Recombinant EB (Epstein-Barr) virus related nucleocapsid antigen as well as preparation method and application thereof

Abstract

The invention relates to a recombinant EB (Epstein-Barr) virus related nucleocapsid antigen, which comprises an expression vector, and an SCP (Sequence Conformation Protocol) protein fragment and a P23 protein fragment of the EB virus capsid antigen, and the SCP protein fragment and the P23 protein fragment are connected through a connecting peptide; the sequence of the SCP protein fragment is as shown in SEQ ID NO.1, the sequence of the P23 protein fragment is as shown in SEQ ID NO.2, and the sequence of the connecting peptide is as shown in SEQ ID NO.5. The invention also relates to a preparation method and application of the antigen. According to the recombinant EB virus related nucleocapsid antigen machine preparation method and application, SCP and P23 proteins in a VCA protein compound are selected, dominant antigen epitopes of the SCP and P23 proteins are analyzed through biological software, chimeric expression is conducted through an escherichia coli expression system, and the recombinant antigen with high antigenicity is obtained; meanwhile, the antibody is applied to a colloidal gold detection kit and is used as a detection antigen of an EB virus capsid antigen IgG antibody.

Description

Technical Field

[0001] The present invention relates to the field of gene recombination and immune diagnosis technology, and in particular to a recombinant Epstein-Barr virus-related nucleocapsid antigen. Background Art

[0002] Epstein-Barr virus (EBV) is a member of the Herpesviridae family, also known as human herpesvirus type 4 (HHV-4). It belongs to the genus Lymphocytic cryptovirus of the subfamily of γ-herpesvirus and is a type of enveloped double-stranded DNA virus. Its genome is about 172kb long, with an outer envelope, a capsid inside the envelope, and a large double-stranded DNA in the innermost layer. EBV is the pathogen of infectious mononucleosis. In addition, EBV is closely related to the occurrence of nasopharyngeal carcinoma and childhood lymphoma, and is listed as one of the human tumor viruses that may cause cancer. At present, the detection of EBV-specific antibodies mainly targets the viral capsid antigen (VCA), early antigen (EA) and nuclear antigen (NA). During primary EBV infection, IgM and IgG against VCA (anti-VCA-IgM / IgG) are first produced; in the late stage of acute infection, anti-EA-IgG appears; in the late recovery period, anti-EBV nuclear antigen (EBNA) IgG is produced. Currently, the EBV-specific antibodies mainly used for laboratory testing include anti-VCA-IgG, VCA-IgM and anti-EBNA-IgG. VCA-IgM can appear in the early stage of infection and disappear after 1-2 weeks; VCA-IgG can appear early after infection and can last for life. The basis for the detection of primary EBV infection: VCA-IgG (+) and VCA-IgM (+) and NA-IgG (-) or VCA-IgM (-) but VCA-IgG (+) with low affinity. At the same time, anti-VCA-IgA has been proven to be a biomarker that helps in the screening and diagnosis of nasopharyngeal carcinoma (NPC). Therefore, effective high-quality antigen raw materials are urgently needed for the detection of antibodies produced after EBV infection.

[0003] Natural antigens are difficult to culture and purify, and there are large batch differences. At present, the coated antigens expressed by genetic engineering cannot achieve satisfactory results in both specificity and sensitivity. The reason is that different EBV fragments have different diagnostic values. Some fragments have poor specificity, causing false positive results; some fragments have high specificity but insufficient sensitivity, resulting in missed detection. Summary of the invention

[0004] The technical problem to be solved by the present invention is: to overcome the shortcomings of the coated antigen raw materials in the previous EBV detection reagents and achieve ideal detection effects in terms of sensitivity and specificity, to provide a recombinant EB virus-related nucleocapsid antigen and its preparation method and application, by selecting SCP and P23 proteins (encoding genes BFRF3 and BLRF2) in the VCA protein complex, and using biological software to analyze their dominant antigenic epitopes, and using an Escherichia coli expression system for chimeric expression to obtain a recombinant antigen with strong antigenicity, and at the same time applying it to a detection kit for preparing colloidal gold, and using it as a detection antigen for EB virus capsid antigen (VCA) IgG antibody.

[0005] The recombinant Epstein-Barr virus-associated nucleocapsid antigen comprises an expression vector, an SCP protein fragment and a P23 protein fragment of the Epstein-Barr virus capsid antigen, wherein the SCP protein fragment and the P23 protein fragment are connected by a connecting peptide; the sequence of the SCP protein fragment is as described in SEQ ID NO.1, the sequence of the P23 protein fragment is as described in SEQ ID NO.2, and the connecting peptide sequence is as described in SEQ ID NO.5.

[0006] Furthermore, the codon-optimized nucleotide sequences corresponding to the amino acid sequences of the SCP protein fragment, the P23 protein fragment and the connecting peptide amino acid sequence are as described in SEQ ID NO.3.

[0007] Furthermore, the sequence of the amino acid sequence fragment of the recombinant Epstein-Barr virus-related nucleocapsid antigen is as described in SEQ ID NO.4.

[0008] The preparation method of the recombinant Epstein-Barr virus-related nucleocapsid antigen is the recombinant Epstein-Barr virus-related nucleocapsid antigen according to any one of claims 1 to 3, and the preparation method comprises the following steps:

[0009] S1-By antigen dominant epitope analysis, the Epstein-Barr virus capsid antigen SCP 24-171aa fragment and the p23 protein 119-154aa fragment were selected, the two fragments were connected with a connecting peptide, and the optimized nucleotide sequence as described in SEQ ID NO.3 for translating the amino acid was obtained by codon optimization. The nucleotide sequence was connected to the prokaryotic expression vector pCold II at the BamH I and EcoR I restriction sites by gene synthesis, and the recombinant expression vector pCold II-rVCA-Ag was successfully obtained;

[0010] S2-transform the recombinant expression vector pCold II-rVCA-Ag obtained in step S1 into the host bacteria, and screen to obtain the recombinant engineered bacteria;

[0011] S3-The recombinant engineered bacteria obtained in step S2 are cultured and induced to express, and the product is separated and purified to obtain recombinant Epstein-Barr virus-related capsid antigens.

[0012] Furthermore, the recombinant expression vector in step S1 is recombined from the expression vector pCold II and the nucleotide sequence SEQ ID NO.3.

[0013] Furthermore, the recombinant engineered bacteria in step S2 are prepared by transforming the recombinant expression vector into a host strain.

[0014] The recombinant Epstein-Barr virus-associated nucleocapsid antigen is applied to an Epstein-Barr virus capsid antigen IgG antibody detection kit, wherein the Epstein-Barr virus capsid antigen is the above-mentioned recombinant Epstein-Barr virus-associated nucleocapsid antigen, and the recombinant Epstein-Barr virus-associated nucleocapsid antigen prepared by the above-mentioned recombinant Epstein-Barr virus-associated nucleocapsid antigen diagnostic antigen preparation method; the recombinant Epstein-Barr virus-associated nucleocapsid antigen is used as the diagnostic antigen of the Epstein-Barr virus capsid antigen IgG antibody detection kit.

[0015] Furthermore, the EB virus capsid antigen IgG antibody detection kit is a colloidal gold detection kit, and its preparation method comprises the following steps:

[0016] S21—Colloidal gold labeling: put 1mL of colloidal gold in a small glass cup, stir, add appropriate amount of 0.2MK2CO3 to adjust pH to 7.6, stir for 1min; add 20μg mouse anti-human IgG monoclonal antibody, stir for 1min; add 100μL 1% PEG20000, stir for 1min; centrifuge at 5000g for 10 minutes, discard the supernatant, and resuspend the precipitate with 100ul colloidal gold diluent; mix thoroughly and store at 4℃, dilute the above gold-labeled complex 10 times with colloidal gold diluent, evenly apply it on the glass fiber, and dry at 37℃ to make a gold-labeled pad;

[0017] S22—Nitrocellulose membrane coating: The coating diluent formula is 0.01M PB, pH 7.4, and the mass percentage concentration is 0.05% trehalose. The test line (rVCA-Ag) and the quality control line (sheep anti-mouse IgG) are coated on the NC membrane from bottom to top at 0.5 mg / ml respectively, and then baked at 37℃ for 30 minutes;

[0018] S23—Assembly: Assemble the solid-phased NC membrane with the sample pad, S pad, gold label pad, PVC base plate, absorbent paper, and Mark sticker, and cut them into 3-4 mm test strips using a strip cutter.

[0019] Furthermore, the colloidal gold dilution solution component parameters in step S21 are 20 mM PB, 150 mM NaCl, 0.1% PEG20000, 0.1% TritonX-100, 2% Sucrose, and 0.01% NaN3.

[0020] The present invention discloses a recombinant Epstein-Barr virus-related nucleocapsid antigen and a preparation method and application thereof, which overcome the shortcomings of the coated antigen raw materials in the previous EBV detection reagents and achieve ideal detection effects in terms of sensitivity and specificity. The SCP and P23 proteins in the VCA protein complex are selected, and the dominant antigen epitopes thereof are analyzed by biological software, and chimeric expression is performed by using an Escherichia coli expression system to obtain a recombinant antigen with strong antigenicity. The recombinant antigen is applied to a detection kit for preparing colloidal gold and is used as a detection antigen for IgG antibodies of the Epstein-Barr virus capsid antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following is a further description of a recombinant Epstein-Barr virus-related nucleocapsid antigen and its preparation method and application according to the present invention in conjunction with the accompanying drawings:

[0022] Figure 1 It is the expression identification gel image described in step 2 of the preparation method of the recombinant Epstein-Barr virus-related nucleocapsid antigen (wherein 1 is the marker, 2 is the whole bacteria image before induction, and 3 is the whole bacteria image after induction);

[0023] Figure 2 This is the purification gel image described in step 3 of the preparation method of the recombinant Epstein-Barr virus-related nucleocapsid antigen (where 1 is the purified rVCA-Ag and 2 is the marker;)

[0024] Figure 3 It is a comparison table for the application of the recombinant Epstein-Barr virus-related nucleocapsid antigen and the testing of product samples. DETAILED DESCRIPTION

[0025] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0026] In the description of the present invention, it should be understood that the terms "left", "right", "front", "back", "top", "bottom", "inside", "outside", etc., indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present invention.

[0027] The technical solution of the present invention is further described below with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0028] Embodiment 1: The recombinant Epstein-Barr virus-associated nucleocapsid antigen comprises an expression vector, an SCP protein fragment and a P23 protein fragment of the Epstein-Barr virus capsid antigen, wherein the SCP protein fragment and the P23 protein fragment are connected via a connecting peptide; the sequence of the SCP protein fragment is as described in SEQ ID NO.1, the sequence of the P23 protein fragment is as described in SEQ ID NO.2, and the connecting peptide sequence is as described in SEQ ID NO.5. The codon-optimized nucleotide sequence corresponding to the amino acid sequence of the SCP protein fragment, the P23 protein fragment and the amino acid sequence of the connecting peptide is as described in SEQ ID NO.3. The sequence of the amino acid sequence fragment of the recombinant Epstein-Barr virus-associated nucleocapsid antigen is as described in SEQ ID NO.4.

[0029] Embodiment 2: Preparation method of the recombinant Epstein-Barr virus-related nucleocapsid antigen The recombinant Epstein-Barr virus-related nucleocapsid antigen is the above-mentioned recombinant Epstein-Barr virus-related nucleocapsid antigen, and the preparation method comprises the following steps:

[0030] S1-By antigen dominant epitope analysis, the Epstein-Barr virus capsid antigen SCP 24-171aa fragment and the p23 protein 119-154aa fragment were selected, the two fragments were connected with a connecting peptide, and the optimized nucleotide sequence as described in SEQ ID NO.3 for translating the amino acid was obtained by codon optimization. The nucleotide sequence was connected to the prokaryotic expression vector pCold II at the BamH I and EcoR I restriction sites by gene synthesis, and the recombinant expression vector pCold II-rVCA-Ag was successfully obtained;

[0031] Specifically, the antigenic epitopes of SCP (reference unprot: P14348) and p23 (reference unprot: P03197) proteins were analyzed by Antheprot software, and SCP: 24-171aa was selected, and the amino acid sequence was shown in SEQ ID No. 1; p23: 119-154aa, and the amino acid sequence was shown in SEQ ID No. 2; the two protein fragments were connected by a linker peptide: SCP (24-171aa)-linker-p23 (119-154aa), and the amino acid sequence was shown in SEQ ID No. 3; the linker amino acid sequence was shown in SEQ ID No. 5. In order to increase the expression of the recombinant protein in Escherichia coli, the preferred codons of Escherichia coli were used to optimize the corresponding nucleotides of SCP (24-171aa)-linker-p23 (119-154aa), and the optimized sequence was shown in SEQ ID No. 3. The nucleotide sequence was synthesized by Qingke Biotechnology Co., Ltd., and the synthesized target gene was cloned between the restriction sites of BamH I and EcoR I in the pColdII vector to construct the recombinant vector pCold II-rVCA-Ag. The protein sequence translated from the target protein reading frame sequence of the recombinant vector is the recombinant Epstein-Barr virus nucleocapsid antigen (rVCA-Ag), and the amino acid sequence is shown in SEQ ID No. 4; the amino acid sequence SEQ ID No. 4 includes the amino acid sequence translated from the part of the multiple cloning site on the N-terminal fusion pCold II vector.

[0032] S2-transform the recombinant expression vector pCold II-rVCA-Ag obtained in step S1 into the host bacteria, and screen to obtain the recombinant engineered bacteria;

[0033] The preparation of recombinant engineered bacteria is as follows: the recombinant vector pCold II-rVCA-Ag constructed in step S1 is transformed into E. coli BL21 (DE3) competent cells, and spread on LB plates containing ampicillin resistance (100 μg / mL), inverted and cultured at 37°C overnight, pick the monoclonal strain on the plate to LB liquid culture medium containing ampicillin resistance (100 μg / mL), culture in a constant temperature shaker at 37°C overnight, induce culture in a constant temperature shaker at 16°C for 6 hours, and collect the bacterial precipitate to prepare the protein electrophoresis sample. Figure 1 As shown, the SDS-PAGE electrophoresis results on the expression identification gel map showed that the recombinant protein was successfully expressed. The results showed that after induction, an obvious target band was expressed near 21 kDa; the recombinant protein expression strain B / pCold II-rVCA-Ag was successfully obtained.

[0034] S3-Cultivate the recombinant engineered bacteria obtained in step S2 and induce expression, and separate and purify the product to obtain recombinant EB virus-related capsid antigen;

[0035] Specifically, the induced expression was as follows: 10 μl of the recombinant plasmid expression bacteria were inoculated into 5 ml LB medium containing 100 ug / ml ampicillin (Amp) (purchased from Solebol), and cultured at 37°C, 200 rpm overnight (about 16 h), 200 μl of the bacterial solution was added to 60 ml LB medium containing the same concentration of Amp, and cultured at 37°C, 200 rpm overnight, all the bacterial solution was inoculated into 1 L LB medium containing the same concentration of Amp, and cultured at 30°C, 150 rpm for 2-2.5 h, and then the OD600 was measured to be between 0.8 and 1.0, and cultured at 16°C, 150 rpm overnight (about 16 h), and then centrifuged at 6000 rpm for 10 min to collect the bacterial precipitate; the bacterial precipitate was resuspended with 50 ml 20 mM PBS (pH 7.4), centrifuged at 6000 rpm for 15 min, and the precipitate was collected.

[0036] The separation and purification of the product is as follows: high-pressure crushing - the bacterial precipitate is resuspended in 30 ml of 20 mM PBS (pH 7.4) and crushed at low temperature and high pressure. The parameter settings are: pressure value 910-980 bar, 3 cycles, temperature setting 4 ° C, and the actual operating temperature shall not be higher than 13 ° C. After the crushing is completed, the crushed product is centrifuged at 9000 rpm for 30 minutes at low temperature, and the supernatant is collected. Protein purification - Take a nickel ion affinity chromatography column: IMAC Bestarose-FF (purchased from Bogelon) for affinity chromatography, balance the column with 10 column volumes of ultrapure water; then balance the column with 10 column volumes of equilibration buffer (20mM Tris-HCl pH 8.0) and add the protein sample; after loading, wash away the unbound impurities with 10 column volumes of equilibration buffer (20mM Tris-HCl pH 8.0); then wash away the impurities with 10 column volumes of washing buffer (20mM Tris-HCl-50mM IM pH8.0), and finally elute the target protein with elution buffer (20mM Tris-HCl-250mM IM pH8.0). Put the collected protein sample into a dialysis bag, put it into a beaker filled with dialysis solution (20mM Tris-HCl pH 8.0), and dialyze it at 4°C overnight. Perform SDS-PAGE to detect the purity of the purified protein sample, such as Figure 2 As shown, the target size of the protein is around 21 kD and the purity is ≥95%.

[0037] In step S1, the recombinant expression vector is recombined from the expression vector pCold II and the nucleotide sequence SEQ ID NO. 3. In step S2, the recombinant engineered bacteria are prepared by transforming the recombinant expression vector into a host strain.

[0038] Embodiment 3: The recombinant Epstein-Barr virus-associated nucleocapsid antigen is applied to an Epstein-Barr virus capsid antigen IgG antibody detection kit, wherein the Epstein-Barr virus capsid antigen is the above-mentioned recombinant Epstein-Barr virus-associated nucleocapsid antigen, and the recombinant Epstein-Barr virus-associated nucleocapsid antigen prepared by the above-mentioned recombinant Epstein-Barr virus-associated nucleocapsid antigen diagnostic antigen preparation method; the recombinant Epstein-Barr virus-associated nucleocapsid antigen is used as the diagnostic antigen of the Epstein-Barr virus capsid antigen IgG antibody detection kit. The Epstein-Barr virus capsid antigen IgG antibody detection kit is a colloidal gold detection kit, and its preparation method comprises the following steps:

[0039] S21—Colloidal gold labeling: Take 1mL of colloidal gold and put it in a small glass cup, stir and add an appropriate amount of 0.2MK2CO3 to adjust the pH to 7.6, and stir for 1 minute; add 20μg mouse anti-human IgG monoclonal antibody and stir for 1 minute; add 100μL 1% PEG20000 and stir for 1 minute; centrifuge at 5000g for 10 minutes, discard the supernatant, and resuspend the precipitate with 100ul colloidal gold diluent; after fully mixing, store at 4°C, dilute the above-mentioned gold-labeled complex 10 times with colloidal gold diluent and evenly apply it on the glass fiber, dry at 37°C to make a gold-labeled pad; the component parameters of the colloidal gold diluent are 20mM PB, 150mM NaCl, 0.1% PEG20000, 0.1% TritonX-100, 2% Sucrose, and 0.01% NaN3.

[0040] S22—Nitrocellulose membrane coating: The coating diluent formula is 0.01M PB, pH 7.4, and the mass percentage concentration is 0.05% trehalose. The test line (rVCA-Ag) and the quality control line (sheep anti-mouse IgG) are coated on the NC membrane from bottom to top at 0.5 mg / ml respectively, and then baked at 37℃ for 30 minutes;

[0041] S23—Assembly: Assemble the solid-phased NC membrane with the sample pad, S pad, gold label pad, PVC base plate, absorbent paper, and Mark sticker, and cut them into 3-4 mm test strips using a strip cutter.

[0042] Product sample testing: The prepared Epstein-Barr virus capsid antigen (VCA) IgG antibody detection reagent was used to test 5 Epstein-Barr virus positive sera and 10 Epstein-Barr virus negative sera. Figure 3 As shown in the table: The positive samples detected by the control A manufacturer antigen 1 were missed, and the sensitivity was low and the color was weak. The positive samples detected by the control B manufacturer antigen 2 were not missed, but the negative samples were false positive. The recombinant Epstein-Barr virus capsid antigen (rVCA-Ag) was used as a coating material in the test strip to detect positive samples, with high sensitivity, deep color, and all negative samples were negative, with excellent specificity.

[0043] The present invention relates to a method for preparing a recombinant Epstein-Barr virus-related nucleocapsid antigen and its application, which overcomes the shortcomings of the coated antigen raw materials in previous EBV detection reagents and achieves ideal detection effects in terms of sensitivity and specificity. The method selects SCP and P23 proteins in the VCA protein complex, analyzes their dominant antigenic epitopes using biological software, and uses an Escherichia coli expression system for chimeric expression to obtain a recombinant antigen with strong antigenicity. The recombinant antigen is then applied to a colloidal gold detection kit and is used as a detection antigen for IgG antibodies to the Epstein-Barr virus capsid antigen.

[0044] The above description shows the main features, basic principles, and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments or embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, the above embodiments or embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0045] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A recombinant Epstein-Barr virus-related nucleocapsid antigen, characterized in that: The recombinant Epstein-Barr virus-related nucleocapsid antigen comprises an expression vector, an SCP protein fragment and a P23 protein fragment of the Epstein-Barr virus capsid antigen, wherein the SCP protein fragment and the P23 protein fragment are connected via a connecting peptide; the sequence of the SCP protein fragment is as described in SEQ ID NO.1, the sequence of the P23 protein fragment is as described in SEQ ID NO.2, and the connecting peptide sequence is as described in SEQ ID NO.

5.

2. The recombinant Epstein-Barr virus-related nucleocapsid antigen according to claim 1, characterized in that: The codon-optimized nucleotide sequence corresponding to the amino acid sequence of the SCP protein fragment, the P23 protein fragment and the amino acid sequence of the connecting peptide, wherein the optimized nucleotide sequence is as described in SEQ ID NO.

3.

3. The recombinant Epstein-Barr virus-related nucleocapsid antigen according to claim 2, characterized in that: The sequence of the amino acid sequence fragment of the recombinant Epstein-Barr virus-related nucleocapsid antigen is as described in SEQ ID NO.

4.

4. A method for preparing a recombinant Epstein-Barr virus-related nucleocapsid antigen, characterized in that: The recombinant Epstein-Barr virus-associated nucleocapsid antigen is the recombinant Epstein-Barr virus-associated nucleocapsid antigen according to any one of claims 1 to 3, and the preparation method comprises the following steps: S1-By antigen dominant epitope analysis, the Epstein-Barr virus capsid antigen SCP 24-171aa fragment and the p23 protein 119-154aa fragment were selected, the two fragments were connected with a connecting peptide, and the optimized nucleotide sequence as described in SEQ ID NO.3 for translating the amino acid was obtained by codon optimization. The nucleotide sequence was connected to the prokaryotic expression vector pCold II at the BamH I and EcoR I restriction sites by gene synthesis, and the recombinant expression vector pCold II-rVCA-Ag was successfully obtained; S2-transform the recombinant expression vector pCold II-rVCA-Ag obtained in step S1 into the host bacteria, and screen to obtain the recombinant engineered bacteria; S3-The recombinant engineered bacteria obtained in step S2 are cultured and induced to express, and the product is separated and purified to obtain recombinant EB virus-related capsid antigens.

5. The method for preparing the recombinant Epstein-Barr virus-related nucleocapsid antigen according to claim 4, characterized in that: The recombinant expression vector in step S1 is recombined from the expression vector pCold II and the nucleotide sequence SEQ ID NO.

3.

6. The method for preparing the recombinant Epstein-Barr virus-related nucleocapsid antigen according to claim 4, characterized in that: The recombinant engineered bacteria in step S2 are prepared by transforming the recombinant expression vector into a host strain.

7. An application of a recombinant Epstein-Barr virus-related nucleocapsid antigen, characterized in that: The recombinant Epstein-Barr virus-associated nucleocapsid antigen is applied to an Epstein-Barr virus capsid antigen IgG antibody detection kit, wherein the Epstein-Barr virus capsid antigen is the recombinant Epstein-Barr virus-associated nucleocapsid antigen described in any one of claims 1 to 3, and the recombinant Epstein-Barr virus-associated nucleocapsid antigen prepared by the recombinant Epstein-Barr virus-associated nucleocapsid antigen diagnostic antigen preparation method described in any one of claims 4 to 6; the recombinant Epstein-Barr virus-associated nucleocapsid antigen is used as the diagnostic antigen of the Epstein-Barr virus capsid antigen IgG antibody detection kit.

8. The use of the recombinant Epstein-Barr virus-related nucleocapsid antigen according to claim 7, characterized in that: The EB virus capsid antigen IgG antibody detection kit is a colloidal gold detection kit, and its preparation method The following steps are included: S21—Colloidal gold labeling: put 1mL of colloidal gold in a small glass cup, stir, add appropriate amount of 0.2MK2CO3 to adjust pH to 7.6, stir for 1min; add 20μg mouse anti-human IgG monoclonal antibody, stir for 1min; add 100μL 1% PEG20000, stir for 1min; centrifuge at 5000g for 10 minutes, discard the supernatant, and resuspend the precipitate with 100ul colloidal gold diluent; mix thoroughly and store at 4℃, dilute the above gold-labeled complex 10 times with colloidal gold diluent, evenly apply it on the glass fiber, and dry at 37℃ to make a gold-labeled pad; S22—Nitrocellulose membrane coating: The coating diluent formula is 0.01M PB, pH 7.4, and the mass percentage concentration is 0.05% trehalose. The test line (rVCA-Ag) and the quality control line (sheep anti-mouse IgG) are coated on the NC membrane from bottom to top at 0.5 mg / ml respectively, and then baked at 37℃ for 30 minutes; S23—Assembly: Assemble the solid-phased NC membrane with the sample pad, S pad, gold label pad, PVC base plate, absorbent paper, and Mark sticker, and cut them into 3-4 mm test strips using a strip cutter.

9. The use of the recombinant Epstein-Barr virus-related nucleocapsid antigen according to claim 8, characterized in that: The colloidal gold dilution solution component parameters in step S21 are 20 mM PB, 150 mM NaCl, 0.1% PEG20000, 0.1% TritonX-100, 2% Sucrose, and 0.01% NaN3.

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