Human hepatitis C virus protein and application thereof

Abstract

The invention provides a human hepatitis C virus protein sequence and application thereof, the human hepatitis C virus protein sequence comprises protein fragments C-1, NS3-b and NS5-a with good specificity, high activity and strong stability, and the human hepatitis C virus protein sequence can be used for preparing a product for detecting an HCV antibody. Meanwhile, the invention relates to an antibody detection kit which is simple to operate, does not need a complex process and professional equipment, and can be quickly operated by ordinary personnel; the detection reaction is rapid, the sensitivity is high (trace antibody signals can be captured), the specificity is strong (misdiagnosis and missed diagnosis are reduced), the kit is suitable for on-site rapid detection, and a professional laboratory environment is not needed. The kit is economical in cost and high in cost performance, high-risk populations such as suspected cases, asymptomatic infected persons and carriers can be rapidly screened in the virus epidemic period, and efficient support is provided for epidemic situation prevention and control.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a human hepatitis C virus (HCV) protein and its application. More specifically, the present invention relates to an HCV protein, an HCV antibody detection test strip based on the protein, a detection kit, and its application in HCV diagnosis. Background Art

[0002] Hepatitis C virus (HCV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the genus Hepatitis C virus in the family Flaviviridae. It is a significant blood-borne human pathogen. Chronic infection often leads to chronic hepatitis, persistent liver damage, and subsequent severe liver diseases such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, placing a heavy health, economic, and psychological burden on society and families.

[0003] Among viral hepatitis, hepatitis C has the highest incidence, second only to hepatitis B. Therefore, increasing the early detection rate of HCV infection and implementing timely early intervention treatment will not only be safer and more effective in shortening clinical treatment time, 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 discover a highly specific hepatitis C protein sequence and 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:

[0006] The first object of the present invention is to provide a human hepatitis C virus protein with high specificity, sensitivity and stability.

[0007] The second object of the present invention is to provide the use of the above-mentioned human hepatitis C virus protein in the preparation of human hepatitis C virus antibody detection products.

[0008] A third object of the present invention is to provide a test paper box to solve at least one of the above problems.

[0009] The fourth object of the present invention is to provide a detection kit for use in detecting human hepatitis C virus.

[0010] 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;

[0011] Protein fragment C-1, the amino acid sequence of which is shown in SEQ ID NO.1;

[0012] Protein fragment NS3-b, the amino acid sequence of which is shown in SEQ ID NO.2;

[0013] Protein fragment NS5-a, the amino acid sequence of which is shown in SEQ ID NO.3;

[0014] The protein fragment C-1, protein fragment NS3-b and protein fragment NS5-a can be combined and connected in any order.

[0015] Instructions are required for:

[0016] The amino acid sequence of protein fragment C-1 is shown in SEQ ID NO. 1, and is specifically as follows:

[0017] MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRAPRKTSERSHP

[0018] RGRRQPIPKARQPEGRTWAQPGYPWPLYGNEGLGWAGWLLSPRGSRPNWGPTDPRRR

[0019] SRNLGKVIDTLTCGFADLMGYIPLVGAPLGGVARALAHGVRVVEDGVNYATGNLPGCS

[0020] FSIFLLALLSCLTIPASA

[0021] The amino acid sequence of the protein fragment NS3-b is shown in SEQ ID NO. 2, and is specifically shown as follows:

[0022] WPAPPGARSLXPCTCGSSDLYLVTRHADVIPVRRRGDGRGSLLSPRPVSYLKGSSGGPLLC

[0023] PSGHAVGIFRAAVCTRGVAKAVDFVPVESMETTMRSPVFTDNSSPPAVPQTFQVAHLHAPT

[0024] GSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGAPITYS

[0025] TYGKFLADGGCSGGAYDIIICDECHSTDSTSILGIGTVLDQAETAGARLVVLATATPPGSVT

[0026] VPHPNIEEVALSNTGEIPFYGKAIPIETIKGGRHLIFCHSKKKCDELAAKLSGLGLNAVAYYR

[0027] GLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTVPQDA

[0028] VSRSQRRGRTGRGRRGIYRFVTPGERPSGMFDSSVLCECYDAGCAWYELTPAETSVRLRA

[0029] YLNTP

[0030] The amino acid sequence of the protein fragment NS5-a is shown in SEQ ID NO. 3, which is specifically as follows: TGPCTPSPAPNYSRALWRVAAEEYVEVTRVGFHYVTGMTTDNVKCPCQVAPAPEFFTELDGVRRHRYAPACKPLLRDEVTFQVLGNQFPVGSQLPCEPEPDVTWTVTSMLTDPSHITAETARRRLARGSPPSLASSASQLSAPSLKATCTTRHDSPDADLIEANLLWRQEMGGNITRVESENKVVILDSFDPLRAEEDEREVSVEAEILRRTRKFPPAIPIWARPDYNPLLESWKDNPYVPPVVHGCPLPPTKAPPIPPPRKKKTVILTESTVSSALAELATKTFGSSESSTVDSGTATAPPNGLFDEGDAGSDAGSYSSMPPLEGEPGDPDLSDGSWSTVSEEAGEDVVCC

[0031] Preferably, the protein fragment combination of the human hepatitis C virus protein includes any one of C-1+NS3-b+NS5-a, C-1+NS5-a+NS3-b, NS3-b+NS5-a+C-1, NS3-b+C-1+NS5-a, NS5-a+NS3-b+C-1 or NS5-a+C-1+NS3-b.

[0032] 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.

[0033] The present invention also 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 stacked on the base plate from bottom to top; wherein the marking pad and / or the detection pad include the human hepatitis C virus protein described above.

[0034] Preferably, the labeling pad contains the human hepatitis C virus protein described above;

[0035] Preferably, the labeling pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker;

[0036] 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.

[0037] Preferably, in any of the above items, the average particle size of the colloidal gold particles is 40 to 100 nm, illustratively including 40 nm, 50 nm, 60 nm, 80 nm, 100 nm and ranges therebetween.

[0038] 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.

[0039] 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.

[0040] Preferably, in any of the above items, the average particle size of the quantum dot microspheres is 100 to 300 nm, exemplarily including 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0041] As a preferred embodiment of the present invention, the mass ratio of the human hepatitis C virus protein to colloidal gold is (0.04-0.32):1, exemplarily 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.

[0042] 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, exemplarily including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0043] 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, exemplarily including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0044] 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, exemplarily including 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0045] 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 goat anti-chicken IgY antibody.

[0046] 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.

[0047] Preferably, any of the above items is coated with 0.5-5 mg / mL of goat anti-chicken IgY antibody, exemplified by 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.

[0048] Preferably, any of the above items is that the diluent for the human hepatitis C virus protein and the goat 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, or a range thereof; 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, or a range thereof.

[0049] 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.

[0050] Preferably, the test pad is provided with a test line and a quality control line; the test line is coated with the human hepatitis C virus protein; the quality control line is coated with goat anti-chicken IgY; the labeling pad contains the same human hepatitis C virus protein, the human hepatitis C virus protein on the labeling pad is labeled with a label; the labeling pad further contains chicken IgY antibody, and the chicken IgY antibody on the labeling pad is labeled with a label.

[0051] The application further provides a shell comprising the human hepatitis C virus antibody test strip according to any one of the above.

[0052] Preferably, the shell comprises an upper cover and a lower cover which are detachably connected.

[0053] Preferably, the upper cover is provided with a viewing window and a sample addition hole.

[0054] Preferably, the lower cover is provided with a test card loading area.

[0055] Compared with the prior art, the application has the following beneficial effects:

[0056] The human hepatitis C virus protein provided by the application has good specificity, high sensitivity, high stability, and high affinity with human hepatitis C virus antibody, and can be used for preparing a product for detecting human hepatitis C virus antibody.

[0057] The human hepatitis C virus antibody test kit provided by the application has the advantages of simple operation, rapid reaction, high sensitivity, high specificity, suitability for on-site rapid detection, economy and practicality, and the like, and has important significance for early diagnosis of HCV infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the curative effect of anti-HCV drugs. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The upper cover of the human hepatitis C virus antibody test kit in Example 1 of the application;

[0059] Figure 2 The lower cover of the human hepatitis C virus antibody test kit in Example 1 of the application;

[0060] Figure 3 The structural diagram of the human hepatitis C virus antibody test strip in Example 1 of the application;

[0061] Figure 4 The test result display diagram in Example 2 of the application;

[0062] Icons: 1-Observation window; 2-Sample loading hole; 3-Test card loading area; 4-Base plate; 5-Test pad; 6-Sample suction pad; 7-Marking pad; 8-Sample pad; 9-Quality control line; 10-Test line. DETAILED DESCRIPTION

[0063] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0064] The embodiment of the present invention provides a human hepatitis C virus protein, comprising protein fragment C-1, protein fragment NS3-b and protein fragment NS5-a;

[0065] The amino acid sequence of protein fragment C-1 is shown in SEQ ID NO.1;

[0066] The amino acid sequence of the protein fragment NS3-b is shown in SEQ ID NO. 2;

[0067] The amino acid sequence of the protein fragment NS5-a is shown in SEQ ID NO. 3;

[0068] The protein fragment C-1, the protein fragment NS3-b and the protein fragment NS5-a can be combined and connected in any order.

[0069] In some embodiments, the protein fragments of human hepatitis C virus protein are combined in any one of the following combinations:

[0070] C-1+NS3-b+NS5-a;

[0071] C-1+NS5-a+NS3-b;

[0072] NS3-b+NS5-a+C-1;

[0073] NS3-b+C-1+NS5-a;

[0074] NS5-a+NS3-b+C-1;

[0075] NS5-a+C-1+NS3-b.

[0076] An 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 stacked on the base plate from top to bottom.

[0077] In some embodiments, the labeling 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.

[0078] 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 economy and practicality. It 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.

[0079] 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;

[0080] 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;

[0081] In some embodiments, the particle size of the colored microspheres, time-resolved fluorescent microspheres, and quantum dot microspheres can be, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm;

[0082] 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.

[0083] In some embodiments, the sensitivity of the test strip can be increased by adjusting the size and amount of the marker particles.

[0084] In some embodiments, a test line (T line) and a quality control line (C line) are provided on the test pad;

[0085] 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;

[0086] 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;

[0087] In some embodiments, the diluent for human hepatitis C virus protein and goat anti-chicken IgY antibody is a trehalose-containing PB buffer, for example, but not limited to 10 mM, 20 mM, 30 mM, 40 mM, or 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, or 1.0 g of trehalose.

[0088] 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.

[0089] The test strip provided by the present invention is based on the principle of antigen-antibody reaction. After the human hepatitis C virus monoclonal protein fragment is labeled 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 a detection pad (such as an NC membrane). Based on the principle of antigen-antibody reaction, it can be detected by the naked eye or a supporting instrument within the detection time. If human hepatitis C 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 are no human hepatitis C virus antibodies 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.

[0090] 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, helping medical personnel to obtain 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.

[0091] An 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 housing, wherein the human hepatitis C virus antibody detection test strip is arranged inside the housing.

[0092] 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.

[0093] In some embodiments, the housing includes an upper cover and a lower cover that are detachably connected;

[0094] The upper cover is provided with an observation window and a sample addition hole, and the lower cover is provided with a test card loading area.

[0095] 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, for observing 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.

[0096] In some embodiments, the test sample of the kit is added:

[0097] 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, with each 100 mL of the diluent containing 0.5 g of trehalose.

[0098] Example 1

[0099] 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 includes a base plate 4 and a sample pad 8, a marking pad 7, a detection pad 5 and a sample suction pad 6 stacked on the base plate in sequence; a detection line 10 and a quality control line 9 are set on the detection pad.

[0100] Example 2

[0101] This example provides a colloidal gold-labeled protein fragment preparation kit for detecting human hepatitis C virus antibodies. The kit structure is shown in Example 1:

[0102] 1 Main Materials

[0103] 1.1 Protein fragments: Human hepatitis C virus protein, used for labeling and test 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;

[0104] 1.2 Nitrocellulose membrane: NC membrane is a product of Sartorius;

[0105] 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.

[0106] 2 Methods

[0107] 2.1 Preparation of colloidal gold labeling pad:

[0108] The steps for preparing the colloidal gold labeling pad are as follows:

[0109] (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;

[0110] (2) Add 25 μg of human hepatitis C virus protein and 5 μg of chicken IgY antibody, with a total protein to colloidal gold particle mass ratio of 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;

[0111] (3) Centrifuge at 12000 rpm for 15 min and discard the supernatant;

[0112] (4) Add 100 μL of colloidal gold solution;

[0113] (5) The above concentrate was diluted in a ratio of 1:7, sprayed with gold, and placed in a drying oven at 37°C to dry for 2 hours.

[0114] 2.2NC membrane coating:

[0115] 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, a film sprayer was used to draw the test line T and the quality control line C on the nitrocellulose membrane. After coating, the NC membrane was dried in an oven at 37°C for 24 h before use.

[0116] 2.3 Assembly of the kit:

[0117] In a drying room, the coated nitrocellulose membrane is placed in the middle of a plastic support plate and pasted. A marker colloidal gold pad is overlapped on one side of the T line of the nitrocellulose membrane (1 / 3 of the colloidal gold pad is overlapped), and a sample pad is overlapped on the other side of the colloidal gold pad (1 / 5 of the colloidal gold pad is overlapped); a sample suction pad is overlapped on one side of the C line of the nitrocellulose membrane (1 / 10 of the sample suction pad is overlapped); and a cutting machine is used to cut the pasted plastic plate into test strips of a certain width, which are then loaded into a test card to form a human hepatitis C virus antibody detection kit.

[0118] 2.4 Detection:

[0119] Step 1: Take out the test kit and the sample to be tested and equilibrate to room temperature;

[0120] Step 2. Open the sealed aluminum foil bag, take out the test kit and place it flat on the table;

[0121] Step 3: Figure 1Add 2 drops of sample (about 80-100 μL) to the sample well;

[0122] Step 4, the timer is timed, and the result is read after 10 min; note that if the sample is not side-chromatographed or percolated within 1 min after being added, it may be due to the sample being too viscous, and the sample needs to be pretreated with normal saline.

[0123] 3Results

[0124] Under the action of side-chromatography, when there is human hepatitis C virus antibody in the sample, the detection line will develop color, and the quality control line will develop color (a) in Fig. 1; Figure 4 When there is no human hepatitis C virus antibody in the sample, the detection line does not develop color, and the quality control line develops color (b) in Fig. 1; Figure 4 After being added, the quality control line does not develop color, and whether the detection line develops color or not, the result is invalid (c, d) in Fig. 1. Figure 4 Figure 4

[0125] 4The kit provided in Example 2 is used to detect HCV patient blood samples, and the results are consistent with those shown in 3, indicating the effectiveness of the human hepatitis C virus protein provided in the application in the detection of HCV antibodies.

[0126] Example 3

[0127] The present embodiment provides a time-resolved fluorescent microsphere-labeled protein fragment preparation human hepatitis C virus antibody detection kit; the human hepatitis C virus monoclonal protein provided in the present embodiment is used to prepare a human hepatitis C virus antibody detection kit, and the kit 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 time-resolved fluorescent microsphere-labeled human hepatitis C virus protein on 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.

[0128] 1Kit preparation operation process:

[0129] The time-resolved fluorescent microspheres are marked on the surface of the human hepatitis C virus protein prepared by the method of the present application. The specific implementation is as follows:

[0130] ​​Time-resolved fluorescent microsphere protein fragment labeling: 1 mL of 1% carboxyl time-resolved fluorescent microspheres was added to 9 mL of MES buffer, followed by 25 μL of EDC solution (10 mg / mL) and 25 μL of NHS solution (10 mg / mL). The mixture was shaken at room temperature for 30 minutes, and the precipitate was collected by centrifugation. After adding HEPES rehydration solution and ultrasonically dispersing the mixture, 1 mL of 1 mg / mL human hepatitis C virus protein was added. The mixture was shaken at room temperature for 120 minutes, and the precipitate was collected by centrifugation. 1 mL of blocking buffer was then added, and the mixture was shaken at room temperature for 120 minutes. The microsphere precipitate was collected by centrifugation and reconstituted with rehydration solution.

[0131] Preparation of fluorescent pad: The labeled time-resolved fluorescent microspheres were diluted with microsphere reconstitution solution and sprayed with the fluorescent pad using a gold sprayer 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.

[0132] NC membrane coated CT line: T line uses human hepatitis C virus protein with a concentration of 1.5 mg / mL, and C line uses goat anti-chicken IgY antibody with a concentration of 1 mg / mL. 1 μL / cm line is drawn, and then placed in 37°C low humidity (<30%) for drying for 24 hours.

[0133] Sample pad treatment: The sample pad treatment solution consists of buffer salt, sustained-release agent, cosolvent, blocking agent, etc. The specific formula is 20mM Tris buffer, and each 100mL Tris buffer contains 1g BSA, 0.5g Tween 20, and 2g sucrose. 2 Treat with 1 mL of sample treatment solution. After evenly treating, place in a 37°C low humidity (<30%) oven dry for 2 hours.

[0134] Test strip assembly: Paste the 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 to 2 mm each, and the sample pad pressing the fluorescent pad 1 to 2 mm; after assembly, cut the test strip into a width of 4±0.4 mm and install the card shell; put the card shell and desiccant into an aluminum foil bag and seal it; label and box it to obtain the finished test card.

[0135] 2 Kit detection process

[0136] Place the test card on a clean, flat surface, draw 80-100 μL of the treated sample and drop it into the sample addition end of the test card. Set up a PBS control group at the same time.

[0137] 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 testing, the titer corresponding to the human hepatitis C virus antibody will be displayed.

[0138] Example 4

[0139] This embodiment provides a kit for detecting human hepatitis C virus antibodies using colored microspheres labeled with protein fragments. The human hepatitis C virus monoclonal protein provided in this embodiment is used to prepare a test strip for detecting human hepatitis C virus antibodies. The test strip has human hepatitis C virus protein labeled with colored microspheres embedded on a labeling pad, and the detection line is coated with human hepatitis C virus protein. A double-protein sandwich method is used to qualitatively detect human hepatitis C virus antibodies in a sample.

[0140] 1. Preparation of test strips:

[0141] 1.1 Colored microspheres labeling protein fragments

[0142] (1) Adjust the pH of 100 nm colored microspheres to 8.0 with 0.1 mol / L K2CO3;

[0143] (2) Human hepatitis C virus protein-labeled microspheres: Take 1 mL of the pH-adjusted solution, add 30 μg of human hepatitis C virus protein, and the mass ratio of total protein to colored microspheres is 0.3:1. After reacting at room temperature for 1 hour, centrifuge and discard the supernatant;

[0144] Chicken IgY protein fragment labeled microspheres: Add 5 μg of chicken IgY to 1 mL of the pH-adjusted solution (mass ratio of protein fragment to colored microspheres is 0.05:1). Incubate at room temperature for 1 hour, then centrifuge and discard the supernatant.

[0145] (3) Add 1 mL of 20% BSA to block for 2 h, then centrifuge and discard the supernatant;

[0146] (4) After the above microspheres were redissolved 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%.

[0147] 1.2 Preparation of labeling pad

[0148] The diluted solution of the labeled colored microglobulin fragment complex was sprayed onto the marking pad using a gold film sprayer at a spraying rate of 7.5 μL / cm and a spraying interval of 6 mm. After spraying, the mixture was dried at 37° C. with low humidity (<30%) for 2 h.

[0149] 1.3 Sample pad preparation

[0150] The sample pad treatment solution consists 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 2 Treat with 1 mL of sample treatment solution. After evenly treating, place in a 37°C low humidity (<30%) oven dry for 2 hours.

[0151] 1.4C / T line coating

[0152] The T line uses a concentration of 1.5 mg / mL human hepatitis C virus protein (the C line uses a concentration of 2 mg / mL goat anti-chicken IgY protein fragment), 1 μL / cm is used for drawing lines, and after completion, it is placed in a 37°C low humidity (<30%) drying for 24 hours.

[0153] 1.5 Test strip assembly

[0154] The sample suction pad, NC film, marking pad and sample pad are sequentially pasted on the PVC board. The sample suction pad and marking pad are each pressed 1 to 2 mm against the NC film, and the sample pad is pressed 1 to 2 mm against the marking pad. 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 placed together in an aluminum foil bag. The finished test kit is obtained by affixing a label and packaging.

[0155] 2. Detection

[0156] Place the test card on a clean, flat surface, draw 80-100 μL of the treated sample and drop it into the sample addition end of the test card, and set up a PBS control group; observe the test card window after 10 minutes. If there are 2 lines in the window, it is positive, and only one C line is negative.

[0157] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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 C-1, the amino acid sequence of which is shown in SEQ ID NO.1; Protein fragment NS3-b, 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 C-1, protein fragment NS3-b and protein fragment NS5-a can be combined and connected in any order.

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

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 to human hepatitis C virus, characterized in that: The human hepatitis C virus antibody 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 from bottom to top; Wherein, the labeling pad and / or 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 labeling pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker; The marker includes any one of colloidal gold, colored 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 comprise 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 to 7.

9. The detection kit according to claim 8, characterized in that The housing comprises an upper cover and a lower cover that are detachably connected; 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 installation area.

10. Use of the detection kit according to claim 8 or 9 in detecting human hepatitis C virus.

Images