Influenza A virus protein and application thereof

Abstract

The invention provides an influenza A virus protein and application thereof. The influenza A virus protein provided by the invention comprises a protein fragment S1, a protein fragment S2 and a protein fragment S3, and the protein fragment S1, the protein fragment S2 and the protein fragment S3 can be combined and connected according to any sequence. The influenza A virus protein disclosed by the invention is good in specificity, high in biological activity and strong in stability, and can be used for preparing products for detecting influenza A virus antibodies. The influenza A virus antibody detection kit provided by the invention has the advantages of being simple and convenient to operate, rapid in reaction, high in sensitivity, strong in specificity, suitable for on-site rapid detection, economical, practical and the like, can be used for rapid screening in high-risk groups, and can be used for rapid, accurate and safe timely detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and relates to an influenza A virus protein and an application thereof, and in particular to an influenza A virus protein, an influenza A virus antibody detection test strip, a detection kit and an application thereof. Background Art

[0002] Influenza A virus is the main influenza virus infection pathogen, which is transmitted through droplets in the air, contact between susceptible and infected persons, or contact with contaminated objects. After influenza A virus infection occurs, the newly replicated progeny virus will spread through the respiratory mucosa, infect other cells, further induce systemic inflammatory response, cytokine storm, etc. and lead to multiple complications. Its typical clinical symptoms are acute high fever, body pain, significant fatigue and respiratory symptoms. Some patients infected with influenza A virus have a serious condition and progress rapidly. If effective intervention is not carried out in time, it may endanger life and health safety. It is worth noting that influenza A has received widespread attention due to its seasonal transmission. According to the announcement released by the WHO, there are 100 million to 1.2 billion cases of influenza worldwide each year, 500,000 to 100,000 deaths, including 300,000 to 500,000 cases of severe influenza, and a mortality rate of 8% to 10%.

[0003] The influenza A virus RNA is divided into 8 segments, of which the 5th segment encodes its nucleocapsid protein (NP). NP is mainly involved in multiple stages of viral replication, affecting viral transcription, replication, assembly and transport functions; in addition, NP is also the main antigen recognized by cytotoxic T lymphocytes. The influenza A virus M1 protein is the structural protein with the highest content in the virus particle, accounting for about 40% of the total viral protein. Each virus particle contains about 3,000 M1 molecules. The high conservation of the sequence makes M1 an ideal broad-spectrum antiviral target. The influenza A virus M2 protein is highly conservative, can form ion channels and affect the formation of the natural conformation of influenza virus hemagglutinin, is conducive to viral uncoating, has strong specificity and is considered to be a structural protein with cross-immune protection. Therefore, the diagnosis of influenza A virus can be achieved based on the above three proteins. Currently established diagnostic methods include virus isolation methods such as virus culture, nucleic acid detection methods such as polymerase chain reaction (PCR) and serological methods such as enzyme-linked immunosorbent assay (ELISA), all of which are widely used in clinical diagnosis. However, although these methods are highly sensitive and specific, they require expensive equipment and professional operators, and it takes a long time to output results, which makes them unsuitable for use in some poor areas or areas with large outbreaks of infectious diseases. Most commercial influenza A virus test strips on the market are prepared by the colloidal gold method, which has problems such as poor sensitivity and false positives. Other detection platforms such as latex method and fluorescence method have problems such as high cost and complicated nanoparticle preparation.

[0004] Based on the above problems, it is very necessary to develop a new detection device and detection method to improve the detection speed, detection sensitivity and specificity of influenza A virus. Summary of the invention

[0005] The objects of the present invention are: The first object of the present invention is to provide an influenza A virus protein having high specificity and sensitivity; The second object of the present invention is to provide the use of the above influenza A virus protein in the preparation of influenza A virus antibody detection products; The third object of the present invention is to provide a detection kit to solve at least one of the above problems; The fourth object of the present invention is to provide a detection kit for use in detecting influenza A virus.

[0006] In order to achieve the above object, the present invention provides influenza A virus protein, comprising a combination of the following protein fragments: Protein fragment S1, whose amino acid sequence is shown in SEQ ID NO.1; Protein fragment S2, whose amino acid sequence is shown in SEQ ID NO.2; Protein fragment S3, the amino acid sequence of which is shown in SEQ ID NO.3; The protein fragment S1, protein fragment S2 and protein fragment S3 can be combined and connected in any order.

[0007] It should be noted that: The amino acid sequence of protein fragment S1 is shown in SEQ ID NO.1, and is specifically as follows: qkasagqisvqptfsvqrnlpferatvmaafsgnnegrtsdmrtevirmmesakpedlsfqgrgvfelsdekatnpivpsfdmsnegsyffgdnae; The amino acid sequence of protein fragment S2 is shown in SEQ ID NO.2, which is specifically as follows: rshrqmatttnplirhenrmvlasttakameqvagsseqaaeamevanktrqmvhamrtigthpsssaglrddllenlqayqkrmgv; The amino acid sequence of protein fragment S3 is shown in SEQ ID NO.3, which is specifically as follows: ptrsewecrcsgssdpppmslltevetptrtgwecncsdssdpppmslltevetltrpgwecncsgssdpppmslltevetpirnewgcrcndssdp.

[0008] Preferably, the combination of protein fragments of the influenza A virus protein includes any one of S1+S2+S3, S1+S3+S2, S2+S3+S1, S2+S1+S3, S3+S2+S1 or S3+S1+S2.

[0009] The present invention also provides a test strip for detecting antibodies to influenza A virus. The influenza A virus antibody detection test strip comprises a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked on the base plate; wherein the marking pad and / or the detection pad comprises the influenza A virus protein described above.

[0010] Preferably, the label pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker; Preferably, the marker comprises any one or a combination of at least two of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres; Preferably, the average particle size of the marker is 40-300 nm.

[0011] Preferably, in any of the above items, the average particle size of the colloidal gold particles is 40 to 100 nm; more preferably, 40 nm, 50 nm, 60 nm, 80 nm, 100 nm and ranges therebetween.

[0012] Preferably, in any of the above items, the average particle size of the colored microspheres is 100-300 nm; more preferably, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0013] Preferably, in any of the above items, the average particle size of the time-resolved fluorescent microspheres is 100-300 nm; more preferably, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0014] Preferably, in any of the above items, the average particle size of the quantum dot microsphere particles is 100 to 300 nm; more preferably, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0015] Preferably, in any of the above items, the mass ratio of the influenza A virus protein to the colloidal gold is (0.04-0.32):1; more preferably, 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1 and ranges therebetween.

[0016] Preferably, in any of the above items, the mass ratio of the influenza A virus protein to the colored microspheres is (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0017] Preferably, in any of the above items, the mass ratio of the influenza A virus protein to the time-resolved fluorescent microspheres is (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the range therebetween.

[0018] Preferably, in any of the above items, the mass ratio of the influenza A virus protein to the quantum dot microspheres is (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the range therebetween.

[0019] Preferably, a detection line and a quality control line are provided on the detection pad, the detection line is coated with the influenza A virus protein according to claim 1 or 2, and the quality control line is coated with sheep anti-chicken IgY antibody.

[0020] Preferably, any of the above items is that the test line is coated with 0.5-5 mg / mL of the influenza A virus protein. Further preferably, the test line is coated with 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL and the range therebetween.

[0021] Preferably, any of the above items is coated with 0.5-5 mg / mL of goat anti-chicken IgY antibody. Further preferably, the quality control line is coated with 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL and the range therebetween.

[0022] Any of the above is preferably that the diluent of influenza A virus protein and sheep anti-chicken IgY antibody is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose. Further preferably, the diluent is a 10 mM, 20 mM, 30 mM, 40 mM, 50 mM PB buffer and the range therebetween containing trehalose; further preferably, each 100 mL of the diluent contains 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g and the range therebetween of trehalose.

[0023] Preferably, both the label pad and the detection pad include the influenza A virus protein described above.

[0024] Any of the above items is preferably that a detection line and a quality control line are provided on the detection pad; the detection line is coated with the influenza A virus protein; the quality control line is coated with sheep anti-chicken IgY; the marking pad contains the same type of influenza A virus protein, and the influenza A virus protein on the marking pad is marked with a marker; the marking pad also contains chicken IgY antibodies, and the chicken IgY antibodies on the marking pad are marked with a marker.

[0025] The present invention also provides a detection kit, which comprises a shell and a detection test paper arranged inside the shell, and the detection test paper is the above-mentioned influenza A virus antibody detection test paper strip.

[0026] Preferably, the housing comprises an upper cover and a lower cover which are detachably connected.

[0027] Preferably, any of the above items is that the upper cover is provided with an observation window and a sample addition hole, and the lower cover is provided with a detection card strip mounting area.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The influenza A virus protein provided by the present invention has good specificity, high sensitivity, strong stability, and high affinity with influenza A virus antibodies, and can be used to prepare products for detecting influenza A virus antibodies.

[0029] The influenza A virus antibody detection kit provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for on-site rapid detection, and economical and practical, and is of great significance for the early diagnosis of influenza A virus infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the efficacy of anti-influenza A virus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The upper cover of the influenza A virus antibody detection kit in Example 1 of the present invention; Figure 2 The lower cover of the influenza A virus antibody detection kit in Example 1 of the present invention; Figure 3 This is a structural diagram of the influenza A virus antibody detection test strip in Example 1 of the present invention; Figure 4 This is a display diagram of the detection results in Example 2 of the present invention; Icons: 1-Observation window; 2-Sample loading hole; 3-Test card strip loading area; 4-Bottom plate; 5-Test pad; 6-Sample suction pad; 7-Marking pad; 8-Sample pad; 9-Quality control line; 10-Test line. DETAILED DESCRIPTION

[0031] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0032] The embodiment of the present invention provides an influenza A virus protein, comprising protein fragment S1, protein fragment S2 and protein fragment S3; Protein fragment S1, whose amino acid sequence is shown in SEQ ID NO.1; Protein fragment S2, whose amino acid sequence is shown in SEQ ID NO.2; Protein fragment S3, the amino acid sequence of which is shown in SEQ ID NO.3; The protein fragment S1, the protein fragment S2 and the protein fragment S3 can be combined and connected in any order.

[0033] In some embodiments, the combination of protein fragments of influenza A virus protein includes any one of S1+S2+S3, S1+S3+S2, S2+S3+S1, S2+S1+S3, S3+S2+S1 or S3+S1+S2.

[0034] The embodiment of the present invention provides an influenza A virus antibody detection test strip, the detection test strip comprising a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked on the base plate; In some embodiments, the label pad contains the influenza A virus protein, and the influenza A virus protein is labeled with a marker, and the marker can be detected to identify the location or concentration of the marker.

[0035] The influenza A virus antibody detection test strip provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for on-site rapid detection, economy and practicality, etc., and is of great significance for the early diagnosis of influenza A virus infection, prediction of disease progression, judgment of prognosis, screening and evaluation of the efficacy of anti-influenza A virus drugs, etc.

[0036] In some embodiments, the label pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker; the marker includes but is not limited to any one or a combination of at least two of colloidal gold, color microspheres, time-resolved fluorescent microspheres or quantum dot microspheres; the average particle size of the marker is 40-300 nm; In some embodiments, the particle size of the colloidal gold may be, for example, but not limited to, 40 nm, 60 nm, 80 nm, or 100 nm; In some embodiments, the particle size of the colored microspheres, time-resolved fluorescent microspheres, and quantum dot microspheres may be, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm; In some embodiments, the mass ratio of influenza A virus protein to colloidal gold can be, for example, but not limited to 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1; the mass ratio of influenza A virus protein to colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres can be, for example, but not limited to 0.1:1, 0.2:1, 0.3:1, 0.4:1.

[0037] In some embodiments, the sensitivity of the test strip is increased by adjusting the size and amount of the marker particles.

[0038] In some embodiments, a test line (T line) and a quality control line (C line) are provided on the test pad; In some embodiments, the test line is coated with, for example, but not limited to, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL of the influenza A virus protein; In some embodiments, the quality control line is coated with, for example, but not limited to, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL of goat anti-chicken IgY antibody; In some embodiments, the diluent of influenza A virus protein and goat anti-chicken IgY antibody is a PB buffer containing trehalose, for example, but not limited to 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, and each 100 mL of the diluent contains, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g of trehalose.

[0039] In some embodiments, the influenza A virus protein on the label pad is the same as the influenza A virus protein on the detection pad.

[0040] The test strip provided by the present invention is based on the principle of antigen-antibody reaction, and after labeling the monoclonal protein fragment of influenza A virus with colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dots, the marker is solidified on a glass cellulose membrane. The same influenza A virus is coated on the detection pad (such as NC membrane), and based on the principle of antigen-antibody reaction, it can be detected by the naked eye or a matching instrument within the detection time. If influenza A virus antibodies are present in the sample, a double protein fragment sandwich structure is formed, forming a band visible to the naked eye or a light intensity signal in the instrument. If there is no influenza A virus antibody in the sample, no band appears on the NC membrane or there is no light intensity signal in the instrument. Negative or positive judgment is made based on the presence or absence of the signal, or the antibody content is predicted based on the intensity of the light intensity signal.

[0041] By using the test strip provided by the present invention for testing, the test results can be obtained within 10-30 minutes of the entire process, which is fast and efficient, and helps medical personnel to obtain the test results in a timely manner, make comprehensive judgments and take timely measures based on the results, avoid panic, and reduce the spread of the epidemic.

[0042] The invention provides an influenza A virus antibody detection kit, comprising a shell and a detection test paper arranged inside the shell, wherein the detection test paper is an influenza A virus antibody detection test paper strip.

[0043] The influenza A virus antibody detection kit provided by the present invention contains the influenza A virus antibody detection test strip, and thus has all the beneficial effects of the influenza A virus antibody detection test strip.

[0044] In some embodiments, the housing includes an upper cover and a lower cover that are detachably connected; In some embodiments, the upper cover is provided with an observation window and a sample loading hole, and the lower cover is provided with a test card loading area.

[0045] The shapes of the observation window and the sample addition hole are not specifically limited in the present invention, and those skilled in the art can adjust them according to actual needs. For example, the observation window can be square, located above the test line and the quality control line of the test strip, and used to observe the test results. The sample addition hole can be a circular hole with a diameter of 0.5-1 cm, located above the sample pad.

[0046] In some embodiments, the test sample of the kit is added: Unless otherwise specified in the following examples, the diluent for influenza A virus protein and goat anti-chicken IgY antibody is 20 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.5 g of trehalose.

[0047] Example 1 This embodiment provides an influenza A virus antibody detection kit, comprising a housing and a detection test strip located inside the housing, wherein the housing comprises a detachably connected upper cover and a lower cover, such as Figure 1 As shown, the upper cover is provided with an observation window 1 and a sample addition hole 2; Figure 2 As shown, the lower cover is provided with a detection card strip area 3; Figure 3 As shown, the influenza A virus antibody detection test strip comprises a base plate 4 and a sample pad 8, a marking pad 7, a detection pad 5 and a sample suction pad 6 which are sequentially stacked on the base plate; a detection line 10 and a quality control line 9 are arranged on the detection pad.

[0048] Example 2 This embodiment provides a colloidal gold-labeled protein fragment for preparing an influenza A virus antibody detection kit, and the structure of the kit is shown in Example 1.

[0049] 1 Main Materials 1.1 Protein fragments: influenza A virus protein (protein fragment combination is S1+S2+S3), used for labeling and detection line coating respectively; chicken IgY antibody: purchased from Nanjing Jingda Biotechnology Co., Ltd., used for labeling; goat anti-chicken IgY antibody: purchased from Nanjing Jingda Biotechnology Co., Ltd., used for nitrocellulose membrane quality control line coating; 1.2 Nitrocellulose membrane: NC membrane is a product of Sartorius; 1.3 Other consumables: PVC boards and other consumables are products of Beacon Labs; commonly used reagents are all analytical grade reagents and can be purchased through platforms such as Aladdin.

[0050] 2 Methods 2.1 Preparation of colloidal gold labeling pad: The steps for preparing the colloidal gold labeling pad are as follows: (1) Take 1 mL of colloidal gold solution with a particle size of 40 nm and use 0.2 M K 2 CO 3Adjust pH to 8.5; (2) Add 25 μg influenza A virus protein and 5 μg chicken IgY antibody, the mass ratio of total protein to colloidal gold particles is 0.12:1, adjust the rotary shaker to a certain speed, rotate at room temperature for 1.5 h, and then add 20 μL of blocking solution; (3) Centrifuge at 12,000 rpm for 15 min and discard the supernatant; (4) Add 100 μL of colloidal gold solution; (5) The above concentrate was diluted in a ratio of 1:7, sprayed with gold, and placed in a drying oven at 37°C for 2 h for later use.

[0051] 2.2 NC membrane coating: Use 0.02M PB containing 0.5% trehalose to dilute influenza A virus protein to 1.5 mg / mL and goat anti-chicken IgY antibody to 2 mg / mL; then use a film sprayer to draw the test line T and the quality control line C on the nitrocellulose membrane respectively. After coating, dry the NC membrane in an oven at 37°C for 24 h for use.

[0052] 2.3 Assembly of the kit: Place the coated nitrocellulose membrane in the middle of the plastic support plate in a drying room and stick it. Overlap the marker colloidal gold pad on one side of the T line of the nitrocellulose membrane (1 / 3 of the colloidal gold pad) and overlap the sample pad on the other side of the colloidal gold pad (1 / 5 of the colloidal gold pad); overlap the sample pad on one side of the C line of the nitrocellulose membrane (1 / 10 of the sample pad); then use a cutting machine to cut the pasted plastic plate into test strips of a certain width, and then load them into the test card to form an influenza A virus antibody test kit.

[0053] 2.4 Detection: Step 1, take out the test kit and the sample to be tested and equilibrate to room temperature; Step 2. Open the sealed aluminum foil bag, take out the test kit and place it flat on the table; Step 3: Figure 1 Add 2 drops of sample (about 80-100 μL) to the sample well; Step 4: Start the timer and read the result after 10 minutes. Note that if the sample does not undergo lateral chromatography or diafiltration within 1 minute after adding the sample, it may be because the sample is too viscous and needs to be pretreated with saline.

[0054] 3 Results Under the action of lateral flow chromatography, when there are influenza A virus antibodies in the sample, the test line will show color, and the quality control line will also show color ( Figure 4 a in the figure); when there is no influenza A virus antibody in the sample, the test line does not show color, but the quality control line shows color ( Figure 4b); After loading, if the quality control line does not show color, the result is considered invalid regardless of whether the test line shows color or not ( Figure 4 c in Figure 4 d) in.

[0055] 4 The test kit provided in Example 2 was used to test the blood samples of patients with influenza A virus, and the results were consistent with those shown in 3. This shows that the influenza A virus protein provided by the present invention is effective in detecting influenza A virus antibodies.

[0056] Example 3 This embodiment provides a time-resolved fluorescent microsphere-labeled protein fragment for preparing an influenza A virus antibody detection kit.

[0057] The influenza A virus monoclonal protein provided in this embodiment is used to prepare an influenza A virus antibody detection kit, which includes a detection card and a test strip. The detection card is divided into an upper cover and a lower bottom. The test strip is embedded with an influenza A virus protein labeled with time-resolved fluorescent microspheres in a fluorescent pad, and the detection line is coated with influenza A virus protein. The double protein sandwich method is used to quantitatively detect influenza A virus antibodies in the sample.

[0058] 1 Kit preparation operation process: The surface of time-resolved fluorescent microspheres is labeled with influenza A virus proteins prepared by the method of the present invention. Specific examples are as follows: Time-resolved fluorescent microsphere protein fragment labeling: 1mL 1% carboxyl time-resolved fluorescent microspheres, add 9mL MES buffer, then add 25μL EDC solution (10mg / mL) and 25μL NHS solution (10mg / mL), shake at room temperature for 30min, and collect the precipitate by centrifugation. Add HEPES reconstitution solution, ultrasonically disperse evenly, then add 1mL 1mg / mL influenza A virus protein (protein fragment combination is S1+S2+S3), shake at room temperature for 120min, and collect the precipitate by centrifugation; then add 1mL blocking solution, shake at room temperature for 120min. Collect the microsphere precipitate by centrifugation and reconstitute with reconstitution solution.

[0059] Preparation of fluorescent pad: The labeled time-resolved fluorescent microspheres were diluted with the microsphere complex solution and sprayed with the fluorescent pad using a gold spray film scribing instrument at a spraying rate of 3 μL / cm and a spraying interval of 6 mm. After spraying, the fluorescent pad was dried at 37°C with low humidity (<30%) for 2 h.

[0060] NC membrane coated CT line: T line uses influenza A virus protein with a concentration of 1.5 mg / mL, C line uses goat anti-chicken IgY antibody with a concentration of 1 mg / mL, 1μL / cm line is drawn, and then placed in 37℃ low humidity (<30%) for drying for 24 hours.

[0061] Sample pad treatment: The sample pad treatment solution is composed of buffer salt, sustained-release agent, cosolvent, blocking agent, etc.; the specific formula is 20 mM Tris buffer, each 100 mL Tris buffer contains 1g BSA, 0.5g Tween 20, 2g sucrose; according to the formula of 20 cm 2 Treat with 1 mL of sample treatment solution; after uniform treatment, place in a 37°C low humidity (<30%) oven to dry for 2 hours.

[0062] Test strip assembly: stick NC film, sample suction pad, fluorescent pad and sample pad on the PVC board in sequence, with the sample suction pad and fluorescent pad pressing the NC film 1~2mm each, and the sample pad pressing the fluorescent pad 1~2mm; after assembly, cut the test strip into a width of 4±0.4mm and put it into a card shell; put the card shell and desiccant into an aluminum foil bag and seal it; label and put it into a box to get the finished test card.

[0063] 2 Kit testing process Place the test card on a clean, flat surface, draw 80-100 μL of the treated sample and drop it into the sample adding end of the test card, and set up a PBS control group at the same time.

[0064] Import the test strip standard curve, and after 10 minutes, use the fluorescent immunoassay analyzer to scan the detection area to obtain the fluorescent signal, and the titer corresponding to the influenza A virus antibody will be displayed after the test.

[0065] 3 Test results Three negative samples and three low-value positive samples were screened, and the samples were diluted 2, 10, 50, 250, and 1250 times with 0.01 M PBS (pH 7.2), respectively. The samples were then tested with the above-mentioned finished test cards. The results showed that the test results of the low-value positive samples after being diluted 250 times were still significantly higher than those of the negative samples. The test results are shown in Table 1.

[0066] Table 1 .

[0067] Example 4 This embodiment provides a kit for detecting influenza A virus antibodies using colored microspheres labeled with protein fragments.

[0068] The influenza A virus monoclonal protein provided in this embodiment is used to prepare an influenza A virus antibody detection test strip. The test strip has an influenza A virus protein labeled with colored microspheres embedded on a marking pad, and the detection line is coated with influenza A virus protein. The double protein sandwich method is used to qualitatively detect influenza A virus antibodies in the sample.

[0069] 1. Preparation of test strips: 1.1 Colored microspheres labeling protein fragments (1) 100 nm colored microspheres were treated with 0.1 mol / L K2 CO 3 Adjust the pH to 8.0; (2) Influenza A virus protein-labeled microspheres: Take 1 mL of the above pH-adjusted solution, add 30 μg of influenza A virus protein (protein fragment combination is S1+S2+S3), the mass ratio of total protein to colored microspheres is 0.3:1, react at room temperature for 1 hour, then centrifuge and discard the supernatant; Chicken IgY protein fragment labeled microspheres: add 5 μg of chicken IgY to 1 mL of the above pH adjusted solution, with the mass ratio of protein fragment to colored microspheres being 0.05:1. After reacting at room temperature for 1 hour, centrifuge and discard the supernatant. (3) Add 1 mL of 20% BSA to block for 2 h, then centrifuge and discard the supernatant; (4) After re-dissolving the above microspheres with 100 μL of pH 8.0 reconstitution solution, the two microspheres were mixed at a ratio of 5:1, and the mixture was diluted with the reconstitution solution at a ratio of 15%.

[0070] 1.2 Preparation of labeling pad The diluted solution of the labeled colored microglobulin fragment complex was sprayed on the marking pad using a gold spray film instrument at a spraying rate of 7.5 μL / cm and a spraying interval of 6 mm. After spraying, it was placed in a 37°C low humidity (<30%) drying chamber for 2 h.

[0071] 1.3 Sample pad preparation The sample pad treatment solution is composed of buffer salt, sustained-release agent, cosolvent, blocking agent, etc.; the specific formula is 20mM Tris, 1% BSA, 0.5% Tween 20, 2% sucrose; according to the formula of 20 cm 2 Treat with 1 mL of sample treatment solution; after uniform treatment, place in a 37°C low humidity (<30%) oven to dry for 2 hours.

[0072] 1.4 C / T line coating The T line uses influenza A virus protein at a concentration of 1.5 mg / mL (the C line uses sheep anti-chicken IgY protein fragment at a concentration of 2 mg / mL), 1 μL / cm is used for drawing, and after the end, it is placed in 37℃ low humidity (<30%) for drying for 24 hours.

[0073] 1.5 Test strip assembly The PVC board is sequentially pasted with the sample suction pad, NC film, marking pad, sample pad, sample suction pad and marking pad, each pressing the NC film by 1-2 mm, and the sample pad pressing the marking pad by 1-2 mm; after assembly, the test strip is cut into a width of 4±0.4 mm and installed in a cartridge; the cartridge and desiccant are put into an aluminum foil bag together; a label is affixed and the box is packed to obtain a finished test kit.

[0074] 2. Detection Methods Place the test card on a clean, flat surface, draw 80-100 μL of the treated sample and drop it into the sample adding end of the test card, and set up a PBS control group; observe the test card window after 10 minutes, if there are 2 positive lines in the window, and only one C line is negative.

[0075] 3 Test results Three negative samples and three low-value positive samples were screened, and the samples were diluted 2, 10, 50, 250, and 1250 times with 0.01 M PBS (pH 7.2), respectively. The samples were then tested with the above-mentioned finished test kit. The results showed that the test results of the low-value positive samples were still positive after being diluted 250 times. The test results are shown in Table 2.

[0076] Table 2 .

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An influenza A virus protein, characterized in that The influenza A virus protein comprises a combination of the following protein fragments: Protein fragment S1, whose amino acid sequence is shown in SEQ ID NO.1; Protein fragment S2, whose amino acid sequence is shown in SEQ ID NO.2; Protein fragment S3, the amino acid sequence of which is shown in SEQ ID NO.3; The protein fragment S1, protein fragment S2 and protein fragment S3 can be combined and connected in any order.

2. The influenza A virus protein according to claim 1, characterized in that The protein fragment combination of the influenza A virus protein includes any one of S1+S2+S3, S1+S3+S2, S2+S3+S1, S2+S1+S3, S3+S2+S1 or S3+S1+S2.

3. Use of the influenza A virus protein according to claim 1 or 2 in the preparation of influenza A virus antibody detection products.

4. A test strip for detecting antibodies to influenza A virus, characterized in that: The influenza A virus antibody detection test strip comprises a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked on the base plate; Wherein, the label pad and / or the detection pad comprises the influenza A virus protein according to claim 1 or 2.

5. The influenza A virus antibody detection test strip according to claim 4, characterized in that: The label pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker; The marker includes any one or a combination of at least two of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres; The average particle size of the marker is 40-300 nm.

6. The influenza A virus antibody detection test strip according to claim 4, characterized in that: The detection pad is provided with a detection line and a quality control line, wherein the detection line is coated with the influenza A virus protein according to claim 1 or 2, and the quality control line is coated with sheep anti-chicken IgY antibody; Based on the concentration of influenza A protein coated on the test line being 0.5-5 mg / mL, the concentration of sheep anti-chicken IgY antibody coated on the quality control line is 0.5-5 mg / mL.

7. The influenza A virus antibody detection test strip according to claim 4, characterized in that: The label pad and the detection pad both include the influenza A virus protein according to claim 1 or 2.

8. A detection kit, characterized in that: The detection kit comprises a shell and a detection test paper arranged inside the shell, and the detection test paper is the influenza A virus antibody detection test paper strip according to any one of claims 4 to 7.

9. The detection kit according to claim 8, characterized in that The housing comprises an upper cover and a lower cover which are detachably connected; The upper cover is provided with an observation window and a sample adding hole, and the lower cover is provided with a detection card strip installation area.

10. Use of the detection kit according to claim 8 or 9 in detecting influenza A virus.

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