Rapid detection test strip for avian influenza virus as well as preparation method and application of rapid detection test strip

Abstract

The invention discloses an avian influenza virus HA protein. The amino acid sequence of the HA protein is as shown in SEQ ID NO. 1. The invention also discloses a monoclonal antibody prepared by using the HA protein, and the monoclonal antibody comprises a monoclonal antibody 1 and a monoclonal antibody 2. By optimizing the design of the HA protein, improving the efficiency of an expression system and combining with an innovative antibody preparation method, the technical bottlenecks of low yield, low activity, poor stability and the like in the traditional avian influenza virus antigen preparation are successfully solved. Meanwhile, the high affinity and specificity of the monoclonal antibody 1 and the monoclonal antibody 2 also provide a basis for developing an efficient and sensitive avian influenza virus detection tool, and the monoclonal antibody 1 and the monoclonal antibody 2 have wide application prospects, and particularly have important significance in the fields of vaccine development, diagnostic reagents and virus detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a test strip for rapid detection of avian influenza virus (AIV) and a preparation method and application thereof. Background Art

[0002] Avian influenza (AI) is an acute infectious disease caused by avian influenza virus, which mainly affects birds. The avian influenza virus has a strong variability. The outbreak of avian influenza not only causes large-scale deaths of poultry, but also has a huge impact on the meat consumption market and agricultural economic stability, and increases the difficulty of prevention and control work and vaccine development. In addition, the avian influenza virus also has a certain ability to spread across species and can infect humans and other animals, further increasing the difficulty of epidemic control and becoming an important issue in the field of global public health.

[0003] At present, the detection of avian influenza mainly relies on RT-PCR and ELISA. Although these methods have high sensitivity and specificity, they have the disadvantages of strong equipment dependence, long time consumption (usually more than 4 hours) and complex operation. The existing colloidal gold test strips are insufficient in sensitivity, and the detection limit is usually greater than 10 3 TCID 50 / mL, which is difficult to meet the needs of real-time and rapid detection. Therefore, it is urgent to develop a convenient, rapid, low-cost and highly sensitive detection tool to facilitate early detection, real-time monitoring and epidemic prevention and control of avian influenza virus. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, one of the purposes of the present invention is to provide an avian influenza virus HA protein and a monoclonal antibody prepared from the protein; a second purpose of the present invention is to provide an avian influenza virus detection kit prepared using the HA protein and monoclonal antibody prepared by the present invention.

[0005] Therefore, the present invention discloses an avian influenza virus HA protein on one hand, and the amino acid sequence of the HA protein is shown in SEQ ID NO.1.

[0006] Preferably, the nucleotide sequence of the HA protein after codon optimization of the present invention is as shown in SEQ ID NO.2.

[0007] Preferably, the HA protein of the present invention has a hydrated particle size of 12.8 nm and a PDI of 0.11 as measured by dynamic light scattering.

[0008] In one aspect, the present invention also discloses a monoclonal antibody prepared using the HA protein, wherein the monoclonal antibodies include monoclonal antibody 1 and monoclonal antibody 2, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown as SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0009] In one aspect, the present invention further discloses a kit for detecting avian influenza virus, the kit comprising an effective amount of the monoclonal antibody 1 and the monoclonal antibody 2 and a matching detection reagent.

[0010] Preferably, the kit of the present invention is a colloidal gold detection test strip, and the colloidal gold detection test strip comprises a test strip containing monoclonal antibody 1 and monoclonal antibody 2 and a sample diluent.

[0011] Preferably, the sample diluent of the present invention is a PBS buffer containing 0.1% Triton X-100+5mM EDTA, and the pH value of the buffer is 7.4.

[0012] Preferably, the test strip in the kit of the present invention comprises a sample pad, a latex microsphere pad, a detection line, a quality control line, and an absorption pad.

[0013] Preferably, the emulsion microsphere pad in the test strip in the kit of the present invention contains monoclonal antibody 1, and the detection line contains monoclonal antibody 2.

[0014] In one aspect, the present invention also discloses a use of the monoclonal antibody 1 and the monoclonal antibody 2 in preparing an avian influenza detection kit.

[0015] The avian influenza virus HA protein prepared by the present invention is suitable for preparing different avian influenza virus diagnostic reagents, such as colloidal gold, ELISA, chemiluminescence detection kits, etc. The monoclonal antibody 1 and monoclonal antibody 2 against the avian influenza virus HA protein prepared by the present invention have good specificity and sensitivity, and are suitable for preparing different avian influenza virus diagnostic reagents, such as colloidal gold, ELISA, chemiluminescence detection kits, etc.

[0016] The avian influenza virus detection kit (colloidal gold detection test strip) provided by the present invention is suitable for the detection of avian influenza virus in serum, tissue, whole blood, anticoagulated blood, anal swabs and oral swabs, etc. It has strong specificity, high sensitivity, good stability and fast detection speed, and can be used for early screening of avian influenza virus, and is particularly suitable for on-site infection diagnosis, etc.

[0017] In summary, the present invention successfully solves the technical bottlenecks of low yield, low activity, poor stability, etc. in the preparation of traditional avian influenza virus antigens by optimizing the design of HA protein, improving the efficiency of the expression system, and combining innovative antibody preparation methods. At the same time, the high affinity and specificity of monoclonal antibody 1 and monoclonal antibody 2 also provide a basis for the development of efficient and sensitive avian influenza virus detection tools, which have broad application prospects, especially in the fields of vaccine development, diagnostic reagents and virus detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 SDS-PAGE and western blot images of HA protein of avian influenza virus after purification. Figure 1 A is the result of SDS-PAGE detection, 1 is the purified HA protein of avian influenza virus; Figure 1 B is the result of western blot detection, and 1 is the purified HA protein of avian influenza virus.

[0019] Figure 2 Schematic diagram of the test strip assembly, where A: sample pad, B: latex microsphere pad, C: test line, D: quality control line, E: absorption pad.

[0020] Figure 3 Diagram of the test strip test result determination model.

[0021] Figure 4 Repeatability test results, 1-5 are the result graphs of 250101 batch of test strips testing 5 specific samples, 6-10 are the result graphs of 250101 batch of test strips testing 5 positive samples. DETAILED DESCRIPTION

[0022] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0023] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0024] Example 1: Preparation of HA protein of avian influenza virus

[0025] 1. HA protein design

[0026] The HA protein sequence of the H5N1 subtype AIV (GenBank accession number: AAO52869.2) was selected, and the receptor binding domain (aa100-300) and the transmembrane region were retained (replaced with the GPI anchor sequence to enhance the membrane surface binding ability, and its amino acid sequence is SNDGNSFSNY QGNFVGSKG). At the same time, a 6×His tag was added to the N-terminus (for easy purification), and an AviTag was fused to the C-terminus (for later directional fixation, the specific amino acid sequence is GLNDIFEAQKIEWHE). The amino acid sequence of the designed HA protein is shown in SEQ ID NO.1. The nucleotides of the above HA protein were then optimized according to the expression preference of HEK293 cells, and the specific codon-optimized nucleotide sequence is shown in SEQ ID NO.2.

[0027] 2. Experimental Materials

[0028] 1. Expression vector: Mammalian Vector.

[0029] 2. Cell line: HEK293F suspension cells, used for large-scale protein expression.

[0030] 3. Main reagents: PEI Max transfection reagent was used for DNA transfection. Expi293 medium (Gibco) was used for HEK293F cell culture. Ni-NTA affinity chromatography column (Cytiva) was used for protein purification.

[0031] 3. Experimental steps

[0032] 1. Construction of recombinant plasmid: Entrust GenScript to synthesize the optimized HA gene (shown in SEQ ID NO.2). During the synthesis process, ensure that there are no extra restriction enzyme sites in the gene sequence to facilitate smooth cloning. Use EcoRI / XhoI double enzyme digestion for gene insertion. Both the vector and the inserted fragment after enzyme digestion are gel-recovered. The inserted gene is connected to the pcDNA3.4 vector together, and T4 DNA ligase (Thermo Fisher) is used for ligation reaction. After connection, Escherichia coli is transformed, and monoclonal colonies are picked for culture and plasmid extraction. The plasmid is submitted to the ABI 3730xl sequencer for sequencing, and the comparison analysis ensures that the gene sequence consistency reaches more than 99.9%, confirming that the inserted gene is correct.

[0033] 2. Protein expression and purification: 1 μg / mL of plasmid DNA was mixed with 3 μg / mL of PEI Max transfection reagent in a ratio of 1:3 and incubated at room temperature for 10 minutes to form a transfection complex. The transfection complex was added to the HEK293F cell culture medium, and the transfection cell density was 2×10^6cells / mL. The transfected cells were cultured at 37°C, 8% CO2, and 120rpm shaking for 5 days to ensure uniform stirring during the culture process. On the fifth day, the cell supernatant was collected by centrifugation (10,000×g, 20 minutes). Filtered with a 0.22μm filter membrane to remove cell debris. The filtered supernatant was loaded onto a Ni-NTA affinity chromatography column (Cytiva) to purify the recombinant protein. The affinity chromatography column was balanced with a solution adjusted with 20mM sodium phosphate, 500mM NaCl, and 20mM imidazole (pH7.4). The same buffer containing 250mM imidazole was used for elution. The peak containing the recombinant protein in the eluate was collected and ultrafiltered and concentrated to about 2 mg / mL using Amicon Ultra-15 (30 kDa filter membrane). After detection and analysis, the expression yield of HA protein was 38.7±2.1 mg / L, and the recovery rate of Ni-NTA purification was 82.4%±3.8%.

[0034] 3. Protein Detection

[0035] (1) SDS-PAGE analysis: Use 12% separation gel and perform electrophoresis under reducing conditions. The results are as follows Figure 1 As shown in A, the molecular weight of HA protein is about 27 kDa, and the purity can reach more than 95%.

[0036] (2) Western Blot verification: Mouse anti-H5 HA monoclonal antibody (1:1000, Abcam ab210953) was used as the primary antibody. HRP-labeled goat anti-mouse IgG (1:5000) was used as the secondary antibody. ECL was used for color development to detect the specific band at about 27 kDa ( Figure 1 B), verify the accuracy of protein expression and purification.

[0037] (3) Red blood cell agglutination test: HA protein was subjected to a multiple dilution reaction with a 1% chicken red blood cell suspension to detect its agglutination activity. The results showed that the minimum agglutination activity unit (HAU) of the HA protein prepared by the present invention was 0.3 μg / mL, and the minimum agglutination concentration of the control group (Abcam ab217654) was 0.5 μg / mL. This indicates that the HA protein prepared by the present invention has better activity.

[0038] IV. Summary

[0039] 1. Structural optimization design: The transmembrane region was replaced by GPI anchoring to allow the HA protein to form a stable trimer (the hydrated particle size measured by dynamic light scattering was 12.8 nm, PDI = 0.11).

[0040] 2. High-efficiency expression system: Mammalian expression ensures correct glycosylation and increases the activity 3 times compared to insect cell expression.

[0041] 3. Directed immobilization technology: The HA protein was pre-coated onto the NC membrane using streptavidin using the AviTag-biotin system, and the binding efficiency was increased by 40% (compared to the protein physical adsorption method without adding the tag).

[0042] In summary, the present invention successfully prepared a recombinant HA protein with high purity (>95%) and high activity (HAU 0.5μg / mL). Through structural optimization and expression system optimization, the technical bottleneck of poor stability and easy aggregation of traditional HA protein was solved, providing a core material basis for the sensitivity and specificity of the test strip.

[0043] Example 2: Preparation of monoclonal antibodies against HA protein of avian influenza virus

[0044] 1. Vaccine preparation and animal immunization

[0045] 1. The avian influenza HA protein prepared in Example 1 was fully emulsified with an equal volume of Freund's complete adjuvant and then subcutaneously injected into 6-8 week old BALB / c mice (100 μg each). Two weeks after immunization, subcutaneous multi-point immunization was performed again using incomplete Freund's adjuvant emulsification (100 μg each), and repeated once two weeks later. After the third immunization, the antibody titer in the mice was detected. To enhance the immune response, mice were boosted with immunization (100 μg each) 3 days before cell fusion by intraperitoneal injection.

[0046] 2. When the ELISA titer of mouse serum is 1:10 5 , then spleen cells were collected for further analysis. The specific method of spleen cell collection is as follows: 3 days after booster immunization, euthanize the mice and disinfect the surface of the body with 75% alcohol, and then remove the spleen under sterile conditions. The spleen was placed in a sterile culture dish containing RPMI 1640 medium (with 100U of triple antibody added), washed twice with RPMI 1640, transferred to a 200-mesh copper mesh, and gently crushed with a sterilized pestle to release spleen cells into the liquid phase. After the cells are dispersed, they are filtered through a 200-mesh copper mesh, collected in a 50ml sterile centrifuge tube, and centrifuged at 1000rpm for 10 minutes. After discarding the supernatant, resuspend the cells with RPMI 1640 and wash once. The cells were counted using trypan blue staining, and the proportion of live cells was greater than 90%, and the cells were set aside.

[0047] 2. Cell fusion: Take out the myeloma cells in the logarithmic growth phase, put them into a 50ml centrifuge tube, centrifuge at 1000rpm for 10 minutes at room temperature, discard the supernatant, wash once with RPMI 1640 and count. Then, mix the myeloma cells and spleen cells at a ratio of 1:10, centrifuge at 1000rpm for 10 minutes, discard the supernatant, and wash once with RPMI 1640. Place the centrifuge tube in a 37℃ water bath. Add 1ml of 50% PEG 1450 solution preheated to 37℃ within 1 minute for cell fusion. After standing for 1 minute, add RPMI 1640 gradually to terminate the fusion. After completion, centrifuge at 1000rpm for 10 minutes at room temperature, discard the supernatant, transfer the cells to RPMI 1640 culture medium containing 20% ​​newborn calf serum containing HAT, and transfer the fused cells to a 96-well plate, adding 100μl culture medium to each well. The culture plate was placed in an incubator at 37°C and 5% CO2 for 15 days, and then the medium was changed to RPMI 1640 medium containing HT and 20% calf serum.

[0048] 3. Monoclonal screening: Hybridoma cells with positive ELISA test results were screened by limiting dilution method, and monoclonal cell lines with high antibody titer and good morphology were selected for further cloning. Finally, 4 positive hybridoma cell lines were obtained, numbered 1, 2, 3, and 4 respectively.

[0049] IV. Preparation of ascites: BALB / c mice aged 6-8 weeks were selected and 1 ml of sterilized liquid paraffin was injected into the peritoneal cavity, 0.5 ml per mouse. After 7 days, 1×10^6 hybridoma cells (1, 2, 3, 4 strains) were injected into each mouse. After another 7 days, the ascites of the mice were collected, centrifuged at 1000 rpm for 10 minutes, the supernatant was collected, and the ascites was divided into 5 ml / tube and stored at -20°C for later use.

[0050] 5. Antibody purification: Take 10ml of ascites, centrifuge at 1000rpm for 10 minutes at 4℃, collect the supernatant and add 40ml acetate buffer (0.06mol / L, pH 4.5), mix evenly with magnetic stirring, add 330μl of caprylic acid at room temperature, and add dropwise while stirring. After reacting for 30 minutes, transfer to 2-8℃ and let stand for 2 hours. Then, centrifuge at 10000rpm for 30 minutes at 2-8℃ and collect the supernatant. Record the volume of the supernatant, and slowly add the same volume of saturated ammonium sulfate solution under an ice bath, and continue to stand at 2-8℃ for 16 hours. Thereafter, centrifuge at 5000rpm for 30 minutes and collect the precipitate. Dissolve the precipitate with 5ml PBS, dialyze with PBS for 16 hours, and change the solution 3 times. The dialyzed monoclonal antibody is sterilized through a 0.22μm microporous filter membrane and dispensed into centrifuge tubes, 0.1ml per tube. Finally, the BCA kit was used to detect the concentrations of the four monoclonal antibodies. The results showed that the concentrations of monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3 and monoclonal antibody 4 were 2.12 mg / ml, 2.34 mg / ml, 3.42 mg / ml and 2.65 mg / ml, respectively.

[0051] Example 3 Systematic identification of monoclonal antibodies

[0052] 1. Class and subclass determination: Monoclonal antibodies 1 to 4 were tested using the mouse monoclonal antibody Ig class / subclass identification ELISA kit (purchased from Beijing Biolong Immunotechnology Co., Ltd.) according to the instructions. After testing, the subclass of the four monoclonal antibodies was IgG1.

[0053] 2. Reactivity determination: The ELISA titer of 4 monoclonal antibodies (1, 2, 3, 4) and the control commercial antibody (Abcam ab210953) was determined by indirect ELISA method (the coating amount of the original avian influenza virus HA protein was 1 μg / ml, 100 μl / well) (the concentration of the monoclonal antibodies was adjusted to 1 μg / ml, and then they were incubated as primary antibodies, and the OD450nm value was detected). The results showed that the OD450nm values ​​of the 4 monoclonal antibodies prepared by the present invention were higher than those of the commercial monoclonal antibodies, which shows that the specificity and sensitivity of the 4 monoclonal antibodies are very good, and better than the control commercial antibodies. Therefore, these 4 monoclonal antibodies can be used for subsequent reagent development. The specific results are shown in Table 1.

[0054] Table 1 ELISA test results

[0055]

[0056] 3. HRP labeling titer determination: The four monoclonal antibodies were labeled using the HRP coupling kit (ab102890), and the labeling titers of the four monoclonal antibodies were determined using the direct ELISA method (the coating amount of the original avian influenza virus HA protein was 1μg / ml, 100μl / well) (positive when the OD value was ≥1.0). The results showed that the highest dilutions of monoclonal antibodies 1 to 4 were 1:20000, 1:10000, 1:1000, and 1:20000, respectively. This shows that the labeling titers of the four monoclonal antibodies are all good and can be used for subsequent reagent development.

[0057] 4. Verification of monoclonal antibody pairing: The 4 selected monoclonal antibodies were coated on the ELISA plate at 1μg / ml and 100μl / well respectively, and the prepared avian influenza virus HA protein was used as the sample (diluted to 50ng / ml, 100μl per well), incubated at 37℃ for 30min; after washing, the paired HRP-labeled monoclonal antibody (1:10000 dilution) was added, incubated at 37℃ for 30min, and after washing, TMB developer (incubated at 37℃ for 10min) and stop solution (2M H2SO4) were added, and the OD450 value was detected. The results showed (Table 2) that when No. 1 antibody was coated and No. 2 antibody was detected, the OD450 value was the highest, indicating that the reaction was the best at this time, so No. 1 coating and No. 2 detection were selected as the paired antibodies for subsequent avian influenza virus detection.

[0058] Table 2 Monoclonal antibody pairing test results

[0059]

[0060] 5. Determination of the sequence of the variable region of the monoclonal antibody: The heavy chain variable region and the light chain variable region of the prepared monoclonal antibodies (No. 1 and No. 2) were determined according to the method of Chinese invention patents (CN 111393525 B, CN113354734A). After sequencing (as shown in Table 3), the sequences of the heavy chain variable region and the light chain variable region of No. 1 antibody are shown in SEQ ID NO.3 and SEQ ID NO.4; the sequences of the heavy chain variable region and the light chain variable region of No. 2 antibody are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0061] Table 3 Monoclonal antibody sequence detection results

[0062]

[0063] Example 4 Detection of avian influenza virus (latex method)

[0064] 1. Preparation of test strips

[0065] 1. Preparation of nitrocellulose membrane: Attach nitrocellulose membrane (purchased from Sartorius) to the corresponding position of PVC base plate (Ruijian, Hangzhou), dilute monoclonal antibody 2 to 1 mg / ml with coating buffer (PBS buffer), adjust the marking position and height of the membrane marking machine, and mark the T line, which is the detection line, and the T line is close to the end of the latex microsphere pad. Dilute goat anti-mouse IgG antibody (purchased from Solebao) to 1 mg / ml with coating buffer, adjust the marking position and height of the membrane marking machine, and mark the C line, which is the quality control line, and the C line is close to the absorption pad, and the distance between the two lines is 5-8 mm. Dry in a 37°C oven for 20 hours, seal in an aluminum foil bag with a desiccant, and store at room temperature for use.

[0066] 2. Preparation of latex pad: Add monoclonal antibody 1 to latex microspheres at 1 mg / ml (purchased from Shanghai Huizhi, 300 nm) and label for 2 hours. Add BSA to a final concentration of 1% and block for 1 hour. Centrifuge at 4°C, 12000rpm for 30 minutes, discard the supernatant, collect the precipitate, resuspend the precipitate with 1ml preservation solution (0.05M Tris buffer containing 1% BSA, pH 8.0) and ultrasonicate for 1 minute. Spread the resuspended latex microsphere labeled antibody evenly on the treated latex microsphere pad, dry in a 37°C oven for 20 hours, seal in an aluminum foil bag, and store at room temperature for later use.

[0067] 3. Sample pad treatment: soak the sample pad (purchased from Tongcheng Paper, 300 mm×20 mm) in blocking solution (10% BSA solution) for 30 minutes, dry it in a 37°C oven for 20 hours, seal it in an aluminum foil bag, and store it at room temperature for later use.

[0068] 4. Assembly: Figure 2 As shown, the sample pad, latex microsphere pad, and absorbent pad are sequentially pasted to corresponding positions of the PVC bottom plate with the nitrocellulose membrane pasted thereon, so that the latex microsphere pad and the absorbent pad are respectively in contact with part of the nitrocellulose membrane, and the sample pad is in contact with part of the latex microsphere pad, to form a large plate.

[0069] 5. Packaging: Use a strip cutter to cut the large plate into 2-3mm wide test strips, install them in the outer shell, and seal them in an aluminum foil bag, which contains a test strip, a straw, and a pack of desiccant. Store them at room temperature away from light for future use. The shelf life is 18 months.

[0070] 6. Preparation of sample diluent: PBS buffer (pH 7.4) containing 0.1% Triton X-100 + 5mM EDTA, stored at room temperature for future use. This diluent can lyse the virus within 2 minutes to release HA protein and inactivate the virus at the same time, so as to prevent the spread of the virus during detection.

[0071] 2. Test strip detection

[0072] 1. Sample processing

[0073] (1) Use a cotton swab to collect a sample from the trachea or cloaca secretions of the animal to be tested, such as an anal swab, oral swab, or nasal swab. Immediately insert the cotton swab into a sample tube (1 mL / tube) containing sample diluent and mix the solution for 2 minutes to dissolve the specimen in the solution as much as possible. After mixing, it can be directly used for testing.

[0074] (2) Grind the tissue sample and sample diluent in a 1:1 ratio, centrifuge and take the supernatant, which can be used directly for detection.

[0075] (3) Blood samples (including whole blood, anticoagulant blood, serum and plasma) are mixed with sample diluent at a ratio of 1:1 for 2 minutes and can be used directly for detection.

[0076] 2. Operation steps

[0077] (1) Take out a test strip bag and tear it open, take out the test strip and place it horizontally on the operating platform.

[0078] (2) Use a pipette to draw up the processed sample solution to be tested, and add 3-4 drops (about 100 μL) into the sample well S.

[0079] (3) After adding the sample, place the test strip flat on the table and observe the results for about 10 minutes. The results will be invalid after 15 minutes.

[0080] 3. Determination (such as Figure 3 (shown)

[0081] (1) If two bands appear on the test strip (T: detection line, C: quality control line), the result is considered positive.

[0082] (2) If only one band (C: quality control line) appears on the test strip, the result is judged as negative.

[0083] (3) If no band appears at the quality control line of the test strip, the test is deemed invalid.

[0084] 3. Test Strip Performance Verification

[0085] 1. Specificity test: The three batches of test strips were used to test five specific poultry samples and ten samples that were negative for avian influenza virus by PCR. The results showed that the test results of the three batches of test strips were all negative. See Table 4 for specific results.

[0086] Table 4 Specificity test results

[0087]

[0088] Note: “-” indicates the test is negative.

[0089] 3. Sensitivity test

[0090] The three batches of test strips were used to test different titers of avian influenza virus (including H5N1 subtype, H7N9 subtype and H9N2 subtype). The results showed that the minimum detection limit of the three batches of test strips for the H5N1 subtype of avian influenza virus could reach 10 1 TCID 50 / ml, and the minimum detection limit for avian influenza virus H7N9 subtype and H9N2 subtype can reach 10 2 TCID 50 The results are shown in Table 5.

[0091] The three batches of test strips prepared were used to detect different concentrations of the HA protein of the avian influenza virus prepared in Example 1. The results showed that the minimum detection limit of the three batches of test strips could reach 2 ng / ml. The results are shown in Table 5.

[0092] The above results show that the prepared test strip has good sensitivity and can simultaneously detect H5N1 subtype, H7N9 subtype and H9N2 subtype of avian influenza.

[0093] Table 5 Sensitivity test results

[0094]

[0095] Note: “-” indicates a negative result, and “+” indicates a positive result.

[0096] 4. Repeatability test: The three batches of test strips were used to test five specific samples and five positive samples. Both between batches and within batches, good repeatability was observed. The results are shown in Table 6 and Figure 4 shown.

[0097] Table 6 Repeatability test results

[0098]

[0099]

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An avian influenza virus HA protein, characterized in that The amino acid sequence of the HA protein is shown in SEQ ID NO.

1.

2. The HA protein according to claim 1, characterized in that The nucleotide sequence of the HA protein after codon optimization is shown in SEQ ID NO.

2.

3. The HA protein according to claim 1, characterized in that The hydrated particle size of the HA protein measured by dynamic light scattering was 12.8 nm, and PDI=0.

11.

4. A monoclonal antibody prepared using the HA protein according to claim 1, characterized in that: The monoclonal antibodies include monoclonal antibody 1 and monoclonal antibody 2, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown as SEQ ID NO.5 and SEQ ID NO.6, respectively.

5. A kit for detecting avian influenza virus, characterized in that: The kit comprises an effective amount of the monoclonal antibody 1 and the monoclonal antibody 2 according to claim 4 and a matching detection reagent.

6. The kit according to claim 5, characterized in that The test kit is a colloidal gold detection test strip, which comprises a test strip containing monoclonal antibody 1 and monoclonal antibody 2 and a sample diluent.

7. The kit according to claim 6, characterized in that The sample diluent is a PBS buffer containing 0.1% Triton X-100+5mM EDTA, and the pH value of the buffer is 7.

4.

8. The kit according to claim 6, characterized in that The test strip in the kit comprises a sample pad, a latex microsphere pad, a detection line, a quality control line and an absorption pad.

9. The kit according to claim 8, characterized in that The emulsion microsphere pad in the test strip in the kit contains monoclonal antibody 1, and the detection line contains monoclonal antibody 2.

10. Use of the monoclonal antibody 1 and the monoclonal antibody 2 as claimed in claim 4 in preparing a kit for detecting avian influenza.

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