Hepatitis C antibody detection antigen composition as well as application, kit and detection method thereof

Abstract

The invention provides a hepatitis C antibody detection antigen composition and application thereof, a kit and a detection method, and relates to the technical field of antibody detection, the hepatitis C antibody detection antigen composition comprises a recombinant antigen for marking and a recombinant antigen for coating, the amino acid sequence of the recombinant antigen for marking comprises Trx and hepatitis C Core regions, NS3 protein, NS4b protein dominant epitopes and a His tag, and the full-length sequence is as shown in SEQ ID NO. 1. According to the recombinant antigen for marking, the NS3 protein is subjected to amino acid mutation to mutate seven cysteines (C) into alanine (A), aggregation and precipitation caused by disulfide bonds of the recombinant protein are reduced, and the protein stability is improved. When colloidal gold is labeled by the antigen, a secondary indirect labeling method is adopted, so that the exposure of antigen epitopes is facilitated, the reaction sensitivity can be improved, and the leakage detection of antibodies is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody detection technology, in particular to a hepatitis C antibody detection antigen composition, application, kit and detection method thereof. BACKGROUND

[0002] Hepatitis C, commonly known as hepatitis C, is an infectious disease caused by hepatitis C virus infection, with a high incidence rate, and can be transmitted through close contact, blood transfusion, needle injection and other routes. Hepatitis C is insidious, and early symptoms are not obvious. As the disease progresses, cirrhosis, ascites, liver fibrosis and liver cancer may occur, posing a great threat to the safety of patients. Hepatitis C antibody is commonly used in the diagnosis of hepatitis C, especially colloidal gold antibody detection.

[0003] The methodology of colloidal gold method for detecting hepatitis C antibody is mainly based on the combination of immunochromatography and colloidal gold labeling technology. The core is to realize detection through antigen-antibody specific binding reaction and color development characteristics of colloidal gold. The existing colloidal gold double antigen sandwich method hepatitis C antibody detection kit, although it improves the specificity of the reaction compared with colloidal gold indirect method, but due to the non-specific adsorption of colloidal gold particles and NC membrane to proteins and components in serum other than HCV antibodies, or due to the non-specific reaction of impurities in the antigens used for coating or labeling, false positives may occur occasionally. Moreover, due to the larger diameter of colloidal gold particles (usually 10-40 nm), if directly coupled to the surface of the detection antigen, the steric hindrance may hinder the combination of antigen and antibody (especially when the antibody binding site is located in the concave area of the antigen surface), which reduces the detection sensitivity and may miss the detection of window period samples. SUMMARY

[0004] The purpose of the present application is to provide a hepatitis C antibody detection antigen composition, application, kit and detection method thereof to improve the above problems. In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0005] In a first aspect, the present application provides a hepatitis C antibody detection antigen composition, comprising a labeled recombinant antigen and a coated recombinant antigen, the amino acid sequence of the labeled recombinant antigen is shown as SEQ ID NO. 1.

[0006] Optionally, the amino acid sequence of the coated recombinant antigen is shown as SEQ ID NO. 2.

[0007] The present application also provides the application of the hepatitis C antibody detection antigen composition described above, which is the application in the preparation of products for detecting hepatitis C antibody.

[0008] Optionally, the product comprises a colloidal gold detection kit or an immunofluorescence chromatography or a latex microsphere immunochromatography kit.

[0009] The application also provides a colloidal gold method for detecting hepatitis C antibody based on secondary indirect labeling, which comprises the hepatitis C antibody detection antigen composition described in the application, and further comprises colloidal gold particles, a polyclonal antibody of a mouse anti-Trx antibody and a mouse anti-Trx antibody.

[0010] Optionally, the polyclonal antibody of the mouse anti-Trx antibody comprises a goat polyclonal antibody or a rabbit polyclonal antibody.

[0011] Optionally, the biological material coated with the recombinant antigen comprises any one of the following:

[0012] (a) a nucleic acid molecule encoding the recombinant antigen as described in claim 2;

[0013] (b) an expression cassette comprising the nucleic acid molecule in (a);

[0014] (c) a recombinant vector comprising the nucleic acid molecule in (a) or the expression cassette in (b);

[0015] (d) a recombinant prokaryotic cell comprising the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c);

[0016] (e) a recombinant eukaryotic cell comprising the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

[0017] Optionally, the amino acid sequence of the recombinant antigen comprises a Trx and dominant epitopes of a hepatitis C Core region, an NS3 protein, an NS4b protein and a His tag.

[0018] The NS3 protein uses the NS3 protein sequence of the 1a subtype HCV strain, and at least one fragment of the NS3 protein is subjected to amino acid mutation, in which cysteine is mutated into alanine.

[0019] Optionally, the amino acid sequence of the recombinant antigen comprises an N-terminal His tag, an NS3 fragment, a Core fragment, an NS4B dominant epitope and a C-terminal His tag.

[0020] The recombinant antigen is coated by connecting the sequence fragments in series, or the protein sequence fragments containing the respective epitopes are finally mixed for coating.

[0021] The application also provides a hepatitis C antibody detection method based on secondary indirect labeling of colloidal gold, which uses the kit described in the application and comprises the following steps:

[0022] The colloidal gold particles are labeled with the polyclonal antibody of the mouse anti-Trx antibody to obtain a first label.

[0023] The mouse anti-Trx antibody is labeled with a first label to obtain a second label;

[0024] The labeled recombinant antigen with a Trx tag is labeled with a second label to obtain a third label;

[0025] The sample to be tested is added to the sample pad to form a first complex with the third label, and moves to the detection line by chromatography, and is captured by the coated recombinant antigen at the detection line to form a second complex, and the detection result is obtained according to the color displayed.

[0026] The beneficial effects of the present application are:

[0027] The labeled recombinant antigen used in the present application is a fusion protein of Trx and the dominant epitopes of Hepatitis C Core region, NS3 protein, NS4b protein and His tag, and a hydrophilic connecting peptide is added between the epitopes, which is beneficial to the exposure of antigen epitopes, can improve the reaction sensitivity, and reduce the missed detection of antibodies. Amino acid mutation is performed on the NS3 protein to mutate 7 cysteines (C) to alanine (A), which reduces the aggregation and precipitation caused by disulfide bonds of the recombinant protein, and improves the stability of the protein. When the antigen is labeled with colloidal gold, a secondary indirect labeling method is adopted, which is beneficial to the exposure of antigen epitopes, can improve the reaction sensitivity, and reduce the missed detection of antibodies.

[0028] Other features and advantages of the present application will be described in the following description, and some will become apparent from the description, or will be understood by those skilled in the art through implementation of the embodiments of the present application. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some embodiments of the present application, but not all embodiments.

[0031] The antibody detection kit usually uses a recombinant protein or a synthetic peptide as a coated antigen or a labeled antigen to simulate the antigen epitopes on the virus protein to capture specific antibodies in the patient's serum. Commonly used proteins include:

[0032] Core protein:

[0033] Diagnostic significance: Anti-core antibody is usually one of the earliest appearing antibodies, and is also one of the antibodies with the longest persistence time after infection. It is a key component in the antibody detection kit.

[0034] NS3 protein:

[0035] Location and function: non-structural protein 3, with protease and helicase activity. The C-terminal portion (about 27 kDa) is often used as an antigen.

[0036] Diagnostic significance: Anti-NS3 antibodies usually appear in the early stages of infection and are one of the important markers for diagnosis. It has high immunogenicity.

[0037] NS4 protein:

[0038] Location and function: non-structural protein 4, often subdivided into NS4A and NS4B. NS4A is a cofactor for NS3 protease, and NS4B is involved in the formation of the viral replication complex.

[0039] Diagnostic significance: Anti-NS4 antibodies usually appear slightly later than anti-core and anti-NS3 antibodies. Recombinant proteins or specific synthetic peptides (such as c100-3, derived from NS4) are often used as antigens. It is an important component of antibody detection, which helps to improve the specificity of detection.

[0040] NS5 protein:

[0041] Location and function: non-structural protein 5, is the RNA-dependent RNA polymerase of the virus, responsible for viral RNA replication. It is often subdivided into NS5A and NS5B.

[0042] Diagnostic significance: Anti-NS5 antibodies appear relatively late. NS5B (polymerase) or specific regions thereof (such as c200, c33c antigens of NS5) are often used as antigens. It is an important component of third-generation and later kits.

[0043] Modern anti-HCV detection kits (especially third-generation, fourth-generation reagents) do not use only a single protein, but a combination of multiple antigens derived from core protein, NS3, NS4 and NS5 (recombinant proteins or synthetic peptides). This multi-antigen combination significantly improves the sensitivity of detection (reduces missed detection, especially in the early stages of infection or immunosuppressed patients) and specificity (reduces false positives).

[0044] Example 1

[0045] This example provides a hepatitis C antibody detection antigen composition, which comprises a labeling recombinant antigen and a coating recombinant antigen, the amino acid sequence of the labeling recombinant antigen is shown in SEQ ID NO. 1; the labeling recombinant antigen is marked as C01, and the protein sequence is:

[0046] Trx + hepatitis C core region + NS4B protein dominant epitope + NS3 protein (1a subtype, 7C→7A) + hydrophilic sequence + His;

[0047] Trx: The commonly used hydrophilic protein tag thioredoxin (Trx protein) was selected as a tag protein that is conducive to the expression of recombinant protein supernatant and indirect labeling.

[0048] Core: The hepatitis C core protein (Core) was subjected to antigen epitope analysis, the region with concentrated antigen epitopes was selected, non-specific binding sites were removed, and 79 aa were selected as the Core epitope.

[0049] Core antigen epitope amino acid sequence:

[0050] STNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPG;

[0051] NS4B: The hepatitis C NS4B protein was subjected to antigen epitope analysis, the region with concentrated antigen epitopes was selected, and amino acid mutations were performed in combination with other subtypes, and 43 aa were selected as the NS4B epitope.

[0052] NS4B antigen epitope amino acid sequence:

[0053] PGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSS;

[0054] NS3 (1a subtype, 7C→7A): The NS3 protein uses the NS3 protein sequence of the 1a subtype HCV strain, and at least one fragment of the NS3 protein is subjected to amino acid mutation, and cysteine is mutated into alanine; specifically, in this embodiment, the hepatitis C NS3 protein was subjected to antigen epitope analysis, the region with concentrated antigen epitopes was selected, and amino acid mutations were performed in combination with other subtypes, and 264 aa were selected as the NS3 epitope. The NS3 protein is subjected to amino acid mutation to mutate 7 cysteines (C) into alanine (A), reduce the aggregation and precipitation caused by disulfide bonds of the recombinant protein, and improve the protein stability.

[0055] NS3 (1a subtype, 7C→7A) antigen epitope amino acid sequence: LETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGASGGAYDI I IADEAHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFAHSKKKADELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVSTDALMTGFTGDFDSVIDANTAVTQTVDFS;

[0056] Hydrophilic sequence: Random hydrophilic amino acids increase the hydrophilicity of the protein, and are conducive to the exposure of the His tag and the binding to the affinity chromatography filler, improving the protein purification efficiency.

[0057] Hydrophilic amino acid sequence:

[0058] GSTQQNTGGPQTTSGGQQNGYPTSQ;

[0059] The multi-antigen combination significantly improves the detection sensitivity and reduces missed detection. The order of each epitope in the C01 recombinant protein is crucial, and the Trx protein tag is located at the N-terminus, which is conducive to its binding with the Trx monoclonal antibody, thereby forming a correct spatial arrangement on the surface of the colloidal gold particles. The NS3 protein is located at the C-terminus of the protein, which is conducive to the exposure of the antigen epitope and improves the activity of the epitope, thereby detecting the NS3 antibody with the highest content in the general positive serum. The Core protein epitope and the NS4B protein epitope are located in the middle position of the antigen amino acid sequence, which plays a supplementary antigen epitope role.

[0060] SEQ ID NO. 1:

[0061] MSDKI IHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAK

[0062] LNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAMAGGSTN

[0063] PKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRR

[0064] QPIPKARRPEGRTWAQPGGGTSPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAAR

[0065] VTQILSSGGEFLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQ

[0066] GYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGA

[0067] SGGAYDI I IADEAHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIE

[0068] EVALSTTGEIPFYGKAIPLEVIKGGRHLIFAHSKKKADELAAKLVALGINAVAYYRGL

[0069] DVSVIPTSGDVVVVSTDALMTGFTGDFDSVIDANTAVTQTVDFSGSTQQNTGGPQTTSGGQQNGYPTSQLEHHHHHH。

[0070] The coating uses the amino acid sequence of the recombinant antigen as shown in SEQ ID NO. 2; the coating with the recombinant antigen is denoted as C02; the C02 protein sequence is: N-terminal His+NS3 fragment+Core fragment+NS4B dominant epitope+C-terminal His;

[0071] His: The His tag with 6 histidines at both ends of the antigen is beneficial for protein purification.

[0072] NS3: Take the hepatitis C NS3 protein for antigen epitope analysis, select the region concentrated with antigen epitopes, and combine other subtypes for amino acid mutation, select 265 aa as the NS3 epitope.

[0073] NS3 antigen white epitope amino acid sequence:

[0074] METTTRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPSVRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDSTTILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIQEVALSNTGEIPFYGKAIPIEAIRGGRHLIFCHSKKKCDELAAKLSSLGLNAVAYYRGLDVSVIPSSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFS;

[0075] Core: The core protein of hepatitis C virus (Core) was subjected to antigen epitope analysis, the region with concentrated antigen epitopes was selected, non-specific binding sites were removed, and 79 aa were selected as the Core epitope.

[0076] NS4B: The NS4B protein of hepatitis C virus was subjected to antigen epitope analysis, the region with concentrated antigen epitopes was selected, and amino acid mutations were made in combination with other subtypes, and 43 aa were selected as the NS4B epitope.

[0077] The order of each epitope in the C02 recombinant protein is crucial, the His tag is located at the N segment and the C terminal, which is conducive to exposure and binding with affinity chromatography filler, and improves the protein purification efficiency; the NS3 protein is close to the N terminal of the protein, which is conducive to the exposure of antigen epitopes and improves the activity of the epitope, so as to detect the NS3 antibody with the highest content in general positive serum. The Core protein epitope and the NS4b protein epitope are located at the C terminal position of the antigen amino acid sequence, which plays a role in supplementing antigen epitopes.

[0078] SEQ ID NO. 2:

[0079] MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMETTTRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPSVRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDI IICDECHSTDSTTILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIQEVALSNTGEIPFYGKAIPIEAIRGGRHLIFCHSKKKCDELAAKLSSLGLNAVAYYRGLDVSVIPSSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSASGGSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGGGTSPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSSGGLEHHHHHH.

[0080] The coating antigen C02 can also be obtained by connecting each sequence fragment to express each epitope, or finally mixed coating by respectively expressing protein sequence fragments containing each epitope.

[0081] Embodiment 2

[0082] The embodiment provides the application of the hepatitis C antibody detection antigen composition of embodiment 1, which is the application in the preparation of the product for detecting hepatitis C antibody.

[0083] As an optional implementation, the product includes a colloidal gold detection kit or an immunofluorescence chromatography or a latex microsphere immunochromatography kit.

[0084] Embodiment 3

[0085] The embodiment is a hepatitis C antibody detection kit based on a secondary indirect labeling colloidal gold method, which includes the hepatitis C antibody detection antigen composition of embodiment 1, and further includes colloidal gold particles, a polyclonal antibody of a mouse anti-Trx antibody and a mouse anti-Trx antibody.

[0086] Optionally, the polyclonal antibody of the mouse anti-Trx antibody includes a goat polyclonal antibody or a rabbit polyclonal antibody.

[0087] The synthesis method of the labeling recombinant antigen includes:

[0088] The Core antigen epitope amino acid sequence, the NS4b antigen epitope amino acid sequence, the NS3 (1a subtype, 7C→7A) antigen epitope amino acid sequence and the hydrophilic amino acid sequence are connected in series, and an NcoI enzyme cutting site is added at the N terminal and an XhoI enzyme cutting site is added at the C terminal to obtain a synthetic gene C01-pUC;

[0089] The C01-pUC is connected to the pET30a-Trx expression vector by NcoI / XhoI double enzyme cutting to construct a recombinant plasmid;

[0090] The recombinant plasmid is transformed into an Escherichia coli cloning bacteria, a single colony is picked for colony PCR, positive single colonies are screened and plasmids are extracted;

[0091] The extracted plasmid is used to transform an Escherichia coli expression bacteria, and the Escherichia coli is induced for expression to obtain an expressed Escherichia coli slurry;

[0092] The expressed Escherichia coli slurry is resuspended and ultrasonically broken, after centrifugal precipitation, the supernatant is taken, and purification treatment is performed to obtain a purified recombinant antigen. The specific operation is as follows:

[0093] S110, C01 gene synthesis;

[0094] The Core antigen epitope amino acid sequence, the NS4b antigen epitope amino acid sequence, the NS3 (1a subtype) antigen epitope amino acid sequence and the hydrophilic amino acid sequence are connected in series, and an NcoI enzyme cutting site is added at the N terminal and an XhoI enzyme cutting site is added at the C terminal to obtain a synthetic gene C01-pUC;

[0095] S120, double enzyme cutting;

[0096] The expression vector adopts pET30a-Trx, which has a Trx sequence at the N terminal, so that the final expression protein has a Trx tag protein at the N terminal.

[0097] The C01-pUC plasmid is double enzyme cut by NcoI and XhoI, and the pET30a-Trx is double enzyme cut by NcoI and XhoI as a skeleton. The enzyme cutting system is according to Table 1, and the enzyme cutting is performed at 30°C for 30 min. The restriction enzyme used in this embodiment is purchased from Beijing Quanshi Gold Biotechnology Co., Ltd., which is a Flycut series fast cutting enzyme.

[0098] Table 1 double enzyme cutting system

[0099]

[0100]

[0101] S130, constructing a recombinant plasmid;

[0102] The enzyme cutting system was subjected to agarose gel electrophoresis and DNA purification recovery, and the gel recovery product was connected with 15-18 hours of connection system to obtain the connection product;

[0103] The gel recovery product was connected with the connection system shown in Table 2 for 16 hours, and the DNA ligase Solution I was purchased from TAKARA company.

[0104] Table 2 Connection system

[0105]

[0106] The connection product was transformed into E. coli Trans5α competent cells to obtain a recombinant plasmid. The E. coli Trans5α competent cells were purchased from Beijing Zoman Biotechnology Co., Ltd.

[0107] The constructed amino acid sequence is:

[0108] MSDKI IHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAMAGGSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGGGTSPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSSGGEFLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGASGGAYDI I IADEAHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFAHSKKKADELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVSTDALMTGFTGDFDSVIDANTAVTQTVDFSGSTQQNTGGPQTTSGGQQNGYPTSQLEHHHHHH*.

[0109] S140, positive spot screening and plasmid extraction;

[0110] The plate growth of the transformed clone bacteria was connected with white E. coli single spot, and the plate single spot was picked for colony PCR. The PCR primers were pET-fw: CACTATAGGGGAATTGTGAGCGGATAAC; and pET-rv: CTCAGCTTCCTTTCGGGCTTTGTTAG. The colony PCR reaction system components are shown in Table 3, and the PCR reaction program is as follows: pre-denaturation: 94°C for 5 min; cycle: 94°C for 30 s, 60°C for 30 s, 72°C for 150 s, for a total of 30 cycles; extension: 72°C for 5 min. The C01 positive spot colony PCR product fragment size is theoretically 1846 bp.

[0111] Table 3 Colony PCR reaction system

[0112] Template DNA 0.5 μl pET-fw (20 μM) 1 μl pET-rv (20 μM) 1 μl 2 x Taq Mix 10 μl ddH2O 7.5 μl Total volume 20 μl

[0113] The positive single spot screened was shaken overnight, and the plasmid was extracted the next day. The plasmid extraction kit was an OMEGA plasmid extraction kit. The obtained C01 plasmid was identified as positive by NdeI and XhoI double enzyme digestion, and was sent to the Beijing Sequencing Department of Shengong Biological Engineering Co., Ltd. for sequencing. The sequence was completely consistent with the design, and could be used for transformation of E. coli expression bacteria.

[0114] S150, C01 plasmid transformed E. coli expression bacteria;

[0115] 1 μl of C01 plasmid was added to 50 μl of E. coli BL21 (DE3) competent cells, placed on ice for 30 min, 42°C heat shock for 90 s, placed on ice for 2 min, added with 500 μl of LB medium (without antibiotics), and cultured at 37°C, 200 rpm for 1 hour. 60 μl of culture solution was used to coat the plate (containing kanamycin sulfate 50 μg / ml). The E. coli BL21 (DE3) competent cells used in this example were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0116] The successfully transformed expression bacteria were stored in sterile glycerol to obtain glycerol bacteria;

[0117] The glycerol bacteria of the expression bacteria were inoculated into 300 ml of culture medium (containing kanamycin sulfate 50 μg / ml), and cultured at 37°C, 200 rpm in a shaking flask until the OD 600 = 0.8, and IPTG was added to induce expression (the final concentration of IPTG was 0.7 mM), and the culture was continued for 4 h. The bacterial solution was centrifuged at 6500 rpm for 10 min, and the supernatant was discarded, and the bacterial slurry was collected.

[0118] S160, ultrasonic disruption of E. coli;

[0119] The expressed E. coli slurry was added to 10 mM Tris-HCl (pH 8.0) at a ratio of 1 g slurry to 40 ml buffer, the bacterial cells were resuspended, and the bacterial cells were broken by ultrasonic wave on an ultrasonic machine. 20 ml of bacterial solution was treated at 200 w for 2 s and stopped for 2 s for 20 min. The bacterial solution was completely clear after ultrasonic wave treatment, and the precipitate was very little after centrifugation at 11000 rpm for 10 min. 30 μl of the supernatant was taken as a sample, and SDS-PAGE was performed to detect whether the target protein was expressed in the supernatant. The C01 protein was expressed in the supernatant, and the whole supernatant was used for subsequent experiments.

[0120] S170, purification of C01 protein;

[0121] The recombinant antigen was purified by using a Ni-NTA affinity column. The operation was performed according to the instructions of the Ni-NTA SefinoseTM Resin Kit, and generally included the following steps: the E. coli supernatant was added to the affinity column, the target protein was combined with the Ni ion, and the target protein remained in the affinity column, and the impurities flowed through; 20 mM Tris-HCl (pH 8.0) + 150 mM NaCl + 50 mM imidazole were passed through the column to remove the non-specifically adsorbed impurities; and 20 mM Tris-HCl (pH 8.0) + 150 mM NaCl + 200 mM imidazole was used to elute the target protein, and the protein solution, i.e. the C01 protein, was obtained. The target protein after purification was subjected to SDS-PAGE to detect the purity of the protein, and the purity of the protein was ≥80%.

[0122] S200, synthesis of coated recombinant antigen

[0123] The synthesis method of the coated recombinant antigen is basically the same as the synthesis method of the labeled recombinant antigen, and the difference lies in that:

[0124] The synthesis of the gene fragment of the coated recombinant antigen includes: the Core antigen epitope amino acid sequence, the NS4b antigen epitope amino acid sequence, and the NS3 antigen epitope amino acid sequence are connected in series, and a NcoI enzyme cutting site is added at the N terminal and a XhoI enzyme cutting site is added at the C terminal, to obtain a synthetic gene C02-pUC.

[0125] The expression vector thereof is pET30a, and His tags are provided at the N terminal and the C terminal of the expression vector, so that the N terminal and the C terminal of the final expression protein are His tags.

[0126] The embodiment also provides a preparation method of the mouse anti-Trx antibody:

[0127] S310, immunization

[0128] The Trx protein was mixed with an equal volume of Freund's adjuvant to prepare a water-in-oil state.

[0129] The mice for monoclonal antibody preparation are derived from BALB / c mice, and the mice are 8-12 weeks old when initially immunized, and the mice are immunized by multiple subcutaneous injections on the back, and the mice are immunized for 3-4 times. After the last immunization, the mice are boosted by intraperitoneal injection of 50 μg of the antigen, and the spleen is removed three days later for fusion.

[0130] S320, fusion;

[0131] The mice are sacrificed, and the spleen cells are taken to prepare a cell suspension. 1×10 8 The spleen cells are fused with 1×10 7 The myeloma cells SP2 / 0 are fused. The 20% calf serum 1640 HAT medium is dispensed into a 96-well cell culture plate in a CO2 incubator, 100 μl per well, and then the culture plate is placed in a 37°C, 5% CO2 incubator for culture.

[0132] S330, screening;

[0133] 100 μl of the cell supernatant is aspirated into a 96-well plate, and a gold-labeled antibody strip is inserted for detection. The positive polyclone is subcloned. The method for cloning is limited dilution, and the positive cells are screened by multiple subcloning.

[0134] S340, expansion culture;

[0135] When the cell coverage reaches 80%, the cells are expanded to a 110 cm 2 The culture bottle is placed in a 37°C, 5% CO2 incubator for static culture.

[0136] S350, monoclonal antibody preparation;

[0137] The mice are sensitized with paraffin oil, and each mouse is intraperitoneally injected with 0.2 ml. The mice can be used for cell inoculation 7-15 days later. The collected monoclonal cells are injected into the abdominal cavity of the mice at 1 ml per mouse. The growth of the inoculated mice is observed regularly, and the ascites is extracted in time when the abdominal cavity is round and the state is poor. The ascites should be collected within 15 days after the cell injection, and then purified.

[0138] S360, purification;

[0139] S361, ascites pretreatment;

[0140] The collected ascites is slowly added with an equal volume of saturated (NH4)2SO4. After completion, the stirring is continued at room temperature for 2 h, and the mixture is statically placed at 2-8°C for 12-16 h. The mixture is centrifuged, and the supernatant is discarded. The precipitate is dissolved and dialyzed in 10 mmol / L PBS for 24 h, and the solution is replaced every 2 hours. The dialyzed antibody is taken out, centrifuged, and the volume and concentration are determined, and the protein amount is calculated. The antibody is stored in a refrigerator at 2-8°C for standby or further purification.

[0141] S362, CNBr-activated Sepharose 4B affinity chromatography purification;

[0142] Column loading: CNBr-activated Sepharose 4B filler coupled with ligand (Trx protein) was loaded into the chromatography column.

[0143] Detection: connect online UV detector, select 280 nm wavelength, 0.5 A gear.

[0144] Equilibrium: after loading, the CNBr-activated Sepharose 4B chromatography column was fully equilibrated with 20 mmol / L PBS (pH 7.2-pH 7.4). Linear flow rate 60 cm / h was used. After the detector baseline was stable, the sample was injected.

[0145] Sample loading: after equilibrium, the sample was injected at a linear flow rate of 60 cm / h, and the breakthrough liquid was collected. After the injection was completed, the pipeline and residual sample in the chromatography column were washed with 20 mmol / L PBS (pH 7.2-pH 7.4) until the detector baseline was stable.

[0146] Elution: After washing, the chromatography column was eluted with 0.1 mol / L glycine solution (pH 2.6) to elute the antibody sample.

[0147] Collection: collect the target peak.

[0148] Sample treatment: the eluted sample was dialyzed for 24 h with 10 mmol / L PBS buffer (pH 7.2-pH 7.4), and the dialysis liquid was replaced twice, every 2-3 hours.

[0149] Storage: the collected antibody pure product was determined for protein concentration and volume, the protein content was calculated, and the mouse anti-Trx antibody was obtained. Add 0.02% NaN3 for storage.

[0150] The present embodiment also provides a preparation method of sheep polyclonal antibody (A01) of the mouse anti-Trx antibody:

[0151] S410, immunize sheep;

[0152] The immunogen is mouse anti-Trx monoclonal antibody (B01).

[0153] First immunization: use Freund's complete adjuvant, invert the bottle several times to resuspend B01 uniformly. Use a 1000 μl pipette to draw the required volume of Freund's complete adjuvant and antigen and physiological saline into a syringe; if it is a second immunization, use Freund's incomplete adjuvant, and the operation method is consistent with the first immunization (the first immunization is the first immunization, and the subsequent immunizations are the second immunizations, and the immunization interval is 3-4 weeks).

[0154] S420, draw blood;

[0155] Weekly sheep polyclonal small sample blood test, using a disposable blood needle and a disposable vacuum blood collection tube to collect 2-3 ml blood from each sheep, and then put the blood collection tube into the centrifuge at 3000 rpm for 30 min, and then suck out the serum to test the serum titer. If the serum titer is above L5, large sample blood can be collected. If the test result does not meet the requirements, the sheep need to be immunized three to four weeks after the last immunization date, and the immunized sheep need to be tested again the next week.

[0156] Sheep large sample serum centrifugation method: put the collected sheep blood into a one-time centrifuge cup, set the centrifuge speed to 8500 rpm and the temperature to 4°C, and centrifuge for 20 min; after centrifugation, prepare a two-time centrifuge cup, put the serum of two sheep of the same variety into the same two-time centrifuge cup and number them, set the centrifuge speed to 8500 rpm and the temperature to 4°C, and centrifuge for 20 min, and obtain the sheep polyclonal serum.

[0157] S430, purification;

[0158] S431, serum pretreatment;

[0159] Thaw the sheep polyclonal serum at 2-8°C and mix it until it is completely melted. Centrifuge the serum at 4°C at 15000g for 15 min, filter the supernatant, and discard the precipitate. Slowly add saturated ammonium sulfate to the supernatant to a final concentration of 50% (V / V) while stirring, continue stirring for 1-2h, and stand at 2-8°C for 18-20h to ensure that all proteins are precipitated. Centrifuge at 4°C for 15 min, discard the supernatant, and dissolve the precipitate in 10 mmol / L PBS (pH 7.2-pH 7.4) buffer, dialyze for 24h, replace the dialysis liquid 5-6 times, and replace it every 2-3h. Take out the dialyzed antibody, centrifuge at 15000g for 15 min, filter to determine the protein concentration, volume, and calculate the protein amount. Store in the 2-8°C refrigerator for standby or continue purification.

[0160] S432, CNBr-activated Sepharose 4B affinity chromatography purification;

[0161] Column loading: load the CNBr-activated Sepharose 4B filler coupled with ligand (B01 protein) into the chromatography column.

[0162] Detection: connect an online ultraviolet detector, select a wavelength of 280nm, and set the 0.5A gear.

[0163] Equilibrium: After the CNBr-activated Sepharose 4B chromatography column is loaded, equilibrate it with 20 mmol / L PBS (pH 7.2-pH 7.4). Use a linear flow rate of 60 cm / h. After the detector baseline is stable, the sample can be injected.

[0164] Load: After equilibration, load at a linear flow rate of 60 cm / h, collect the breakthrough. After loading, rinse the line and column of residual sample with 20 mM PBS (pH 7.2-pH 7.4) until the detector baseline is stable.

[0165] Elution: After equilibration, elute the column of antibody sample with 0.1 M glycine (pH 2.6).

[0166] Collection: Collect the peak of interest.

[0167] Sample treatment: Eluted sample is dialyzed against 10 mM PBS (pH 7.2-pH 7.4) for 24 h, changing the dialysis solution twice, every 2-3 hours.

[0168] Storage: The collected antibody sample is determined for protein concentration and volume, and the protein content is calculated. Add 0.02% NaN3 for storage.

[0169] Example 4

[0170] The embodiment provides a colloidal gold method for detecting hepatitis C antibodies based on secondary indirect labeling, and the method adopts the kit in the embodiment 3, and comprises the following steps.

[0171] S510, colloidal gold labeling (preparing a colloidal gold-A01-B01-C01 compound);

[0172] S511, labeling the colloidal gold particles with a goat polyclonal antibody of a mouse anti-Trx antibody to obtain a first label;

[0173] 10 μl of 0.2 mol / L K2CO3 solution is added to 1 ml of colloidal gold solution, and after uniform mixing, 25 μg of A01 protein is added and uniformly mixed, and reaction is performed for 15 min; 10 μl of 20% BSA solution is added to 1 ml of colloidal gold solution, and after uniform mixing, reaction is performed for 10 min; centrifugation is performed at 10000 rpm for 10 min, and the supernatant is discarded; the precipitate is resuspended with 1 / 10 volume of TB9 solution of the original colloidal gold solution to obtain the first label (colloidal gold-A01).

[0174] S512, labeling the mouse anti-Trx antibody with the first label to obtain a second label;

[0175] 25 μg of B01 protein is added to 100 μl of the resuspension, and after uniform mixing, reaction is performed for 10 min; centrifugation is performed at 10000 rpm for 10 min, and the supernatant is discarded; the precipitate is resuspended with 1 / 10 volume of TB9 solution of the original colloidal gold solution. The second label (colloidal gold-A01-B01) is obtained.

[0176] S513. Label the recombinant antigen with the Trx tag using the second marker to obtain the third marker;

[0177] Add 0.1-0.4 μg of C01 protein to each 100 μl resuspension, mix thoroughly, and react for 10 min; centrifuge at 10000 rpm for 10 min, discard the supernatant; resuspend the precipitate in 1 / 10 volume of TB9 solution of the original colloidal gold solution. The third label (colloidal gold-A01-B01-C01) is obtained.

[0178] After each labeling step, a thorough washing process can be performed to preferentially remove unbound polyclonal antibodies and free monoclonal antibodies, avoiding false positives caused by residues; at the same time, it reduces the possibility of false positives caused by the electrostatic adsorption of positively charged impurities in the sample by the negative charge on the surface of colloidal gold particles.

[0179] S514, the colloidal gold-A01-B01-C01 resuspension was reconstituted with 9 / 10 volume of the original colloidal gold solution in DOA solution, and the solution was evenly coated onto a glass cellulose membrane (1200 glass fiber), 42ml / sheet.

[0180] S515. Freeze-dry or oven-dry the gold pads. After drying, pack the gold pads into aluminum foil bags, add desiccant, vacuum-pack them, and store them at 4-30℃.

[0181] S520, Coating CO2;

[0182] CO2 protein was diluted to 1.5 mg / ml with HCV coating buffer and uniformly coated onto an NC membrane (13502, 2.0 cm). The NC membrane (nitrocellulose membrane) was then dried in a drying room (temperature 24±4℃, humidity <25%) for 1-2 hours. After drying, the NC membrane was placed in an aluminum foil bag, desiccant was added, and the membrane was vacuum-packed and stored at 4-30℃.

[0183] S530, Assemble colloidal gold test strips;

[0184] Assemble the marked gold pad, coated nitrocellulose membrane, absorbent paper, PVC base plate, sample pad, and other auxiliary materials into a test strip. 7.35cm plate, 2.8cm absorbent paper, 4mm x 2 gold leaf, whole blood indirect method SP, 3.4mm wide strips.

[0185] S540. The sample to be tested is added to the sample pad and forms a first complex with the third marker. As the sample moves to the detection line by chromatography, the sample at the detection line is captured by the recombinant antigen to form a second complex. The detection result is obtained based on the displayed color.

[0186] Comparative Example 1

[0187] S610, preparation of control group;

[0188] Control group colloidal gold labeling (preparation of colloidal gold-B01-C01 complex):

[0189] Add 10 μl of 0.2 mol / L K2CO3 solution to 1 ml of colloidal gold solution, mix well, then add 25 μg of B01 protein, mix well, and react for 15 min; add 10 μl of 20% BSA solution to 1 ml of colloidal gold solution, mix well, and react for 10 min; centrifuge at 10000 rpm for 10 min, discard the supernatant; resuspend the precipitate with 1 / 10 volume of TB9 solution of the original colloidal gold solution. Obtain colloidal gold-B01.

[0190] Add 4 μg of C01 protein to 100 μl of resuspension, mix well, and react for 10 min; centrifuge at 10000 rpm for 10 min, discard the supernatant; resuspend the precipitate with 1 / 10 volume of TB9 solution of the original colloidal gold solution. Obtain colloidal gold-B01-C01.

[0191] Resuspend the colloidal gold-B01-C01 solution with 9 / 10 volume of DDA solution of the original colloidal gold solution; evenly coat the solution on glass fiber membrane (1200 glass fiber), 42 ml per sheet.

[0192] Freeze-dry or dry the gold pad with a freeze dryer. After drying, the gold pad is placed in an aluminum foil bag, dry agent is added, vacuum packaged, and stored at 4-30°C.

[0193] Then use the same method as in Example 4 to coat C02 and assemble into a colloidal gold test strip as a control kit.

[0194] S620, sensitivity experiment;

[0195] Sensitivity experiment samples: HCV-HP (strong positive), HCV-MP (moderate positive), and HCV-COV (weak positive) three positive sera;

[0196] Use the kit of Example 4 and the control kit to detect the samples, record the color development time of the samples and the final color development intensity at 15 min, and the color development intensity is determined according to the color development card. The color development card has color development levels L0-L10, with L0 being the lightest color and L10 being the darkest color. The color development results are shown in Table 4.

[0197]

[0198]

[0199] According to the above experimental data, the kit set of the application (using a two-step indirect labeling method) has shorter color development time and deeper final color development intensity compared with the control kit set (using a one-step indirect labeling method), so it can be known that the sensitivity of the kit set is higher than that of the control set.

[0200] S630, specificity experiment;

[0201] Specificity samples: 10 enterprise positive samples P1-P10, 20 enterprise negative samples N1-N20, and 1000 random clinical samples. The kit was used for detection, and the results are shown in Table 5:

[0202] Table 5: Detection results of specificity experiment

[0203]

[0204] 59# sample was detected as negative by the HCV antibody confirmation kit, so it can be known that the clinical specificity of the kit set of the application is better than that of the control kit set.

[0205] S640, stability experiment

[0206] The same batch of kit of Example 4 was divided into two parts, one was placed in a 4℃ refrigerator, and the other was placed in a 37℃ constant temperature box for 6 days and then taken out and placed in a 4℃ refrigerator overnight. The stability was detected by using a sensitivity sample, and the results are shown in Table 6:

[0207] Table 6: Detection results of stability experiment

[0208]

[0209]

[0210] The kit of the application was placed at 37℃ for 6 days, and the sensitivity judgment was consistent with that of the kit placed at 4℃ for initial detection, and the stability was good.

[0211] Performance evaluation:

[0212] From the above experiments, it can be known that the sensitivity of the kit set of the application is higher than that of the control set, the specificity is better than that of the control set, and the stability is qualified. Therefore, it can be known that the two-step indirect labeling has better effect than the one-step indirect labeling.

[0213] The indirect labeling is equivalent to adding an "arm" between the nanoparticle and the labeled antigen, which is beneficial to expose the antigen epitope. The secondary indirect labeling is equivalent to adding another "arm", which makes the exposure more sufficient and can reduce the spatial steric hindrance interference between antigens, so as to improve the sensitivity.

[0214] The binding of the tag antibody to the tag protein and the binding of the antibody of the tag antibody to the tag antibody are specific binding, the antigen is equivalent to being screened by secondary specificity, the possibility of non-specific impurity proteins being labeled on the nanoparticles is reduced, and the specificity can be improved;

[0215] Compared with the primary indirect labeling, the colloidal gold labeling complex has a larger particle size and a deeper color development, and the sensitivity can be improved.

[0216] The number of recombinant antigens used for labeling can be reduced, and the post-labeling effect caused by excessive antigens can be avoided.

[0217] The kit provided by the application is simple to operate, does not require instruments, and can directly add whole blood (serum) samples to manually interpret the results. The detection time is short, and the results can be interpreted in only 15 minutes. The labeling antibody used is a mouse anti-Trx monoclonal antibody (B01) and a goat polyclonal antibody (A01) of a mouse anti-Trx monoclonal antibody. The labeling antigen used is a fusion protein of Trx and hepatitis C Core region, NS3 protein (1a subtype, 7C→7A), NS4b protein dominant epitope and His tag in series, and a hydrophilic connecting peptide is added between the epitopes, which is beneficial to the exposure of the antigen epitopes, can improve the reaction sensitivity and reduce the antibody missed detection. The coating antigen used is a fusion protein of hepatitis C NS3 protein, Core region, NS4b protein dominant epitope and His tag in series, which is beneficial to protein purification, reduces the influence of impurity proteins on the reaction, and simultaneously detects multiple epitope antibodies, which can improve the reaction sensitivity and reduce the antibody missed detection.

[0218] The colloidal gold secondary indirect labeling method is adopted, the high specificity of the monoclonal antibody is used, the active epitopes of the detected antigens are ensured to be completely exposed, the antigen epitope shielding or conformational change caused by direct labeling is avoided, the detection specificity can be effectively improved, the unbound polyclonal antibody and free monoclonal antibody are preferentially removed after each labeling step, false positives caused by residues are avoided, the negative charge on the surface of the colloidal gold particles is reduced, and false positives caused by electrostatic adsorption of the positive impurities in the sample are avoided. The polyclonal antibody and the monoclonal antibody serve as a flexible connecting arm to reduce steric hindrance, the detection signal is amplified by the cascade of the goat polyclonal antibody, the detection sensitivity is improved, the number of recombinant antigens used for labeling is reduced, the missed detection caused by the post-labeling effect caused by excessive antigens is avoided, and the kit has high sensitivity and specificity.

[0219] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0220] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A hepatitis C antibody detection antigen composition, characterized in that, The recombinant antigen for labeling has an amino acid sequence as shown in SEQ ID NO.

1.

2. The Hepatitis C antibody detection antigen composition according to claim 1, wherein, The recombinant antigen for coating has an amino acid sequence as shown in SEQ ID NO.

2.

3. Use of a Hepatitis C antibody detection antigen composition according to any one of claims 1-2, characterized in that, The application is used in the preparation of a product for detecting hepatitis C antibody.

4. The use of an antigen composition for detecting an antibody to hepatitis C according to claim 3, wherein The product comprises at least one of colloidal gold detection kit, immunofluorescence chromatography kit or latex microsphere immunochromatography kit.

5. A colloidal gold method based on secondary indirect labeling for detecting hepatitis C antibody, characterized in that, The hepatitis C antibody detection antigen composition of claim 2 further comprises colloidal gold particles, polyclonal antibodies of mouse anti-Trx antibodies and mouse anti-Trx antibodies.

6. The colloidal gold method based on secondary indirect labeling for hepatitis C antibody test kit according to claim 5, characterized in that, The polyclonal antibodies of mouse anti-Trx antibodies comprise goat polyclonal antibodies or rabbit polyclonal antibodies.

7. The colloidal gold-based method for detecting hepatitis C antibody according to claim 5, characterized in that, The biological material related to the recombinant antigen for labeling and the recombinant antigen for coating is any one of the following: (a) a nucleic acid molecule encoding the recombinant antigen of claim 2; (b) an expression cassette comprising the nucleic acid molecule of (a); (c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b); (d) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b) or the recombinant vector of (c); (e) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b) or the recombinant vector of (c).

8. The colloidal gold-based method for detecting hepatitis C antibody according to claim 5, characterized in that, The amino acid sequence of the recombinant antigen for labeling comprises Trx and dominant epitopes of hepatitis C Core region, NS3 protein, NS4b protein and His tag; The NS3 protein uses the NS3 protein sequence of HCV strain 1a subtype, and at least one fragment of the NS3 protein is mutated to mutate cysteine into alanine.

9. The colloidal gold-based method for detecting hepatitis C antibody according to claim 5, wherein, The amino acid sequence of the recombinant antigen for coating comprises N-terminal His tag, NS3 fragment, Core fragment, NS4B dominant epitope and C-terminal His tag. The recombinant antigen for coating is obtained by concatenating each sequence fragment, or the protein sequence fragments containing each epitope are finally mixed and coated by being expressed respectively.

10. A colloidal gold method for detecting hepatitis C antibody based on secondary indirect labeling, characterized in that, The method uses the kit of claim 5, comprising: The colloidal gold particles are labeled with polyclonal antibodies of mouse anti-Trx antibodies to obtain a first label; The mouse anti-Trx antibodies are labeled with the first label to obtain a second label; The recombinant antigen for labeling with Trx tag is labeled with the second label to obtain a third label; The sample to be tested is added to the sample pad to form a first complex with the third label, and moves to the detection line by chromatography, is captured by the recombinant antigen for coating at the detection line to form a second complex, and the detection result is obtained according to the color displayed.

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