong specificity is explored, and a new and rapid method for detecting dengue virus is provided. By immunizing New Zealand white rabbits, polyclonal antibodies with strong specificity and high titer (1:256,000) are prepared; the present invention establishes an enzyme-linked immunosorbent assay (ELISA) based on the polyclonal antibodies for the detection of dengue virus type 2, and there is no serum cross-reaction between the polyclonal antibodies and flaviviruses of other types (Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus); the kit prepared based on the polyclonal antibodies has the advantages of simple and convenient operation, high sensitivity, and strong specificity, providing a basis and direction for the detection and research of related virus proteins such as dengue virus type 2. Brief Description of the Drawings

[0040] Figure 1 It is the SDS-PAGE electrophoresis diagram before and after the purification of the recombinant dengue virus type 2 envelope protein EDⅢ expressed by the present invention. (M: Protein standard molecular weight; CL: Lysate; FT: Flow-through; W1-2: Denaturing solution; W3-5: Washing solution; E1-5: Eluate).

[0041] Figure 2 It is the result diagram of the serum titer of the polyclonal antibody against the dengue virus type 2 envelope protein EDⅢ prepared by the present invention.

[0042] Figure 3SDS-PAGE electrophoresis pattern of the purified anti-Dengue virus type 2 envelope protein EDⅢ polyclonal antibody of the present invention (M: protein standard molecular weight; 1: polyclonal antibody before purification; 2: polyclonal antibody after purification).

[0043] Figure 4 Standard curve of the double antibody sandwich enzyme-linked immunosorbent assay established by the present invention.

[0044] Figure 5 Specificity test result diagram of the double antibody sandwich enzyme-linked immunosorbent assay used in the present invention (DENV: Dengue virus; ZIKV: Zika virus E protein; JEV: Japanese encephalitis virus protein; CHIKV: Chikungunya virus protein; YEV: Yellow fever virus protein). Detailed implementation mode

[0045] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0046] In the embodiments of the present invention, the test methods used are all conventional methods without special instructions; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels without special instructions.

[0047] Example 1 Preparation of recombinant Dengue virus type 2 envelope protein EDⅢ antigen

[0048] A method for preparing recombinant Dengue virus type 2 envelope protein EDⅢ antigen is specifically described as follows:

[0049] (1) By inserting the coding target gene sequence of Dengue virus type 2 EDⅢ protein into the pET22b+ expression vector through restriction enzymes NcoI / XhoI, an engineering bacterium of the target protein expression vector is constructed;

[0050] (2) The EDⅢ-pET22b+ protein expression vector obtained in step (1) is transformed into the host cell Escherichia coli BL 21(DE3)pLysS competent cells by heat shock method, coated on a medium resistant to sodium carbenicillin (CA), cultured at 37°C for 18 hours, and a positive single colony is picked into 5 mL of CA-resistant LB medium, and shaken overnight at 37°C and 200 rpm. The next day, the bacterial solution is added to 200 mL of CA-resistant LB medium at a ratio of 1:100. When the OD of the culture solution 600When = 0.6 - 0.8, add isopropyl - β - D - thiogalactoside (IPTG) to a final concentration of 0.5 mM, and culture the expression host cells to produce recombinant dengue type 2 virus EDⅢ protein;

[0051] (3) Centrifuge the bacterial solution obtained in step (2) at 4 °C for 5 minutes at a speed of 11,000 rpm, discard the supernatant to obtain a cell pellet. Add 25 mM Tris - HCl and wash 3 times. Add 25 mM Tris - HCl containing 2 M urea and 10 mg lysozyme, and lyse the bacteria by ice - bath sonication (sonication power is set to 20%, total sonication time is 20 minutes, sonication on - time is 2 seconds, sonication off - time is 3 seconds). Centrifuge for 5 minutes at a speed of 11,000 rpm, discard the supernatant to obtain an inclusion body pellet;

[0052] (4) Wash the inclusion body pellet obtained in step (3) 3 times with 25 mM Tris - HCl containing 2% Triton X - 100, resuspend it in 25 mM Tris - HCl buffer containing 8 M urea, and suspend it at 4 °C for 12 - 16 hours;

[0053] (5) Centrifuge the suspension obtained in step (4) for 30 minutes at a speed of 11,000 rpm, take the supernatant and filter it through a 0.22 μm filter membrane; Pack the Ni - NTA column, and equilibrate the purification column 2 times with a denaturing solution (including 25 mM tris(hydroxymethyl)aminomethane hydrochloride, 8 M urea, 300 mM sodium chloride), load the solution after the filter membrane onto the column, and repeat 5 times; Wash the column 10 times with 1 column volume of washing solution (including 25 mM tris(hydroxymethyl)aminomethane hydrochloride, 8 M urea, 300 mM sodium chloride, 20 mM imidazole); Wash the column 5 times with 1 column volume of elution solution (including 25 mM tris(hydroxymethyl)aminomethane hydrochloride, 8 M urea, 300 mM sodium chloride, 250 mM imidazole), and collect the eluate;

[0054] (6) Place the eluate obtained in step (5) into Tris - HCl composite buffers containing 4 M urea, 2 M urea, 1 M urea, and Tris - HCl composite buffer respectively for dialysis for 4 hours. Take the lysis solution, flow - through solution, denaturing solution, washing solution, eluate, and renaturation solution (including 25 mM tris(hydroxymethyl)aminomethane hydrochloride, 8 M urea, 300 mM sodium chloride, 100 mM sodium dihydrogen phosphate, 10% (mass concentration) glycerol, 1 mM ethylenediaminetetraacetic acid, 0.1% (mass concentration) polyethyleneglycol octylphenyl ether, 1 mM dithiothreitol) for SDS - PAGE electrophoresis analysis. After electrophoresis, stain with Coomassie Brilliant Blue to verify the expression of the recombinant protein;

[0055] (7) Centrifuge and ultrafilter the renaturation solution obtained in step (6) using a 3 kDa ultrafiltration tube until the volume of the solution in the ultrafiltration collection tube is approximately 500 μL at a rotation speed of 3,500 rpm. Measure the protein concentration of the ultrafiltered protein using a BCA protein quantification kit, aliquot it into EP tubes at 1 mL / tube, and store it at -80 °C.

[0056] As Figure 1 shown, SDS-PAGE experiments and Western blot were performed to verify the reaction specificity of the expressed antigen, and a genetic engineering strain capable of highly expressing the recombinant dengue type 2 virus EDⅢ protein antigen was obtained. Through the above-mentioned recombinant antigen protein expression and purification experiments using this highly expressing strain, a recombinant antigen protein suitable for ELISA experiments was obtained, and the nucleotide sequence of its coding gene is as shown in SEQ ID NO.1.

[0057] Example 2 Preparation of Polyclonal Antibody Against Dengue Type 2 Virus Envelope Protein EDⅢ

[0058] A method for preparing a polyclonal antibody against dengue type 2 virus envelope protein EDⅢ is specifically described as follows:

[0059] (1) Dilute the dengue type 2 virus envelope protein EDⅢ antigen prepared in Example 1 with PBS solution to 1 mg / mL. Take 1 mL of the diluted antigen and mix it with 1 mL of Freund's adjuvant (Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used after the second immunization), and fully emulsify it to form a "water-in-oil" emulsion. Transfer the emulsion to a 1 mL syringe and evacuate the air.

[0060] (2) Perform multiple-point injection subcutaneously in New Zealand white rabbits. Before injection, disinfect the injection site with 75% alcohol. Inject approximately 200 μL of the emulsion at each point, and a total of 0.5 mg of immunizing antigen is injected. Observe for swelling after injection.

[0061] (3) After the first round of immunization, a new round of immunization is carried out every 14 - 15 days for a total of 5 rounds. Seven days after immunization, collect blood from the rabbit ear vein, let it stand at 37 °C for 1 - 2 hours, centrifuge to obtain the supernatant, and detect the antibody titer of the obtained rabbit serum by enzyme-linked immunosorbent assay.

[0062] (4) Purify the serum with a higher titer using the caprylic acid-ammonium sulfate method, perform SDS-PAGE electrophoresis analysis, and after electrophoresis, stain with Coomassie brilliant blue to verify the purification of the polyclonal antibody.

[0063] The results of serum titer detection are as Figure 2 shown, and the antibody titer of the obtained rabbit serum is 1:256,000. Figure 3 The polyclonal antibody against dengue type 2 virus envelope protein EDⅢ obtained after purification.

[0064] Example 3 Enzyme-linked Immunosorbent Assay (ELISA) Method Based on Dengue Virus Type 2 Envelope Protein EDⅢ Antibody

[0065] 1. The ELISA method based on the dengue virus type 2 envelope protein EDⅢ antibody, and the optimization steps for selecting antibodies are as follows:

[0066] (1) Coating: Dilute the polyclonal antibody with coating buffer to 500, 1,000, 2,000, 4,000, 8,000, 16,000, and 32,000 times respectively, 100 μL / well. The PBS solution is used as the blank control group, and coat at 4°C for 12 - 16 hours;

[0067] (2) Washing: Wash the plate 5 times with PBST solution, soak with PBST solution for 1 minute each time when washing the plate, and finally shake off the water;

[0068] (3) Blocking: Add PBS solution containing 3% skim milk powder, 350 μL / well, and incubate at 37°C for 1 hour;

[0069] (4) Incubating standard sample: Repeat step (2), add 1 μg / mL dengue virus type 2 envelope protein EDⅢ, 100 μL / well, and incubate at 37°C for 1 hour;

[0070] (5) Incubating monoclonal antibody: Repeat step (2), dilute the monoclonal antibody with PBS solution to 200, 400, 800, 1,600, 3,200, 6,400, and 12,800 times respectively, 100 μL / well, and incubate at 37°C for 1 hour;

[0071] (6) Incubating secondary antibody: Repeat step (2), dilute the HRP-labeled goat anti-mouse antibody with PBS solution at a ratio of 1:8,000, 100 μL / well, and incubate at 37°C for 1 hour;

[0072] (7) Color development: Repeat step (2), add freshly prepared substrate chromogenic solution, 100 μL / well, and incubate at 37°C for 15 minutes;

[0073] (8) Termination: Add 2M sulfuric acid termination solution, 50 μL / well, and observe the solution change from blue to yellow;

[0074] (9) Detection: Detect the absorbance value of each well of the enzyme-linked immunosorbent assay plate at 450 nm with an enzyme-linked immunosorbent assay reader.

[0075] The dilution factors of the optimal polyclonal antibody and monoclonal antibody were determined using the matrix method, and the combination with the largest P / N value was selected as the optimal combination. The checkerboard titration method was used to optimize the concentrations of the polyclonal antibody and monoclonal antibody against the envelope protein EDⅢ of dengue type 2 virus (see Table 1). According to the results shown in Table 1, the optimal dilution factor of the polyclonal antibody was 1:2,000, and the optimal dilution factor of the monoclonal antibody was 1:400.

[0076] Table 1

[0077]

[0078]

[0079] 2. The double antibody sandwich ELISA method was used to detect the envelope protein EDⅢ of dengue type 2 virus, and the specific optimization steps were as follows:

[0080] (1) Coating: The polyclonal antibody was diluted 2,000-fold with coating buffer, 100 μL / well. The PBS solution was used as the blank control group, and coated at 4°C for 12 - 16 hours;

[0081] (2) Washing: The plate was washed 5 times with PBST solution, soaked with PBST solution for 1 minute each time, and the water was finally shaken off;

[0082] (3) Blocking: Add PBS solution containing 3% skim milk powder, 350 μL / well, and incubate at 37°C for 1 hour;

[0083] (4) Incubating samples: Repeat step (2), add dengue type 2 virus envelope protein EDⅢ diluted 1,000 - 256,000-fold in a serial dilution (starting concentration of 1 mg / mL), 100 μL / well, and incubate at 37°C for 1 hour;

[0084] (5) Incubating monoclonal antibody: Repeat step (2), dilute the monoclonal antibody 400-fold with PBS solution, 100 μL / well, and incubate at 37°C for 1 hour;

[0085] (6) Incubating secondary antibody: Repeat step (2), dilute the HRP-labeled goat anti-mouse antibody with PBS solution at a ratio of 1:8,000, 100 μL / well, and incubate at 37°C for 1 hour;

[0086] (7) Color development: Repeat step (2), add freshly prepared substrate chromogenic solution, 100 μL / well, and incubate at 37°C for 15 minutes;

[0087] (8) Termination: Add 2M sulfuric acid termination solution, 50 μL / well, and observe the solution change from blue to yellow;

[0088] (9) Detection: Use an enzyme-labeled instrument to detect the absorbance values of each well of the enzyme-labeled plate at 450 nm.

[0089] The results are as Figure 4 shown. The detection linear range of the established ELISA method for detecting dengue type 2 virus envelope protein EDⅢ is 7.8 - 50 ng / mL, the detection limit is 7.8 ng / mL, and it has good detection sensitivity.

[0090] Example 4 Specificity experiment of the ELISA method based on the antibody against dengue type 2 virus envelope protein EDⅢ

[0091] Carry out the ELISA experiment under the optimized conditions in Example 3, and select Zika virus E protein (ZIKV), Japanese encephalitis virus protein (JEV), Chikungunya virus protein (CHIKV), and yellow fever virus protein (YEV) as reference samples for the specificity experiment. The results are as Figure 5 shown. It can be seen that the polyclonal antibody and ELISA method prepared by the present invention have good specificity for dengue type 2 virus and have no cross-reaction with a variety of different flaviviruses.

[0092] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, rather than limitations on the specific implementation manners of the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A recombinant dengue 2 virus envelope protein EDⅢ antigen, characterized in that: The nucleotide sequence of the antigen encoding gene is shown in SEQ ID NO.

1.

2. Use of the antigen according to claim 1 in the preparation of antibodies against dengue 2 virus envelope protein EDⅢ.

3. A polyclonal antibody against dengue type 2 virus envelope protein EDⅢ, characterized in that: The polyclonal antibody is obtained by the following method: emulsifying the antigen according to claim 1 with Freund's adjuvant, immunizing non-human animals by subcutaneous multi-point injection, using Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the booster immunization.

4. Use of the anti-dengue type 2 virus envelope protein EDIII polyclonal antibody according to claim 3 in the preparation of a reagent for detecting dengue type 2 virus.

5. A method for detecting dengue 2 virus envelope protein EDⅢ, characterized in that: The method is used for non-disease diagnosis, and comprises the step of using the anti-dengue type 2 virus envelope protein EDⅢ polyclonal antibody according to claim 3.

6. An ELISA detection kit for dengue type 2 virus envelope protein EDⅢ, characterized in that: The invention comprises a dengue 2 virus envelope protein EDⅢ standard product, the anti-dengue 2 virus envelope protein EDⅢ specific polyclonal antibody according to claim 3, the dengue 2 virus envelope protein EDⅢ specific monoclonal antibody, a coating solution, a washing solution, a blocking solution, an HRP-labeled goat anti-mouse antibody, a substrate color developing solution and a stop solution.

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