Human hepatitis C virus protein and application thereof

Abstract

The invention provides a human hepatitis C virus protein and application thereof. The human hepatitis C virus protein provided by the invention comprises a protein fragment C-a, a protein fragment NS3-a and a protein fragment NS5-a, and the amino acid sequence of the protein fragments is defined. The human hepatitis C 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, and can be used for carrying out rapid screening when viruses are epidemic; and a large number of people including suspected cases, asymptomatic infection cases in an incubation period and asymptomatic virus carriers with higher risk can be rapidly, accurately and safely detected in time.

Description

Technical Field

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

[0002] Hepatitis C virus (HCV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the genus Hepatitis C virus of the family Flaviviridae. It is an important human pathogen transmitted through blood. Chronic infection often leads to chronic hepatitis, persistent liver damage, and then to serious liver diseases such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, which brings heavy health, economic, and psychological burdens to society and families.

[0003] According to 2020 data, there are about 70 million HCV-infected people in the world, of which only 20% are diagnosed and 7% receive treatment. In the past five years, my country has reported about 200,000 new cases of HCV infection each year, making it one of the countries with a high burden of HCV infection. In 2020, there were 9.487 million HCV-infected people in my country, but the cumulative number of reported cases was only 2.7 million, and about 2 / 3 of HCV-infected people were undiagnosed. Therefore, expanding the early detection rate of HCV infection and conducting early intervention treatment in a timely manner can not only shorten the clinical treatment time more safely and effectively, but also is an important way to control HCV infection and an effective means to reduce the burden of late-stage disease.

[0004] Based on the above background, it is very necessary to develop a new detection device and detection method to improve the sensitivity of HCV detection and assist clinical diagnosis. Summary of the invention

[0005] The objects of the present invention are: The first object of the present invention is to provide a human hepatitis C virus protein; The second object of the present invention is to provide the use of the above human hepatitis C virus protein in the preparation of human hepatitis C virus antibody detection products; The third object of the present invention is to provide a test paper box 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 human hepatitis C virus.

[0006] In order to achieve the above object, the present invention provides a human hepatitis C virus protein, comprising a combination of the following protein fragments; Protein fragment Ca, whose amino acid sequence is shown in SEQ ID NO.1; Protein fragment NS3-a, the amino acid sequence of which is shown in SEQ ID NO.2; Protein fragment NS5-a, the amino acid sequence of which is shown in SEQ ID NO.3; The protein fragment Ca, protein fragment NS3-a and protein fragment NS5-a can be combined and connected in any order.

[0007] The instructions required are: The amino acid sequence of the protein fragment Ca is shown in SEQ ID NO.1, and is specifically as follows: mstnpkpqrktkrntnrrpqdvkfpgggqivggvyllprrgprlgvratrktsersqprgrrqpipkarrpegrawaqpgypwplygnegmgwagwllsprgsrpswgptdprrrsrnlgkvidtltcgfadlmgyiplvgaplggaaralahgvrvledgvnyatgnl.

[0008] The amino acid sequence of the protein fragment NS3-a is shown in SEQ ID NO.2, and is specifically expressed as follows: egevqvvstatqsflatcvngvcwtvyhgagsktlagpkgpitqmytnvdqdlvgwqappgarsmtpctcgssdlylvtrhadvipvrrrgdgrgsllsprpisylkgssggpllcplghvvgifraavctrgvakavdfvpvesmett.

[0009] The amino acid sequence of the protein fragment NS5-a is shown in SEQ ID NO.3, and is specifically shown as follows: sgswlrdvwdwictvlsdfktwlqskllpqlpgvpffscqrgykgvwrgdgimqttcpcgaqitghvkngsmritgpktcsntwhgtfpvnayttgpc tpspapnysralwrvaaeeyvevtrvgdfhyvtgmttdnlkcpcqvpapefftevdgvrlhryapacrpllreevvfqvglnqylvgsqlpcepepd.

[0010] Preferably, the protein fragment combination of the human hepatitis C virus protein includes any one of C-a+NS3-a+NS5-a, C-a+NS5-a+NS3-a, NS3-a+NS5-a+Ca, NS3-a+C-a+NS5-a, NS5-a+NS3-a+Ca or NS5-a+C-a+NS3-a.

[0011] The present invention does not make further optimization for the combination of protein fragments of human hepatitis C virus protein, and those skilled in the art can select or adjust according to actual needs.

[0012] The present invention also provides a human hepatitis C virus antibody detection test strip, which comprises a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked on the base plate from bottom to top; wherein the marking pad and / or the detection pad comprises the human hepatitis C virus protein described above.

[0013] Preferably, the label pad contains the human hepatitis C virus protein described above; Preferably, the label pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker; Preferably, the marker comprises any one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres, or a combination of at least two of them.

[0014] Preferably, in any of the above items, the average particle size of the colloidal gold particles is 40-100 nm, illustratively including 40 nm, 50 nm, 60 nm, 80 nm, 100 nm and ranges therebetween.

[0015] Preferably, in any of the above items, the average particle size of the colored microspheres is 100 to 300 nm, exemplarily including 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0016] Preferably, in any of the above items, the average particle size of the time-resolved fluorescent microspheres is 100-300 nm, exemplarily including 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0017] Preferably, in any of the above items, the average particle size of the quantum dot microsphere particles is 100-300 nm, exemplarily including 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0018] As a preferred embodiment of the present invention, the mass ratio of human hepatitis C virus protein to colloidal gold is (0.04-0.32):1; illustratively including 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1 and ranges therebetween.

[0019] As a preferred embodiment of the present invention, the mass ratio of the human hepatitis C virus protein to the colored microspheres is (0.1-0.4):1; illustratively including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0020] As a preferred embodiment of the present invention, the mass ratio of the human hepatitis C virus protein to the time-resolved fluorescent microspheres is (0.1-0.4):1; illustratively including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0021] As a preferred embodiment of the present invention, the mass ratio of the human hepatitis C virus protein to the quantum dot microspheres is (0.1-0.4):1; illustratively including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0022] Preferably, a detection line and a quality control line are provided on the detection pad; the detection line is coated with the human hepatitis C virus protein; and the quality control line is coated with sheep anti-chicken IgY antibody.

[0023] Preferably, any of the above items is that the test line is coated with 0.5-5 mg / mL of the human hepatitis C virus protein, exemplarily including 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 ranges therebetween.

[0024] Preferably, any of the above items is that the quality control line is coated with 0.5-5 mg / mL of goat anti-chicken IgY antibody, exemplarily including 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 ranges therebetween.

[0025] Any of the above is preferably that the diluent of human hepatitis C 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. Exemplarily, the diluent is a 10 mM, 20 mM, 30 mM, 40 mM, 50 mM PB buffer and the range therebetween containing trehalose; Exemplarily, each 100 mL of the diluent contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g and the range therebetween of trehalose.

[0026] Preferably, the human hepatitis C virus protein on the labeling pad is the same as the human hepatitis C virus protein on the detection pad.

[0027] 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 human hepatitis C virus protein; the quality control line is coated with sheep anti-chicken IgY; the marking pad contains the same kind of human hepatitis C virus protein, and the human hepatitis C 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.

[0028] The present invention also provides a human hepatitis C virus antibody detection test strip comprising any one of the above items and a shell, wherein the human hepatitis C virus antibody detection test strip is arranged inside the shell.

[0029] Preferably, the housing comprises an upper cover and a lower cover which are detachably connected.

[0030] Preferably, any one of the above items is that the upper cover is provided with an observation window and a sample addition hole.

[0031] Preferably, any of the above items is that the lower cover is provided with a detection card mounting strip area.

[0032] Compared with the prior art, the present invention has the following beneficial effects: The human hepatitis C 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 economy and practicality, and is of great significance for the early diagnosis of HCV infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the efficacy of anti-HCV drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The upper cover of the human hepatitis C virus antibody detection kit in Example 1 of the present invention; Figure 2 The lower cover of the human hepatitis C virus antibody detection kit in Example 1 of the present invention; Figure 3 This is a structural diagram of a test strip for detecting antibodies against human hepatitis C virus 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

[0034] 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.

[0035] The embodiment of the present invention provides a human hepatitis C virus protein, comprising a protein fragment Ca, a protein fragment NS3-a and a protein fragment NS5-a; The amino acid sequence of protein fragment Ca is shown in SEQ ID NO.1; The amino acid sequence of protein fragment NS3-a is shown in SEQ ID NO.2; The amino acid sequence of protein fragment NS5-a is shown in SEQ ID NO.3; Among them, protein fragment Ca, protein fragment NS3-a and protein fragment NS5-a can be combined and connected in any order.

[0036] In some embodiments, the protein fragments of human hepatitis C virus protein are combined in any one of the following combinations: C-a+NS3-a+NS5-a; C-a+NS5-a+NS3-a; NS3-a+NS5-a+Ca; NS3-a+C-a+NS5-a; NS5-a+NS3-a+Ca; NS5-a+C-a+NS3-a.

[0037] The embodiment of the present invention provides a human hepatitis C virus antibody detection test strip, which includes a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad which are sequentially stacked on the base plate from top to bottom.

[0038] In some embodiments, the label pad contains the human hepatitis C virus protein, and the human hepatitis C virus protein is labeled with a marker, and the marker can be detected to identify the location or concentration of the marker.

[0039] The human hepatitis C 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, and economical and practical, and is of great significance for the early diagnosis of HCV infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the efficacy of anti-HCV drugs.

[0040] In some embodiments, the label includes but is not limited to at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres, or quantum dot microspheres; 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 human hepatitis C virus protein coupled 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 human hepatitis C 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.

[0041] In some embodiments, the sensitivity of the test strip is increased by adjusting the size and amount of the marker particles.

[0042] In some embodiments, a test line (T line) and a quality control line (C line) are provided on the test pad; 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, and 5.0 mg / mL of the human hepatitis C virus protein; 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, and 5.0 mg / mL of goat anti-chicken IgY antibody; In some embodiments, the diluent of human hepatitis C 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.

[0043] In some preferred embodiments, the human hepatitis C virus protein on the label pad is the same as the human hepatitis C virus protein on the detection pad.

[0044] The test strip provided by the present invention is based on the principle of antigen-antibody reaction, and after labeling the human hepatitis C virus monoclonal protein fragment with colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dots, the marker is solidified on a glass cellulose membrane. The same human hepatitis C 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 human hepatitis C virus antibodies exist in the sample, a double protein fragment sandwich structure is formed, forming a strip visible to the naked eye or a light intensity signal in the instrument. If there is no human hepatitis C virus antibody in the sample, no strip 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.

[0045] 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.

[0046] The embodiment of the present invention provides a human hepatitis C virus antibody detection kit, comprising a human hepatitis C virus antibody detection test strip and a shell, wherein the human hepatitis C virus antibody detection test strip is arranged inside the shell.

[0047] The human hepatitis C virus antibody detection kit provided by the present invention contains a human hepatitis C virus antibody detection test strip, and thus has all the beneficial effects of the human hepatitis C virus antibody detection test strip.

[0048] In some embodiments, the housing includes an upper cover and a lower cover that 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 loading area.

[0049] 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 is used to observe the test results. The sample addition hole can be, for example, a circular hole with a diameter of 0.5-1 cm, located above the sample pad.

[0050] In some embodiments, the test sample of the kit is added: Unless otherwise specified in the following examples, the diluent for human hepatitis C 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.

[0051] Example 1 This embodiment provides a human hepatitis C virus antibody detection kit, including a housing and a detection test strip located inside the housing, wherein the housing includes 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 human hepatitis C 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.

[0052] Example 2 This embodiment provides a colloidal gold-labeled protein fragment for preparing a human hepatitis C virus antibody detection kit, and the structure of the kit is shown in Example 1.

[0053] 1 Main Materials 1.1 Protein fragments: human hepatitis C virus protein (protein fragment combination is C-a+NS3-a+NS5-a), 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.

[0054] 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 adjust the pH to 8.5 with 0.2 M K2CO3; (2) Add 25 μg of human hepatitis C virus protein and 5 μg of 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 and label at room temperature for 1.5 h. 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.

[0055] 2.2 NC membrane coating: Human hepatitis C virus protein was diluted to 1.5 mg / mL and goat anti-chicken IgY antibody was diluted to 2 mg / mL using 0.02 M PB containing 0.5% trehalose. Then, the detection line T and the quality control line C were drawn on the nitrocellulose membrane using a film sprayer. After coating, the NC membrane was dried in an oven at 37°C for 24 h for later use.

[0056] 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 a human hepatitis C virus antibody detection kit.

[0057] 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.

[0058] 3 Results Under the action of lateral flow chromatography, when there are human hepatitis C 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 human hepatitis C 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.

[0059] 4 The blood samples of HCV patients were tested using the kit provided in Example 2, and the results were consistent with those shown in 3. This indicates the effectiveness of the human hepatitis C virus protein provided by the present invention in the detection of HCV antibodies.

[0060] Example 3 The present embodiment provides a human hepatitis C virus antibody detection kit prepared by using a time-resolved fluorescent microsphere-labeled protein fragment; a human hepatitis C virus antibody detection kit is prepared using the human hepatitis C virus monoclonal protein provided in the present embodiment, the kit comprising a detection card and a test strip, the detection card is divided into an upper cover and a lower bottom, the test strip has human hepatitis C virus protein labeled with time-resolved fluorescent microspheres embedded in a fluorescent pad, the detection line is coated with human hepatitis C virus protein, and a double protein sandwich method is used to quantitatively detect human hepatitis C virus antibodies in a sample.

[0061] 1 Kit preparation operation process: Time-resolved fluorescent microsphere surface labeling using human hepatitis C virus protein (protein fragment combination is C-a+NS3-a+NS5-a) 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, add 1mL 1mg / mL human hepatitis C virus protein, 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.

[0062] 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.

[0063] NC membrane coated CT line: T line uses human hepatitis C 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.

[0064] Sample pad treatment: The sample pad treatment solution is composed of buffer salt, slow-release agent, cosolvent, blocking agent, etc. The specific formula is 20 mM Tris buffer, and each 100 mL Tris buffer contains 1g BSA, 0.5g Tween 20, and 2g sucrose. 2 Use 1mL of sample treatment solution to treat. After evenly treating, place in a 37℃ low humidity (<30%) oven to dry for 2h.

[0065] 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.

[0066] 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.

[0067] Import the test strip standard curve, and after 10 minutes, use a fluorescent immunoassay analyzer to scan the detection area to obtain a fluorescent signal. After the test, the titer corresponding to the human hepatitis C virus antibody will be displayed.

[0068] 3 Test results Three negative samples and three low-value positive samples were screened, and they were diluted 2, 10, 50, 250, and 1250 times with 0.01 M PBS (pH 7.2), respectively. Then, the above-mentioned finished test cards were used for testing. The results showed that the test results of the low-value positive samples after dilution 250 times were still significantly different from those of the negative samples. The test results are shown in Table 1.

[0069] Table 1 .

[0070] Example 4 The present embodiment provides a kit for detecting human hepatitis C virus antibodies by using colored microspheres labeled protein fragments. The human hepatitis C virus monoclonal protein provided in the present embodiment is used to prepare a test strip for detecting human hepatitis C virus antibodies. The test strip has human hepatitis C virus proteins labeled with colored microspheres embedded on a marking pad, and the detection line is coated with human hepatitis C virus proteins. The double protein sandwich method is used to qualitatively detect human hepatitis C virus antibodies in a sample.

[0071] 1. Preparation of test strips: 1.1 Colored microspheres labeling protein fragments (1) Adjust the pH of 100 nm colored microspheres to 8.0 with 0.1 mol / L K2CO3; (2) Human hepatitis C virus protein-labeled microspheres: Take 1 mL of the above pH-adjusted solution, add 30 μg of human hepatitis C virus protein (protein fragment combination is C-a+NS3-a+NS5-a), 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%.

[0072] 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.

[0073] 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 Use 1mL of sample treatment solution to treat. After evenly treating, place in a 37℃ low humidity (<30%) oven to dry for 2h.

[0074] 1.4 C / T line coating The T line uses human hepatitis C 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.

[0075] 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.

[0076] 2. Detection 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.

[0077] 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.

[0078] Table 2 .

[0079] 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. A human hepatitis C virus protein, characterized in that The human hepatitis C virus protein comprises a combination of the following protein fragments: Protein fragment Ca, whose amino acid sequence is shown in SEQ ID NO.1; Protein fragment NS3-a, the amino acid sequence of which is shown in SEQ ID NO.2; Protein fragment NS5-a, the amino acid sequence of which is shown in SEQ ID NO.3; The protein fragment Ca, protein fragment NS3-a and protein fragment NS5-a can be combined and connected in any order.

2. The human hepatitis C virus protein according to claim 1, characterized in that The protein fragment combination of the human hepatitis C virus protein includes any one of C-a+NS3-a+NS5-a, C-a+NS5-a+NS3-a, NS3-a+NS5-a+Ca, NS3-a+C-a+NS5-a, NS5-a+NS3-a+Ca or NS5-a+C-a+NS3-a.

3. Use of the human hepatitis C virus protein according to claim 1 or 2 in the preparation of a human hepatitis C virus antibody detection product.

4. A test strip for detecting antibodies against human hepatitis C virus, characterized in that: The human hepatitis C virus antibody detection test strip comprises a bottom plate and a sample pad, a marking pad, a detection pad and a sample suction pad which are sequentially stacked on the bottom plate from bottom to top; Wherein, the labeling pad and / or the detection pad comprises the human hepatitis C virus protein according to claim 1 or 2.

5. The human hepatitis C 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 of colloidal gold, color microspheres, time-resolved fluorescent microspheres or quantum dot microspheres, or a combination of at least two of them.

6. The human hepatitis C 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; The detection pad includes the human hepatitis C virus protein according to claim 1 or 2, and the detection line is coated with the human hepatitis C virus protein at a concentration of 0.5 to 5 mg / mL; The quality control line is coated with goat anti-chicken IgY antibody with a concentration of 0.5-5 mg / mL.

7. The human hepatitis C virus antibody detection test strip according to claim 4, characterized in that: The labeling pad and the detection pad both include the human hepatitis C 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 human hepatitis C virus antibody detection test strip according to any one of claims 4-7.

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 human hepatitis C virus.

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