EB virus BNLF2b gene-encoded polypeptide and its detection applications

Abstract

This invention relates to immunological detection, particularly to the field of immunological diagnosis of tumors. Specifically, this invention provides a method for diagnosing nasopharyngeal carcinoma based on anti-EBV antibody levels, and a kit for using said method. This invention also provides a polypeptide encoded by the EBV BNLF2b gene for the above-mentioned diagnosis and its use in the diagnosis of nasopharyngeal carcinoma.

Description

Technical Field

[0001] This invention relates to immunological detection, particularly to the field of immunological diagnosis of tumors. Specifically, this invention provides a method for diagnosing nasopharyngeal carcinoma based on anti-EBV antibody levels, and a kit for using said method. This invention also provides a polypeptide encoded by the EBV BNLF2b gene for the above-mentioned diagnosis and its use in the diagnosis of nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma is a malignant tumor that commonly occurs in the top and lateral walls of the nasopharynx, and its incidence rate is the highest among ENT malignancies. my country is a high-incidence area for nasopharyngeal carcinoma, with the highest incidence rates in Guangdong and Guangxi provinces[1]. The occurrence of nasopharyngeal carcinoma is related to a variety of factors, including EB virus infection, genetic factors, and environmental factors[2]. Because the early symptoms of nasopharyngeal carcinoma are not obvious and the location of the disease is relatively hidden, most nasopharyngeal carcinomas are only discovered in the late stage[3]. However, the five-year survival rate of early-stage nasopharyngeal carcinoma can reach more than 90%, while the prognosis of late-stage nasopharyngeal carcinoma is significantly reduced[3]. At the same time, early-stage nasopharyngeal carcinoma can be treated with local radiotherapy, while late-stage nasopharyngeal carcinoma requires chemotherapy. Therefore, early diagnosis is the key to improving the survival rate and quality of life of patients with nasopharyngeal carcinoma.

[0003] As early as 1976, Werner Henle et al. found that EBV IgA antibodies were significantly elevated in the serum of nasopharyngeal carcinoma patients[4]. Subsequently, studies found that antibody levels against multiple proteins such as EBNA1, EA-D, and VCA were significantly elevated in the serum of nasopharyngeal carcinoma patients[5-12]. Early EBV antibody detection was mainly based on immunofluorescence, which had low throughput, long processing time, and highly subjective results, making it difficult to meet the needs of large-scale screening[13-15]. With the development of enzyme-linked immunosorbent assay (ELISA), researchers in various countries have developed a large number of EB virus antibody ELISA detection kits, which have significantly improved the throughput and objectivity of detection[16-23]. In addition, studies have found that by using different serological markers in combination, the sensitivity and specificity of nasopharyngeal carcinoma screening can be further improved, among which the most commonly used combination is EBNA1-IgA and VCA-IgA[24-26]. The results of prospective cohort studies show that the early diagnosis rate of nasopharyngeal carcinoma can be increased from 10-20% to more than 60% through screening[27-29]. Based on this, our research group proposed a two-step screening strategy, namely: on the basis of combined screening of EBNA1-IgA and VCA-IgA, EAD-IgA is further tested on the high-risk population in the first step screening, and those who are positive for EAD-IgA are then diagnosed by nasopharyngoscopy. Through the two-step screening, the positive predictive value of nasopharyngeal carcinoma screening increased from the original 4.69% to 18.52%

[30] .

[0004] In addition to EBV antibody levels, the levels of EBV DNA and microRNA in the blood of nasopharyngeal carcinoma patients were also significantly elevated [31-33]. The results of a prospective study in Hong Kong by Allan Chan et al. showed that using persistent EBV DNA positivity as the standard for nasopharyngeal carcinoma screening can significantly improve the specificity of nasopharyngeal carcinoma screening. Among the 1112 people (5.5%) who were initially screened as having positive EBV DNA, only 309 people had persistent EBV DNA positivity, of whom 34 were eventually diagnosed with nasopharyngeal carcinoma

[34] . Using persistent EBV positivity as the screening standard, the sensitivity and specificity reached 97.1% and 98.6%, respectively, and the positive predictive value increased from 3.06% to 11%. The early diagnosis rate of nasopharyngeal carcinoma through screening increased from 20% to 71%

[34] .

[0005] Compared with the one-step screening, the positive predictive value of the two-step screening is significantly lower, which can meet the needs of a larger scale of nasopharyngeal carcinoma screening. However, whether it is serological screening or DNA screening, the two-step screening operation is more complicated and the cost is higher. In addition, persistent DNA positivity requires blood to be drawn again, which further increases the difficulty of screening. Therefore, although the one-step serological antibody screening has significant cost-effectiveness

[35] , whether the two-step screening has higher cost-effectiveness needs to be further evaluated. In addition, the highest positive predictive value of the currently reported nasopharyngeal carcinoma screening method is only 18.52%, that is, at least 71.48% of non-nasopharyngeal carcinoma patients in the high-risk population need to undergo nasopharyngoscopy. Nasopharyngoscopy takes a long time, which directly limits the scale of nasopharyngeal carcinoma screening, and at the same time, it will also cause psychological burden to non-nasopharyngeal carcinoma patients who are positive in the screening.

[0006] Therefore, there is still a need in this field to develop new nasopharyngeal carcinoma screening methods that have higher specificity and positive predictive value than existing methods, while also being high-throughput and low-cost, to meet the needs of large-scale screening and enable more people to benefit from screening. Summary of the Invention

[0007] The inventors of this application, after extensive research, unexpectedly discovered that the antibody levels in the serum of subjects targeting the protein encoded by the EBV BNLF2b gene (which can be detected, for example, using the BNLF2b gene-encoded protein or polypeptide fragment as a coating antigen via an indirect method or a double-antigen sandwich method) can effectively distinguish nasopharyngeal carcinoma patients from healthy controls. Furthermore, the inventors have devoted considerable inventive effort to obtaining a BNLF2b gene-encoded polypeptide fragment particularly suitable as a capture reagent (e.g., a coating antigen in ELISA). Therefore, based on the detection of antibody levels against the EBV BNLF2b gene-encoded protein, the inventors have successfully established a method and platform for diagnosing nasopharyngeal carcinoma, which can significantly improve the specificity and positive predictive value of nasopharyngeal carcinoma screening.

[0008] Isolated polypeptides or their variants

[0009] In a first aspect, the present invention provides an isolated polypeptide or a variant thereof, wherein the polypeptide comprises at least seven consecutive amino acid residues of a wild-type protein encoded by the BNLF2b gene, and includes at least one (e.g., at least two, at least three, or all four) sequence selected from the following: amino acid residues 5-11, 16-23, 31-39, or 53-60 (e.g., amino acid residues 53-61) of the wild-type protein encoded by the BNLF2b gene;

[0010] The variant differs from its source polypeptide only in the substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid residues (e.g., conserved or non-conserved substitution), and retains the biological function of its source polypeptide (e.g., the activity of being recognized and bound by anti-EBV antibodies).

[0011] In some embodiments, the wild-type protein encoded by the BNLF2b gene has a sequence as shown in SEQ ID NO:101.

[0012] In this document, the biological functions of the polypeptides or variants thereof of the present invention include, but are not limited to, the activity of being recognized and bound as epitope peptides by anti-EBV antibodies (e.g., antibodies specific to the protein encoded by the BNLF2b gene).

[0013] In some embodiments, the isolated polypeptide comprises: amino acid residues 53-60 (e.g., amino acid residues 53-61), 5-23, 16-39, 31-60 (e.g., amino acid residues 31-61), 5-39, 16-60 (e.g., amino acid residues 16-61), or 5-60 (e.g., amino acid residues 5-61) of the wild-type protein encoded by the BNLF2b gene.

[0014] In one embodiment, the isolated polypeptide consists of at least eight (e.g., at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and includes amino acid residues 53-60 of the wild-type protein encoded by the BNLF2b gene.

[0015] In some embodiments, the polypeptide comprises amino acid residues 53-61 of the wild-type protein encoded by the BNLF2b gene.

[0016] In some embodiments, the polypeptide comprises amino acid residues 51-65 of the wild-type protein encoded by the BNLF2b gene.

[0017] In some embodiments, the amino acid residues at positions 51-65 have the sequence shown in SEQ ID NO:97.

[0018] In some embodiments, the polypeptide consists of at least 15 consecutive amino acid residues of a wild-type protein encoded by the BNLF2b gene.

[0019] In one embodiment, the isolated polypeptide consists of at least 19 (e.g., at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and includes amino acid residues 5-23 of the wild-type protein encoded by the BNLF2b gene.

[0020] In some embodiments, the polypeptide comprises amino acid residues 1-25 of the wild-type protein encoded by the BNLF2b gene.

[0021] In some embodiments, the amino acid residues 1-25 have the sequence shown in SEQ ID NO:102.

[0022] In some embodiments, the polypeptide consists of at least 25 consecutive amino acid residues of a wild-type protein encoded by the BNLF2b gene.

[0023] In one embodiment, the isolated polypeptide consists of at least 24 (e.g., at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, or at least 39) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and includes amino acid residues 16-39 of the wild-type protein encoded by the BNLF2b gene.

[0024] In some embodiments, the polypeptide comprises amino acid residues 14-52 of the wild-type protein encoded by the BNLF2b gene.

[0025] In some embodiments, the amino acid residues at positions 14-52 have the sequence shown in SEQ ID NO:88.

[0026] In some embodiments, the polypeptide consists of at least 39 consecutive amino acid residues of a wild-type protein encoded by the BNLF2b gene.

[0027] In one embodiment, the isolated polypeptide consists of at least 30 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and includes amino acid residues 31-60 of the wild-type protein encoded by the BNLF2b gene.

[0028] In some embodiments, the isolated polypeptide contains amino acid residues 31-61 of the wild-type protein encoded by the BNLF2b gene.

[0029] In one embodiment, the isolated polypeptide consists of at least 35 (e.g., at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, or at least 53) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and includes amino acid residues 5-39 of the wild-type protein encoded by the BNLF2b gene.

[0030] In some embodiments, the polypeptide comprises amino acid residues 1-52 of the wild-type protein encoded by the BNLF2b gene.

[0031] In some embodiments, the amino acid residues 1-52 have the sequence shown in SEQ ID NO:91.

[0032] In some embodiments, the polypeptide consists of at least 53 consecutive amino acid residues of a wild-type protein encoded by the BNLF2b gene.

[0033] In one embodiment, the isolated polypeptide consists of at least 45 (e.g., at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, or at least 64) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and includes amino acid residues 16-60 of the wild-type protein encoded by the BNLF2b gene.

[0034] In some embodiments, the polypeptide comprises amino acid residues 16-61 of the wild-type protein encoded by the BNLF2b gene.

[0035] In some embodiments, the polypeptide comprises amino acid residues 14-74 of the wild-type protein encoded by the BNLF2b gene (e.g., the sequence shown in SEQ ID NO: 90). In some embodiments, the polypeptide consists of at least 61 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.

[0036] In some embodiments, the polypeptide comprises amino acid residues 11-65 of the wild-type protein encoded by the BNLF2b gene (e.g., the sequence shown in SEQ ID NO: 103). In some embodiments, the polypeptide consists of at least 55 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.

[0037] In some embodiments, the polypeptide comprises amino acid residues 11-74 of the wild-type protein encoded by the BNLF2b gene (e.g., the sequence shown in SEQ ID NO: 104). In some embodiments, the polypeptide consists of at least 64 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.

[0038] In one embodiment, the isolated polypeptide consists of at least 56 (e.g., at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, or at least 74) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and includes amino acid residues 5-60 of the wild-type protein encoded by the BNLF2b gene.

[0039] In some embodiments, the polypeptide comprises amino acid residues 5-61 of the wild-type protein encoded by the BNLF2b gene.

[0040] In some embodiments, the polypeptide comprises amino acid residues 1-74 of the wild-type protein encoded by the BNLF2b gene.

[0041] In some embodiments, the amino acid residues 1-74 have the sequence shown in SEQ ID NO:89.

[0042] In some embodiments, the polypeptide consists of at least 74 consecutive amino acid residues of a wild-type protein encoded by the BNLF2b gene.

[0043] In some exemplary embodiments, the isolated polypeptide consists of sequences selected from the following: amino acid residues 51-56, 1-25, 14-52, 14-74, 11-65, 11-74, or 1-74 of the wild-type protein encoded by the BNLF2b gene. In some exemplary embodiments, the isolated polypeptide consists of sequences selected from the following: SEQ ID NOs: 88-91, 97, 102-104.

[0044] In some embodiments, the variants of the present invention differ from the polypeptide from which they are derived only in the substitution of one, two, three, or four amino acid residues (e.g., conservative or non-conservative substitutions) and retain the biological function of the polypeptide from which it is derived (e.g., the activity of being recognized and bound by anti-EBV antibodies).

[0045] In some embodiments, the variant does not include amino acid substitutions at positions corresponding to the amino acid positions shown in the wild-type protein encoded by the BNLF2b gene: 6, 9, 10, 11, 16, 31, 33, 38, 39, 53, 54, 56, 57, 58, 59, 95, 96, 97.

[0046] In some embodiments, the variant contains amino acid substitutions at one or more (e.g., position 1, position 2, position 3, or position 4) corresponding to amino acid positions shown in the wild-type protein encoded by the BNLF2b gene, wherein the positions are: 5, 7, 8, 12, 13, 14, 15, 19, 22, 24, 25, 32, 34, 35, 36, 37, 40, 41, 42, 52, 55, 60, 61, 89, 91, 93, or 98.

[0047] In some embodiments, the variant contains amino acid substitutions at one or more of the following positions (e.g., position 1, position 2, position 3, or position 4), referring to the amino acid positions shown in the wild-type protein encoded by the BNLF2b gene: 5, 7, 8, 12, 13, 14, 22, 25, 32, 34, 35, 36, 40, 41, 42, 52, 55, 60, 61, 89, 91, 93, or 98.

[0048] In some embodiments, the variant includes one or more (e.g., 1, 2, 3, or 4) amino acid substitutions selected from the following: an amino acid substitution of A at position 5, an amino acid substitution of A at position 7, an amino acid substitution of G at position 8, an amino acid substitution of G, T, D, or S at position 12, an amino acid substitution of A at position 13, an amino acid substitution of G at position 14, an amino acid substitution of A at position 15, an amino acid substitution of A at position 22, an amino acid substitution of A at position 24, an amino acid substitution of A at position 25, an amino acid substitution of A at position 32, an amino acid substitution of A at position 34, and an amino acid substitution of A at position 35. The amino acid at position 36 is replaced with A; the amino acid at position 37 is replaced with A, N, Q, S, or R; the amino acid at position 40 is replaced with A; the amino acid at position 41 is replaced with A; the amino acid at position 42 is replaced with A; the amino acid at position 52 is replaced with K, H, A, S, or D; the amino acid at position 55 is replaced with S; the amino acid at position 60 is replaced with A; the amino acid at position 61 is replaced with K, H, S, or A; the amino acid at position 89 is replaced with A or T; the amino acid at position 91 is replaced with A; the amino acid at position 93 is replaced with Q; and the amino acid at position 98 is replaced with A.

[0049] In some embodiments, the variant includes one or more (e.g., 1, 2, 3, or 4) amino acid substitutions selected from the following: an amino acid substitution of A at position 5, an amino acid substitution of A at position 7, an amino acid substitution of G at position 8, an amino acid substitution of G, T, D, or S at position 12, an amino acid substitution of A at position 13, an amino acid substitution of G at position 14, an amino acid substitution of A at position 22, an amino acid substitution of A at position 25, an amino acid substitution of A at position 32, an amino acid substitution of A at position 34, and an amino acid substitution of A at position 35. The amino acid is replaced with A at position 36, at position 40, at position 41, at position 42, at position 52, at position 55, at position 60, at position 61, at position 61, at position 89, at position 91, at position 93, at position 98, and at position 98.

[0050] In some embodiments, the isolated polypeptide consists of no more than 97 (e.g., no more than 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 76, 75, or 74) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.

[0051] In some embodiments, the isolated polypeptide consists of 15 to 80 (e.g., 15 to 74) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene. In some embodiments, the isolated polypeptide consists of 39 to 74 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.

[0052] In one embodiment, the isolated polypeptide or its variant is attached to the surface of a solid support or has a modifying group that can be attached to the solid support. In some embodiments, the C-terminus of the isolated polypeptide or its variant is attached to the surface of the solid support or has a modifying group that can be attached to the solid support. In some embodiments, the modifying group is biotin or avidin. In some embodiments, the solid support is selected from magnetic beads or microtiter plates (e.g., microplates or ELISA plates).

[0053] In another embodiment, the isolated polypeptide or a variant thereof carries a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin.

[0054] Preparation of peptides

[0055] The polypeptides or variants thereof of the present invention are not limited by their manner of production; for example, they can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0056] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide or a variant thereof of the present invention.

[0057] In another aspect, the present invention also provides a vector comprising the isolated nucleic acid molecules as described above. The vector of the present invention can be a cloning vector or an expression vector. In a preferred embodiment, the vector of the present invention is, for example, a plasmid, a granulocyte, a bacteriophage, a Cosmid, etc.

[0058] In another aspect, the present invention also provides host cells comprising the isolated nucleic acid molecules or vectors of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The cells of the present invention can also be cell lines, such as 293T cells.

[0059] In another aspect, the present invention also provides a method for preparing the polypeptide or variant thereof of the present invention, comprising culturing the host cell of the present invention under conditions that allow expression of the polypeptide or variant thereof, and recovering the polypeptide or variant thereof from the cultured host cell culture.

[0060] Test kit

[0061] In a second aspect, the present invention provides a kit comprising a capture reagent selected from the isolated peptides or variants thereof described in the first aspect. In some embodiments, the kit further comprises instructions for using the isolated peptides or variants thereof as capture reagents to detect antibodies specific to the protein encoded by the BNLF2b gene in a sample, or instructions for using the isolated peptides or variants thereof as capture reagents to determine the level of antibodies specific to the protein encoded by the BNLF2b gene in a sample from a subject, thereby determining whether the subject has nasopharyngeal carcinoma or is at risk of nasopharyngeal carcinoma.

[0062] In some implementations, the subject is a mammal, such as a human.

[0063] In some embodiments, the capture reagent is attached to the surface of a solid support. In some embodiments, the C-terminus of the capture reagent is attached to the solid support. Hereinafter, the solid support includes well-drained plates, test tubes, beads (e.g., latex particles), or films (e.g., nitrocellulose membranes) made of or coated with polymeric materials (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose), or magnetic beads pre-coated with functional groups (e.g., amino, carboxyl, biotin, or avidin). In some embodiments, the solid support is selected from magnetic beads or microtiter plates (e.g., microplates or ELISA plates).

[0064] In other embodiments, the capture reagent has a modifying group that can be linked to a solid support. In some embodiments, the C-terminus of the capture reagent has a modifying group that can be linked to a solid support. In such embodiments, the kit may further comprise a coating reagent for coating the capture reagent onto the solid support, such as a coating buffer (e.g., carbonate buffer, phosphate buffer, Tris-HCl buffer, or borate buffer). Methods for coating proteins or peptides onto solid supports are well known in the art, such as physical adsorption, covalent coupling via aminated or carboxylated surfaces, or mediated binding via avidin-biotin systems, polylysine pre-coated surfaces, protein A or protein G pre-coated surfaces. In some embodiments, the modifying group is biotin or avidin, and the solid support surface has a corresponding linker group.

[0065] In some embodiments, the kit contains at least a solid-phase carrier coated with avidin or streptoavidin, and the above-described capture reagent, in a separate container or in a separate compartment of a single container unit.

[0066] In some embodiments, the method for determining whether a subject has nasopharyngeal carcinoma or is at risk of nasopharyngeal carcinoma by measuring the level of anti-EBV antibodies in a sample is performed in a double-antigen sandwich configuration. Determining antibody levels in a sample using a double-antigen sandwich method is well known to those skilled in the art. In such assays, the "capture antigen" and "detect antigen" processes create a bridge between two specific antigens, which are typically identical, have the same core epitope, or exhibit immune cross-reactivity, allowing one antibody to bind to both antigens.

[0067] Therefore, in some embodiments, the kit further comprises a detection reagent selected from the isolated peptides or variants thereof described in the first aspect.

[0068] In some embodiments, the detection reagent carries a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0069] In some embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core fragment selected from amino acid residues 5-11, 16-23, 31-39, and / or amino acid residues 53-60 (e.g., amino acid residues 53-61) of the wild-type protein encoded by the BNLF2b gene. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 53-60 (e.g., amino acid residues 53-61), 5-23, 16-39, 5-39, 16-60 (e.g., amino acid residues 16-61), or 5-60 (e.g., amino acid residues 5-61) of the wild-type protein encoded by the BNLF2b gene. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 51-56, 1-25, 14-52, 1-52, 14-74, 11-65, 11-74, or 1-74 of the wild-type protein encoded by the BNLF2b gene.

[0070] In other embodiments, the method for determining whether a subject has nasopharyngeal carcinoma or is at risk of nasopharyngeal carcinoma by measuring the level of anti-EBV antibodies in a sample is performed in an indirect manner. Measuring antibody levels in a sample indirectly is well known to those skilled in the art. In such assays, a "capture antigen" first forms an immune complex with antibodies in the sample, and the captured antibodies are then detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0071] Therefore, in some embodiments, the kit further comprises a detection reagent selected from secondary antibodies with a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0072] In some implementations, the secondary antibody is specific to the species (e.g., human) from which the test antibody originates.

[0073] In some implementations, the secondary antibody is an anti-immunoglobulin antibody.

[0074] In some exemplary embodiments, the kit is used to detect anti-EBV IgG antibodies. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0075] In some exemplary embodiments, the kit is used to detect anti-EBV IgM antibodies. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0076] In some exemplary embodiments, the kit is used to detect anti-EBV IgA antibodies. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0077] In some embodiments, the kit of the present invention may also contain one or more reagents or devices selected from: (i) a device for collecting or storing samples from a subject (e.g., a blood collection device); and (ii) other reagents required for performing the assay (e.g., buffer solutions, diluents, blocking solutions, and / or standards).

[0078] Detection uses and methods

[0079] In a third aspect, the present invention provides a method for detecting antibodies specific to a protein encoded by the BNLF2b gene in a sample, comprising the following steps:

[0080] (1) The sample is contacted with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptides or variants thereof described in the first aspect;

[0081] (2) Determine the amount of antigen-antibody immune complex obtained in step (1).

[0082] In some implementations, in step (2), the amount of the immune complex is determined by immunological detection.

[0083] In some embodiments, the immunological assay is selected from enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0084] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0085] In other embodiments, the capturing agent has a modifying group that can be linked to a solid support. In such embodiments, prior to step (1), the method further includes the step of coating the capturing agent onto the surface of the solid support.

[0086] In some embodiments, the C-terminus of the capture reagent is attached to the surface of the solid support, or has a modifying group that can be attached to the solid support.

[0087] In some embodiments, the assay is performed in a double-antigen sandwich configuration. Assessing antibody levels in a sample using a double-antigen sandwich method is well known to those skilled in the art. In such assays, the "capture antigen" and "detect antigen" processes create a bridge between two specific antigens, which are typically identical, have the same core epitope, or exhibit immune cross-reactivity, allowing one antibody to bind to both antigens.

[0088] Therefore, in some embodiments, in step (2), a detection reagent is used to detect the amount of the immune complex, the detection reagent being selected from the isolated polypeptides or variants thereof described in the first aspect.

[0089] In some embodiments, the detection reagent is selected from the isolated polypeptide or variants thereof described in the first aspect, the isolated polypeptide or variant thereof bearing a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0090] In some embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core fragment selected from amino acid residues 5-11, 16-23, 31-39, and / or amino acid residues 53-60 (e.g., amino acid residues 53-61) of the wild-type protein encoded by the BNLF2b gene. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 53-60 (e.g., amino acid residues 53-61), 5-23, 16-39, 5-39, 16-60 (e.g., amino acid residues 16-61), or 5-60 (e.g., amino acid residues 5-61) of the wild-type protein encoded by the BNLF2b gene. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 51-56, 1-25, 14-52, 1-52, 14-74, 11-65, 11-74, or 1-74 of the wild-type protein encoded by the BNLF2b gene.

[0091] In other embodiments, the assay is performed in an indirect manner. Determining antibody levels in a sample using indirect methods is well known to those skilled in the art. In such assays, a "capture antigen" first forms an immune complex with antibodies in the sample, and the captured antibodies are then detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0092] Therefore, in some embodiments, in step (2), a detection reagent is used to detect the amount of the immune complex, the detection reagent being selected from secondary antibodies with detectable labels.

[0093] In some embodiments, the detectable marker is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin.

[0094] In some implementations, the secondary antibody is specific to the species (e.g., human) from which the test antibody originates.

[0095] In some implementations, the secondary antibody is an anti-immunoglobulin antibody.

[0096] In some exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0097] In some exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0098] In some exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0099] In another aspect, the invention also relates to the use of the isolated polypeptide or variants thereof described in the first aspect in the preparation of a kit for detecting antibodies specific to the protein encoded by the BNLF2b gene in a sample. In some embodiments, the kit detects antibodies specific to the protein encoded by the BNLF2b gene in a sample by means of the method described in the third aspect.

[0100] Diagnostic uses and methods

[0101] In a fourth aspect, the present invention provides a method for determining whether a subject has nasopharyngeal carcinoma or is at risk of nasopharyngeal carcinoma, comprising:

[0102] (1) Determine the level of antibodies specific to the protein encoded by the BNLF2b gene in samples from the subjects; and,

[0103] (2) Compare the level with the reference value.

[0104] In some implementations, if the level is higher than a reference value, the subject is determined to have nasopharyngeal carcinoma or be at risk of nasopharyngeal carcinoma.

[0105] In this document, the reference values ​​are derived from subjects or healthy individuals who do not have nasopharyngeal carcinoma (e.g., subjects without detectable disease and without a history of cancer or nasopharyngeal carcinoma), or indicate the level of antibody-specific antibodies against the protein encoded by the BNLF2b gene in the corresponding samples of subjects or healthy individuals who do not have nasopharyngeal carcinoma.

[0106] In some embodiments, the sample is a blood sample, such as whole blood, plasma, or serum.

[0107] In some implementations, the subject is a mammal, such as a human.

[0108] In some embodiments, the level of antibodies specific to the protein encoded by the BNLF2b gene in the sample is determined by an immunoassay. In some embodiments, the immunoassay is selected from enzyme immunoassays (e.g., ELISA), chemiluminescent immunoassays, fluorescence immunoassays, or radioimmunoassays.

[0109] In some embodiments, the assay includes using the isolated polypeptide or a variant thereof described in the first aspect as a capture reagent.

[0110] In some implementations, step (1) includes the following steps:

[0111] (1a) A sample from the subject is contacted with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptides or variants thereof described in the first aspect;

[0112] (1b) Determine the amount of antigen-antibody immune complex obtained in step (1a).

[0113] In some embodiments, in step (1b), the amount of the immune complex is determined by an immunological assay. In some embodiments, the immunological assay is selected from enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0114] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0115] In other embodiments, the capturing agent has a modifying group that can be linked to a solid support. In such embodiments, prior to step (1), the method further includes the step of coating the capturing agent onto the surface of the solid support.

[0116] In some embodiments, the C-terminus of the capture reagent is attached to the surface of the solid support, or has a modifying group that can be attached to the solid support.

[0117] In some embodiments, the assay is performed in a double-antigen sandwich configuration. Assessing antibody levels in a sample using a double-antigen sandwich method is well known to those skilled in the art. In such assays, the "capture antigen" and "detect antigen" processes create a bridge between two specific antigens, which are typically identical, have the same core epitope, or exhibit immune cross-reactivity, allowing one antibody to bind to both antigens.

[0118] Therefore, in some embodiments, in step (1b), a detection reagent is used to detect the amount of the immune complex, the detection reagent being selected from the isolated polypeptides or variants thereof described in the first aspect.

[0119] In some embodiments, the detection reagent is selected from the isolated polypeptide or variants thereof described in the first aspect, the isolated polypeptide or variant thereof bearing a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0120] In some embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core fragment selected from amino acid residues 5-11, 16-23, 31-39, and / or amino acid residues 53-60 (e.g., amino acid residues 53-61) of the wild-type protein encoded by the BNLF2b gene. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 53-60 (e.g., amino acid residues 53-61), 5-23, 16-39, 5-39, 16-60 (e.g., amino acid residues 16-61), or 5-60 (e.g., amino acid residues 5-61) of the wild-type protein encoded by the BNLF2b gene. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 51-56, 1-25, 14-52, 1-52, 14-74, 11-65, 11-74, or 1-74 of the wild-type protein encoded by the BNLF2b gene.

[0121] In other embodiments, the assay is performed in an indirect manner. Determining antibody levels in a sample using indirect methods is well known to those skilled in the art. In such assays, a "capture antigen" first forms an immune complex with antibodies in the sample, and the captured antibodies are then detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0122] Therefore, in some embodiments, in step (1b), a detection reagent is used to detect the amount of the immune complex, the detection reagent being selected from secondary antibodies with detectable labels.

[0123] In some embodiments, the detectable marker is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin.

[0124] In some implementations, the secondary antibody is specific to the species (e.g., human) from which the test antibody originates.

[0125] In some implementations, the secondary antibody is an anti-immunoglobulin antibody.

[0126] In some exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0127] In some exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0128] In some exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0129] In some embodiments, the method further includes providing a sample from the subject prior to step (1).

[0130] In some embodiments, the method further includes, after step (2), administering a therapeutically effective dose of an antitumor therapy (e.g., chemotherapy, radiotherapy, and / or immunotherapy) to a subject who is determined to have nasopharyngeal carcinoma or is at risk of having nasopharyngeal carcinoma.

[0131] In some implementations, the antitumor therapy for nasopharyngeal carcinoma is selected from surgical treatment, radiotherapy (such as external beam radiotherapy, EBRT, brachytherapy), or chemotherapy (such as carboplatin, paclitaxel, docetaxel, gemcitabine, doxorubicin, epirubicin, bleomycin, methotrexate), targeted therapy (such as cetuximab), immunotherapy (such as PD-1 monoclonal antibody), and combination therapy (such as radiotherapy + chemotherapy, radiotherapy + surgery).

[0132] In another aspect, the present invention also relates to the use of reagents capable of measuring levels of antibodies specific to the protein encoded by the BNLF2b gene in the preparation of a kit for determining whether a subject has nasopharyngeal carcinoma or is at risk of nasopharyngeal carcinoma.

[0133] In some embodiments, the reagent is capable of measuring the level of antibodies specific to the protein encoded by the BNLF2b gene via an immunoassay. In some embodiments, the immunoassay is selected from enzyme immunoassays (e.g., ELISA), chemiluminescent immunoassays, fluorescence immunoassays, or radioimmunoassays.

[0134] In some implementations, the subject is a mammal, such as a human.

[0135] In some embodiments, the sample is a blood sample, such as whole blood, plasma, or serum.

[0136] In some implementations, the kit determines whether a subject has nasopharyngeal carcinoma or is at risk of developing it by:

[0137] (1) Determine the level of antibodies specific to the protein encoded by the BNLF2b gene in samples from the subjects; and,

[0138] (2) Compare the level with the reference value.

[0139] In some implementations, if the level is higher than a reference value, the subject is determined to have nasopharyngeal carcinoma or be at risk of nasopharyngeal carcinoma.

[0140] In some embodiments, the reagent capable of determining the level of an antibody specific to the protein encoded by the BNLF2b gene comprises a capture reagent selected from the isolated polypeptides or variants thereof described in the first aspect.

[0141] In some implementations, step (1) above includes the following steps:

[0142] (1a) A sample from the subject is contacted with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptides or variants thereof described in the first aspect;

[0143] (1b) Determine the amount of antigen-antibody immune complex obtained in step (1a).

[0144] In some embodiments, in step (1b), the amount of the immune complex is determined by an immunological assay. In some embodiments, the immunological assay is selected from enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0145] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0146] In other embodiments, the capturing agent has a modifying group that can be linked to a solid support. In such embodiments, prior to step (1) above, the method further includes coating the capturing agent onto the surface of the solid support.

[0147] In some embodiments, the C-terminus of the capture reagent is attached to the surface of the solid support, or has a modifying group that can be attached to the solid support.

[0148] In some embodiments, the reagent capable of determining the level of an antibody specific to the protein encoded by the BNLF2b gene further comprises a detection reagent selected from the isolated polypeptide or variants thereof described in the first aspect.

[0149] In some implementations, in step (1b) above, the detection reagent is used to detect the amount of the immune complex.

[0150] In some embodiments, the detection reagent is selected from the isolated polypeptide or variants thereof described in the first aspect, the isolated polypeptide or variant thereof bearing a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0151] In some embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core fragment selected from amino acid residues 5-11, 16-23, 31-39, and / or amino acid residues 53-60 (e.g., amino acid residues 53-61) of the wild-type protein encoded by the BNLF2b gene. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 53-60 (e.g., amino acid residues 53-61), 5-23, 16-39, 5-39, 16-60 (e.g., amino acid residues 16-61), or 5-60 (e.g., amino acid residues 5-61) of the wild-type protein encoded by the BNLF2b gene. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 51-56, 1-25, 14-52, 1-52, 14-74, 11-65, 11-74, or 1-74 of the wild-type protein encoded by the BNLF2b gene.

[0152] In other embodiments, the reagent capable of determining the level of an antibody specific to the protein encoded by the BNLF2b gene further comprises a detection reagent selected from secondary antibodies with a detectable label.

[0153] In some implementations, in step (1b) above, the detection reagent is used to detect the amount of the immune complex.

[0154] In some embodiments, the detectable marker is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin.

[0155] In some implementations, the secondary antibody is specific to the species (e.g., human) from which the test antibody originates.

[0156] In some implementations, the secondary antibody is an anti-immunoglobulin antibody.

[0157] In some exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0158] In some exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0159] In some exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0160] Terminology Definition

[0161] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0162] As used herein, the term "BNLF2b" refers to the BNLF2b gene of Epstein-Barr virus (EBV), which is well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: CAA24811.1). The full-length protein encoded by the BNLF2b gene contains 98 amino acids, and its sequence is shown in SEQ ID NO:101. In this document, the full-length protein encoded by the BNLF2b gene may also be referred to simply as the wild-type protein encoded by the BNLF2b gene, or the protein encoded by the BNLF2b gene.

[0163] As used herein, when referring to the amino acid sequence of the protein encoded by the BNLF2b gene, the sequence shown in SEQ ID NO: 101 is used for description. For example, the expression "amino acid residues 53-60 of the wild-type protein encoded by the BNLF2b gene" refers to amino acid residues 53-60 of the polypeptide shown in SEQ ID NO: 101. However, those skilled in the art will understand that mutations or variations can be naturally generated or artificially introduced into the amino acid sequence of the protein encoded by the BNLF2b gene without affecting its biological function. Therefore, in this invention, the term "protein encoded by the BNLF2b gene" and similar expressions should include all such sequences, including, for example, the sequence shown in SEQ ID NO: 101 and its natural or artificial variants. Furthermore, when describing a sequence fragment of the protein encoded by the BNLF2b gene, it includes not only the sequence fragment of SEQ ID NO: 101 but also the corresponding sequence fragments in its natural or artificial variants. For example, the expression "amino acid residues 53-60 of the protein encoded by the BNLF2b gene" includes amino acid residues 53-60 of SEQ ID NO: 101, and the corresponding fragment in its variants (natural or artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at an equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0164] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its targeted antigen. The binding affinity between two molecules can be measured using Kx. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as molar concentration (M). D The smaller the value, the tighter the binding between the two molecules, and the higher the affinity. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) refers to an antibody with a binding affinity of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to this antigen. K D The value can be determined by methods well known in the art, such as using surface plasmon resonance (SPR) in a BIACORE instrument.

[0165] As used herein, the term "immunological assay" refers to an assay that utilizes the specific interaction / binding affinity between an antigen and antibody, and is generally used to detect the presence or level of a specific antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art and include, but are not limited to, enzyme immunoassays (EIA), chemiluminescent immunoassays (CLIA), radioimmunoassays (RIA), fluorescence immunoassays (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, etc. In some embodiments, the immunological assay is an enzyme immunoassay (EIA), such as an ELISA, Elispot assay, or CLEIA assay. For a detailed description of immunological assays, see, for example, Fundamental Immunology, Ch. 7, Paul W., ed., 2nd edition, Raven Press, NY (1989).

[0166] As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to in the art as an "antigenic determinant." For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-continuous amino acids in a distinctive spatial conformation, and can be "linear" or "conformal." In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are present linearly along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are present across protein amino acid residues that are separated from each other.

[0167] As used herein, the term "detectable label" can refer to any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Particularly preferred are such labels that are suitable for immunological assays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescence immunoassays, chemiluminescent immunoassays, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters), magnetic beads (e.g., The invention includes, but is not limited to, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of these markers include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). The markers covered in this invention can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing an enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers. In some implementations, the detectable markers described above can be connected to the detection antibodies or antigens via connectors of varying lengths to reduce potential steric hindrance.

[0168] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). H and V LIt consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding sites.

[0169] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0170] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0171] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0172] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0173] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0174] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0175] As used herein, the term “subject” includes, but is not limited to, various animals, particularly mammals such as humans.

[0176] Beneficial effects

[0177] This invention is the first to discover that antibody levels targeting the protein encoded by the Epstein-Barr virus (EBV) BNLF2b gene can be used to diagnose nasopharyngeal carcinoma (NPC) or assess NPC risk, and its diagnostic efficacy is significantly superior to existing biomarkers, significantly improving the specificity and positive predictive value of NPC screening. This invention also provides for the first time a serological screening kit for NPC based on the protein or peptide fragment encoded by the EBV BNLF2b gene.

[0178] Compared with existing technologies, the technical solution of the present invention can achieve detection sensitivity comparable to, or even better than, known EB virus antibody combined detection methods, and can achieve rapid, high-throughput detection, thus possessing significant clinical application value.

[0179] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0180] Figure 1 The diagram shows the reactivity of IgA and IgG antibodies in one nasopharyngeal carcinoma pooled serum sample (NPC) and three healthy control serum samples (H1-H3) from Examples 2 and 3 with 87 EBV peptides (numbered 1-87). The left graph shows IgA antibodies, and the right graph shows IgG antibodies. The horizontal axis represents different serum samples, and the vertical axis represents different EBV peptides. The darker the color, the higher the reactivity.

[0181] Figure 2 The reactivity of antibodies in the serum of nasopharyngeal carcinoma patients and healthy controls in Examples 2 and 3 with different EBV peptides is shown. The horizontal axis represents the EBV peptide, and the vertical axis represents the OD value (logarithmic scale) of the reaction.

[0182] Figure 3 The reactivity of IgG antibodies in the serum of nasopharyngeal carcinoma patients and healthy controls in Example 4 with the protein aa14-52 encoded by BNLF2b is shown. Figure 3 A represents the detection OD values ​​(logarithmic scale) for different samples; Figure 3 B represents the results of ROC curve analysis, with the horizontal axis representing 100% specificity and the vertical axis representing sensitivity.

[0183] Figure 4The reactivity of IgA, IgM, and IgG antibodies in the serum of nasopharyngeal carcinoma patients and healthy controls in Example 4 with the BNLF2b-encoded protein aa1-74 is shown. Figure A shows the detected OD values, with the horizontal axis representing serum classification and the vertical axis representing OD values ​​(logarithmic scale); Figure B shows the results of ROC curve analysis, with the horizontal axis representing 100% specificity and the vertical axis representing sensitivity; Figure C shows the reactivity of IgA and IgM antibodies in the serum of healthy controls with the BNLF2b-encoded protein aa1-74, with the horizontal axis representing antibody type and the vertical axis representing OD values ​​(logarithmic scale).

[0184] Figure 5 The results of the BNLF2b antibody double-antigen sandwich assay in Example 5 are shown. These results indicate the presence of 50 nasopharyngeal carcinoma serum samples and 500 healthy control serum samples. Figure 5 A represents the detected OD value, with the horizontal axis representing serum differential and the vertical axis representing the OD value (logarithmic scale). Figure 5 B represents the ROC curve analysis results, with the horizontal axis representing 100% specificity and the vertical axis representing sensitivity.

[0185] Figure 6 This shows the reactivity of 74 nasopharyngeal carcinoma serum samples and 250 healthy control serum samples in Example 6 with EBNA1 / IgA and VCA / IgA, as well as the risk rate of developing nasopharyngeal carcinoma calculated by combining the two. The horizontal axis represents serum classification. Figure 6 A and Figure 6 In B, the vertical axis represents the detected OD value (logarithmic scale). Figure 6 The vertical axis C represents the risk of developing nasopharyngeal carcinoma.

[0186] Figure 7 The results of the BNLF2b antibody double-antigen sandwich assay in Example 6 are shown, comparing serum from nasopharyngeal carcinoma patients and healthy controls. The horizontal axis represents serum classification, and the vertical axis represents the OD value of the reaction.

[0187] Figure 8 The ROC curves for the BNLF2b antibody double antigen sandwich assay and the combined detection of EBNA1 / IgA+VCA / IgA in Example 6 are shown. The horizontal axis represents 100% specificity, and the vertical axis represents sensitivity.

[0188] Figure 9 The diagram shows the reactivity of IgG antibodies in nasopharyngeal carcinoma serum with different peptides encoded by the BNLF2b protein in Example 10. The horizontal axis represents different BNLF2b peptides, and the vertical axis represents the mixed nasopharyngeal carcinoma serum number; the darker the color, the higher the reactivity.

[0189] Figure 10The reactivity of IgG antibodies in two nasopharyngeal carcinoma mixed serum samples with different peptides encoded by the BNLF2b protein is shown in Example 10. AD represents the reactivity of nasopharyngeal carcinoma serum with truncated peptides in the aa1-25, aa31-45, aa51-65, and aa81-98 regions, respectively. The horizontal axis represents the peptide position (e.g., 1-25 represents amino acids 1-25), and the vertical axis represents the ratio of the detected OD value to the detected OD value of the untruncated peptide.

[0190] Figure 11 The reactivity of nasopharyngeal carcinoma serum with different truncated polypeptides within the aa1-25 region was shown in Example 10. Figure 11 A shows the reactivity of serum from different nasopharyngeal carcinoma patients with aa1-25, aa1-15, and aa11-25. Figure 11 B shows the reactivity of these sera with aa1-25 and its truncated polypeptide. The horizontal axis represents the amino acid position of the synthetic peptide, and the vertical axis represents the nasopharyngeal carcinoma serum number. The darker the color, the higher the reactivity.

[0191] Figure 12 The reactivity of IgG antibodies against BNLF2b-encoded protein mutants in nasopharyngeal carcinoma patients in Example 10 is shown. AD represents the reactivity of nasopharyngeal carcinoma serum with mutants in the aa1-25, aa31-45, aa51-65, and aa81-98 regions, respectively. The horizontal axis represents the mutation location, and the vertical axis represents the ratio of the detected OD value to the detected OD value of the corresponding unmutated peptide.

[0192] Figure 13 The reactivity of IgG antibodies in the serum of nasopharyngeal carcinoma patients and healthy controls in Example 11 with different BNLF2b-encoded peptides is shown. The horizontal axis represents peptide fragments, and the vertical axis represents the detected OD value.

[0193] Sequence information

[0194] Table 1: Information about the sequences involved in this application is described in the table below.

[0195]

[0196]

[0197]

[0198] Example 1: Synthesis of EB virus gene-encoded polypeptides

[0199] Based on the amino acid sequence information of the proteins encoded by 86 open reading frames (ORFs) of the EBV B95-8 strain in GenBank (GenBank ID: V01555.2), B-cell epitopes for each protein were predicted online using bioinformatics tools. Based on the prediction results, 1-2 potential B-cell epitope peptides for each protein were selected and commissioned to Xiamen Jingju Biotechnology Co., Ltd. for synthesis. During synthesis, biotin was conjugated to the N-terminus of the peptides to facilitate subsequent experiments. Ultimately, 87 EBV gene-encoded peptides (SEQ ID NO: 1-87) were successfully synthesized. These peptides originated from 68 ORFs, and their specific information is shown in Table 1.

[0200] Example 2: Evaluation of the reactivity of EB virus peptides with serum IgA antibodies

[0201] The biotin-labeled peptides (numbered 1-87) obtained in Example 1 were diluted to 500 ng / ml and added to streptavidin-coated 96-well microplates at a standard ratio of 100 μL per well. The plates were incubated at 37°C for 2 hours. After the reaction, the plates were washed twice with PBST, and 200 μL of blocking buffer was added to each well. The plates were then blocked at 37°C for 2 hours. After blocking, the blocking buffer was discarded, and 100 μL of a 1:20 diluted mixture of nasopharyngeal carcinoma patient serum or negative control serum was added to each well. The plates were incubated at 37°C for 30 minutes. After the reaction, the plates were washed five times with PBST, and 1:20000 diluted HRP-labeled goat anti-human IgA (KPL, Gaithersburg, MD) was added. The plates were then incubated at 37°C for another 30 minutes. After washing with PBST 5 times, add 100 μL of TMB chromogenic solution to each well, incubate at 37 degrees Celsius for 15 minutes, add 50 μL of stop solution to each well, mix well, and then measure the absorbance at 450 and 630 nm using a microplate reader.

[0202] The results are as follows Figure 1 As shown in Table 2-1, among the 87 peptides, 8 peptides had OD values ​​greater than 0.3 in response to mixed serum of nasopharyngeal carcinoma. Of these 8 peptides, 5 peptides encoded by the BSRL1, BLLF1b, BGLF3, BDLF2, and BVRF2 genes had not been reported in previous studies for their use in the adjunctive diagnosis of nasopharyngeal carcinoma.

[0203] We further evaluated the specificity of these 5 peptides using 36 negative serum samples, and the results are as follows: Figure 2 As shown in Table 2-2, the three polypeptides encoded by the BSRF1, BGLF3, and BVRF2 genes exhibited relatively good specificity. Furthermore, we evaluated the detection sensitivity of these three polypeptides using 12 nasopharyngeal carcinoma serum samples, and the results are as follows: Figure 2As shown, the reactivity of these three peptides was low. Among them, the peptides encoded by BSRF1 and BVRF2 showed a reactivity of more than 0.1 with 6 serum samples, while the peptide encoded by BGLF3 showed a reactivity of more than 0.1 with only 3 serum samples.

[0204] Table 2-1: Reactivity of EB virus peptides with serum IgA antibodies in nasopharyngeal carcinoma patients

[0205]

[0206] Table 2-2: Reactivity of EB virus peptides with negative control serum IgA antibodies

[0207]

[0208] Example 3: Evaluation of the reactivity of EB virus peptides with serum IgG antibodies

[0209] The reactivity of the 87 polypeptides (1-87) synthesized in Example 1 with serum IgG was tested according to the method in Example 2, wherein HRP-labeled mouse anti-human IgG (Wanyu Meilan, Beijing) diluted 1:5000 was used instead of goat anti-human IgA. The results are as follows. Figure 1 As shown, among these polypeptides, five genes encoding polypeptides, including BZLF1 and BRLF1, showed a reactivity of more than 0.1 with mixed serum of nasopharyngeal carcinoma (Table 3-1). Among them, BNLF1 (ZTA), BRLF1 (RTA), and BILF2 (gp78) have been reported.

[0210] We further evaluated the specificity of the BVRF2 and BNLF2b encoded peptides using 36 negative serum samples, and the results are as follows: Figure 2 As shown in Table 3-2, the OD values ​​of the BNLF2b gene-encoded peptide reacting with 36 negative serum samples were all below 0.034, indicating good specificity. Furthermore, we evaluated the detection sensitivity of the BNLF2b gene-encoded peptide using 12 nasopharyngeal carcinoma serum samples, and the results are as follows... Figure 2 As shown, among the 12 serum samples from nasopharyngeal carcinoma patients, 6 samples had a reaction OD value higher than 0.1.

[0211] Table 3-1: Reactivity of EB virus peptides with positive serum IgG antibodies

[0212]

[0213] Table 3-2: Reactivity of EB virus peptides with negative serum IgG antibodies

[0214]

[0215] Example 4: Establishment and preliminary performance evaluation of the indirect method for anti-BNLF2b antibody

[0216] We further analyzed the hydrophilicity, hydrophobicity, and antigenicity of the BNLF2b-encoded protein (SEQ ID NO:101) using the Protean software in the DNASTAR package, and synthesized two polypeptides, aa14-52 (SEQ ID NO:88) and aa1-74 (SEQ ID NO:89), which were used as coating antigens for indirect detection.

[0217] 4.1 Indirect detection based on peptide aa14-52

[0218] The polypeptide aa14-52 was diluted to 125 ng / ml with carbonate buffer (pH 9.6), and 100 μL was coated per well. Serum samples from 86 nasopharyngeal carcinoma patients and 195 healthy individuals were analyzed according to the method described in Example 3. Results are as follows... Figure 3 As shown in Figure A, when aa14-52 was used as the coating antigen, 57 out of 86 serum samples from nasopharyngeal carcinoma patients had OD values ​​higher than 0.1, while the OD values ​​of all 195 serum samples from healthy individuals were lower than 0.025. Simultaneously, the same method was used to test the serum of 122 individuals who were identified as high-risk under combined EBNA1 / IgA+VCA / IgA screening (this combined detection method can be found in Example 6) but were ultimately diagnosed with non-nasopharyngeal carcinoma. The results are as follows... Figure 3 As shown in Figure A, only 3 cases had OD values ​​exceeding 0.1. The data from the indirect method based on peptide aa14-52 were analyzed using ROC curves with MedCalc 16.2.1 software (MedCalc Software, Ostend, Belgium). The results are as follows: Figure 3 As shown in B, the area under the curve (AUC) is 0.942. When the cut-off value is 0.025, the Youden index is 0.80, the specificity is 100%, and the sensitivity is 80.23% (69 / 86).

[0219] 4.2 Indirect detection based on peptide aa1-74

[0220] Using polypeptide aa1-74 as the coating antigen, 63 serum samples from healthy individuals and 221 serum samples from nasopharyngeal carcinoma were tested according to the same method as in section 4.1. The results are as follows: Figure 4 As shown in Figure A, 55 out of 63 serum samples from nasopharyngeal carcinoma patients had OD values ​​exceeding 0.1, while only 10 out of 221 serum samples from healthy controls had OD values ​​exceeding 0.1. Further ROC curve analysis of the above-mentioned indirect method based on peptide aa1-74 yielded the following results: Figure 4 As shown in B, this method can effectively distinguish between nasopharyngeal carcinoma patients and healthy individuals, with an AUC of 0.950. When the cut-off value is set to 0.1, the sensitivity is 87.30% (55 / 63) and the specificity is 95.48% (211 / 221).

[0221] In addition, using peptide aa1-74 as the coating antigen, the levels of IgA and IgM antibodies in 221 healthy control serum samples were detected indirectly. IgA antibody detection used HRP-labeled goat anti-human IgA (KPL, Gaithersburg, MD) diluted 1:20,000, and IgM antibody detection used HRP-labeled goat anti-human IgM (Wanyu Meilan, Beijing) diluted 1:50,000. Results are as follows... Figure 4 As shown in C, the detection of anti-BNLF2b IgA antibody and anti-BNLF2b IgM antibody both showed good specificity, with only 4 and 7 serum samples having detection values ​​exceeding 0.1, respectively. The specificities were 98.19% (217 / 221) and 96.83% (214 / 221), respectively.

[0222] Table 4 shows the performance of the above methods in distinguishing between nasopharyngeal carcinoma and non-nasopharyngeal carcinoma. The results show that both the indirect method using IgG antibodies with peptide aa14-52 as the coating antigen and the indirect method using IgG, IgA and IgM antibodies with peptide aa1-74 as the coating antigen can efficiently distinguish between nasopharyngeal carcinoma and healthy controls, and have good detection sensitivity and specificity.

[0223] Table 4: Performance of anti-BNLF2b antibody in indirect method for differentiating nasopharyngeal carcinoma from non-nasopharyngeal carcinoma

[0224]

[0225]

[0226] Example 5: Establishment of the anti-BNLF2b antibody double antigen sandwich method

[0227] Since the results of Example 4 showed that IgA, IgM, and IgG antibodies all had good specificity in predicting nasopharyngeal carcinoma risk, nasopharyngeal carcinoma risk can be predicted by detecting total antibodies against BNLF2b. Based on this, this example establishes a double-antigen sandwich method for detecting anti-BNLF2b antibodies to evaluate its performance in screening for nasopharyngeal carcinoma.

[0228] The BNLF2b-encoded peptide aa1-74 was diluted to 100 ng / mL with carbonate buffer (pH 9.6) and added to each well of a standard 96-well microplate at 100 μL. The reaction was carried out at 37°C for 2 hours. After the reaction, the plates were washed twice with PBST, and 200 μL of blocking buffer was added to each well. Blocking was carried out at 37°C for 2 hours. After blocking, the blocking buffer was discarded, and 50 μL of a diluted buffer containing 67 ng / mL biotinylated peptide aa1-74 and 50 μL of nasopharyngeal carcinoma patient serum or negative control serum were added to each well. The reaction was carried out at 37°C for 60 minutes. After the reaction, the plates were washed five times with PBST, and 1:5000 dilution of HRP-labeled streptavidin and 1:15000 dilution of HRP-labeled aa1-74 were added. The reaction was carried out at 37°C for another 30 minutes. After washing five times with PBST, 100 μL of TMB chromogenic solution was added to each well. The mixture was incubated at 37°C for 15 minutes, followed by the addition of 50 μL of stop solution to each well. After mixing, the absorbance at 450 nm and 630 nm was measured using a microplate reader. This method was used to detect the absorbance in 50 serum samples from nasopharyngeal carcinoma patients and 500 serum samples from healthy individuals. The results are as follows: Figure 5 As shown in Figure A, among 50 serum samples from nasopharyngeal carcinoma patients, 46 had reactive OD values ​​higher than 0.1, while among 500 negative serum samples, only 1 had an OD value exceeding 0.1. Further ROC curve analysis of these results yielded the following results: Figure 5 As shown in Figure B, the area under the curve is 0.977. When the threshold is 0.1, the sensitivity and specificity of the reagent are 92.0% and 99.8%, respectively, and the Youden index is 0.91.

[0229] In addition, to compare the performance of different BNLF2b peptides in the diagnosis of nasopharyngeal carcinoma, besides aa1-74, we synthesized two other peptides, aa1-52 (SEQ ID NO:91) and aa14-74 (SEQ ID NO:90), both of which were C-terminally labeled with biotin. Serum samples from another 175 nasopharyngeal carcinoma patients were collected and tested using the same method. The results are shown in the table below. With 0.1 as the cutoff value, the sensitivity of all three peptides was above 85%, indicating that this method possesses high detection sensitivity and specificity when using aa1-52, aa14-74, or aa1-74 as the coating antigen.

[0230] Table 5: Comparison of detection sensitivity when using different BNLF2b peptides to label antigens

[0231]

[0232] Example 6: Comparison of the anti-BNLF2b antibody double-antigen sandwich method with existing nasopharyngeal carcinoma screening reagents

[0233] Seventy-four serum samples from nasopharyngeal carcinoma patients and 250 serum samples from healthy individuals undergoing physical examinations were selected and tested in parallel using the Zhongshan Bio EBNA1 / IgA Detection Kit, the EU VCA / IgA Detection Kit (catalog number: EI 2791-9601A), and the double-antigen sandwich method (aa1-74) described in Example 5. The EBNA1 / IgA test results are as follows: Figure 6 As shown in Figure A, the detection results of VCA / IgA are as follows: Figure 6 As shown in B. EBNA1 / IgA and VCA / IgA are usually used together as screening indicators. When both are detected together, the risk of nasopharyngeal carcinoma is calculated using the formula: LogitP = -3.934 + 2.203 × VCA / IgA + 4.797 × EBNA1 / IgA (Liu, Z., et al. 2013, Am J Epidemiol), with a threshold of 0.98. The results of the combined detection of EBNA1 / IgA and VCA / IgA are shown in Figure B. Figure 6 As shown in C, among 74 nasopharyngeal carcinoma patients and 250 healthy individuals undergoing physical examinations, 69 and 8 samples, respectively, had a risk rate exceeding 0.98, with sensitivity and specificity of 93.64% and 96.80%, respectively.

[0234] The results of the anti-BNLF2b antibody test are as follows: Figure 7 As shown, 70 serum samples from nasopharyngeal carcinoma patients and 1 sample from healthy individuals had OD values ​​exceeding 0.1. Using 0.1 as the cut-off value, the sensitivity and specificity of the anti-BNLF2b antibody detection were 94.59% and 99.60%, respectively. Further analysis using SPSS software and a chi-square test revealed no significant difference in sensitivity between the anti-BNLF2b antibody detection and the combined EBNA1 / IgA+VCA / IgA detection (P = 1.00), but the specificity of the anti-BNLF2b antibody detection was significantly higher than that of the combined EBNA1 / IgA+VCA / IgA detection (P = 0.037).

[0235] ROC curve analysis was performed on the results of the above-mentioned anti-BNLF2b antibody detection and EBNA1 / IgA+VCA / IgA combined detection, and the results are as follows: Figure 8 As shown, the AUCs for anti-BNLF2b antibody detection and the combined EBNA1 / IgA+VCA / IgA detection were 0.97 and 0.99, respectively, with no significant difference overall (P = 0.316). However, when the sensitivity was 94.59% or lower, the specificity of anti-BNLF2b antibody detection was higher than that of the combined detection. Furthermore, among the 74 nasopharyngeal carcinoma patients, 28 were stage I / II. In these 28 patients, both BNLF2b and the combined EBNA1 / IgA+VCA / IgA detection resulted in one missed detection, with a sensitivity of 96.43%.

[0236] Combining the data from Examples 5 and 6, the sensitivity of the anti-BNLF2b antibody detection was 93.55% (116 / 124), and the specificity was 99.73% (748 / 750). According to the incidence rate calculation formula in the literature (Liu, Z., et al. 2013, Am J Epidemiol), the positive predictive value of the anti-BNLF2b antibody detection was 33.2%, significantly higher than the positive predictive value of the combined EBNA1 / IgA + VCA / IgA detection (4.4%, 38 / 862) (P<0.0001). These results indicate that the anti-BNLF2b antibody detection significantly improves the specificity and positive predictive value of nasopharyngeal carcinoma screening compared to the existing combined EBNA1 / IgA + VCA / IgA screening method.

[0237] Example 7: Application of the anti-BNLF2b antibody double-antigen sandwich method in nasopharyngeal carcinoma screening

[0238] Population screening was conducted in Fusha and Nanlang towns of Zhongshan City, Guangdong Province, a high-incidence area of ​​nasopharyngeal carcinoma. 496 people were enrolled in Fusha and 829 in Nanlang, totaling 1325 people. Following the method described in Example 6 (hereinafter referred to as anti-BNLF2b antibody detection), EBNA1 / IgA, VCA / IgA, and anti-BNLF2b antibodies were simultaneously detected in these samples. The risk factor for nasopharyngeal carcinoma was calculated using the combined EBNA1 / IgA and VCA / IgA detection formula (LogitP = -3.934 + 2.203 × VCA / IgA + 4.797 × EBNA1 / IgA). The results are shown in the table below. Among the 1325 people, 163 were positive for EBNA1 / IgA, 218 for VCA / IgA, and 32 for anti-BNLF2b antibodies. Of the 126 individuals with a risk factor exceeding 0.98, 5 were ultimately diagnosed with nasopharyngeal carcinoma (NPC). Among those with a risk factor not exceeding 0.98, no prematurely diagnosed NPC cases were found in the tumor registry system. Of the 5 NPC-diagnosed specimens, only one showed VCA / IgA detection, while the other methods achieved 100% detection. In contrast, among 1320 non-NPC specimens, the specificity and positive predictive value of anti-BNLF2b antibody detection were 97.95% and 15.63%, respectively, both higher than EBNA1 / IgA, VCA / IgA, and the combined detection of both.

[0239] Table 6: Performance comparison of anti-BNLF2b antibody with existing biomarkers in population screening

[0240]

[0241] Example 8: Combining the anti-BNLF2b antibody double-antigen sandwich method with existing nasopharyngeal carcinoma screening reagents

[0242] The data from Examples 6 and 7 were combined, resulting in a total of 79 nasopharyngeal carcinoma (NPC) cases and 1570 non-NPC cases. The detection results of the 79 NPC samples are shown in Table 7-1. 70, 67, and 72 samples were positive for anti-BNLF2b antibody along with EBNA1 / IgA, VCA / IgA, and risk factor, respectively. The detection results of the 1570 non-NPC samples are shown in Table 7-2. Only 5, 5, and 4 samples were positive for anti-BNLF2b antibody along with EBNA / IgA, VCA / IgA, and risk factor, respectively. The results of the anti-BNLF2b antibody detection combined with further combined detection of EBNA / IgA, VCA / IgA, and EBNA / IgA+VCA / IgA are shown in Table 7-3. After combined detection with EBNA / IgA, the positive predictive value increased from 15.15% to 50%, and after combined detection with the risk factor, the positive predictive value increased from 15.15% to 55.56%.

[0243] Table 7-1: Detection results of 79 nasopharyngeal carcinoma specimens

[0244]

[0245] Table 7-2: Detection results of 1570 non-nasopharyngeal carcinoma specimens

[0246]

[0247]

[0248] Table 7-3: Performance of anti-BNLF2b antibody detection and its combination with existing biomarkers for nasopharyngeal carcinoma screening

[0249]

[0250] We further selected 227 specimens (sample 2) from the initial screening cohort at Zhongshan People's Hospital that were identified as high-risk by combined EBNA1 / IgA and VCA / IgA testing. Among these, 8 specimens were confirmed as nasopharyngeal carcinoma by nasopharyngoscopy. The results are shown in Table 8. 24 specimens were positive for anti-BNLF2b antibody, of which 7 were nasopharyngeal carcinoma. The sensitivity and positive predictive value of the combined detection were 87.50% and 29.17%, respectively.

[0251] Sample 2 was combined with Sample 1, which included 8 cases with a risk factor >0.98 from 250 healthy controls in Example 6 and 126 cases with a risk factor >0.98 (5 of which were nasopharyngeal carcinoma) from a screening cohort of 1325 people in Example 7. After combining the two samples, a total of 13 nasopharyngeal carcinoma samples and 348 non-nasopharyngeal carcinoma samples were obtained. The positive predictive value of the combined EBNA1 / IgA + VCA / IgA detection was 3.60%. Based on this, after anti-BNLF2b antibody testing, 12 of the 13 nasopharyngeal carcinoma samples were positive, while only 21 of the 348 non-nasopharyngeal carcinoma samples were positive, increasing the positive predictive value of the combined method to 36.36% (Table 8). Therefore, the combined use of anti-BNLF2b antibody testing and EBNA1 / IgA + VCA / IgA can further improve the specificity and positive predictive value of nasopharyngeal carcinoma screening. To reduce workload, anti-BNLF2b antibody can be detected first, followed by further detection of EBNA1 / IgA and VCA / IgA.

[0252] Table 8: Performance of anti-BNLF2b antibody detection in high-risk populations

[0253]

[0254]

[0255] Example 9: Application of the anti-BNLF2b antibody double-antigen sandwich method in the auxiliary diagnosis of nasopharyngeal carcinoma

[0256] Nasopharyngeal carcinoma (NPC) presents with nonspecific clinical symptoms, making it difficult to distinguish from other head and neck diseases. Clinically, suspected cases are primarily diagnosed using nasopharyngoscopy and pathological examination. We collected 63 suspected NPC cases, of which 31 were ultimately confirmed as NPC. We tested these 63 specimens using the double-antigen sandwich method described in Example 6. The results showed that, with a cut-off value of 0.1, 30 out of the 31 NPC cases tested positive for BNLF2b antibody, while only 2 out of 32 non-NPC cases tested positive. The sensitivity of the BNLF2b antibody test was 96.78% (30 / 31), the specificity was 93.75% (30 / 32), and the positive predictive value was 93.75% (30 / 32). These results indicate that in outpatient cases, BNLF2b antibody testing can significantly reduce the number of unnecessary nasopharyngoscopy examinations, alleviating the economic and physical burden on patients.

[0257] Example 10: Study on the immunodominant epitopes of the BNLF2b encoded protein

[0258] The BNLF2b gene encodes 98 amino acids (SEQ ID NO: 101). To analyze the immunodominant epitopes of the BNLF2b-encoded protein, we designed nine peptides using a walking assay. Each peptide contains 15 amino acids (the last peptide contains 18 amino acids), with a 5-amino acid overlap between adjacent peptides. Biotin was labeled at the C-terminus for subsequent detection (SEQ ID NOs: 92-100). Following the method described in Example 3, IgG in nasopharyngeal carcinoma patient serum diluted 1:300 was detected using the above peptide fragments as the coating antigen. The results are as follows... Figure 9 As shown, the BNLF2b-encoded protein epitopes are mainly located in four regions: aa1-25, aa31-45, aa51-65, and aa81-98.

[0259] Following the method described in Example 3, we further evaluated the sensitivity of four peptide segments—aa1-25 (SEQ ID NO:102), aa31-45 (SEQ ID NO:95), aa51-65 (SEQ ID NO:97), and aa81-98 (SEQ ID NO:100)—in nasopharyngeal carcinoma screening using 43 nasopharyngeal carcinoma serum samples. The results showed that, with 0.1 as the cutoff value, the detection sensitivity of aa51-65 was 76.7% (33 / 43), aa1-25 was 72.1% (31 / 43), and the sensitivity of the other two peptides was 55.8% (24 / 43). The sensitivities for AA14-52 and AA1-74 were 90.7% (39 / 43) and 93.0% (40 / 43), respectively. The three serum samples that were missed by AA1-74 were all negative for all four peptides, while AA14-52 was negative. The OD value of the AA1-74 positive serum reacting with AA51-65 was 0.393.

[0260] To further identify the key amino acids constituting these epitopes, we further truncated the N-terminus and C-terminus of aa1-25, aa31-45, aa51-65, and aa81-98, synthesizing a series of peptides. In this experiment, 40 nasopharyngeal carcinoma serum samples were mixed to form two mixed serum samples, diluted 1:300, and then IgG was detected according to the method described in Example 3. The results are as follows... Figure 10 As shown.

[0261] (1) Section aa1-25 ( Figure 10A): With the C-terminus fixed, the N-terminus truncated to position 16 can still bind to IgG in nasopharyngeal carcinoma serum, while truncating to position 19 completely eliminates its reactivity with IgG. Similarly, with the N-terminus fixed, the peptide can still react with IgG in nasopharyngeal carcinoma serum when the C-terminus is truncated to position 7, but completely loses its reactivity with IgG when the C-terminus is truncated to position 4. Therefore, aa1-15 and aa11-25 contain two independent epitopes, with key amino acids aa5-7 and aa16-18, respectively.

[0262] (2) Section aa31-45 ( Figure 10 B): Regardless of whether the N-terminus is truncated or the C-terminus is truncated, if the amino acid EDR at position 37-39 is not present, the polypeptide will completely lose its reactivity with IgG in nasopharyngeal carcinoma serum. Therefore, KER (aa31-33) and EDR (aa37-39) are the key amino acids that constitute this epitope.

[0263] (3) Section aa51-65 ( Figure 10 C): When the N-terminus does not contain aa54-56 or the C-terminus does not contain aa60, the polypeptide completely loses its reactivity with nasopharyngeal carcinoma serum.

[0264] (4) Section aa81-98 Figure 10 D): When the N-terminus does not contain aa87-89 or the C-terminus does not contain aa96-98, the polypeptide completely loses its reactivity with nasopharyngeal carcinoma serum.

[0265] To analyze whether the aa1-25 region contains epitopes spanning both aa1-15 and aa11-25 regions, we detected IgG in 43 nasopharyngeal carcinoma serum samples using aa1-15, aa11-25, and aa1-25 as coating antigens, respectively. The results are as follows: Figure 11 As shown in Figure A, the OD values ​​of the 14 serum samples reacting to aa1-25 were higher than the sum of those reacting to aa1-15 and aa11-25. Further analysis was conducted on these 14 serum samples using different truncated polypeptides within the aa1-25 region as coating antigens, and the results are as follows. Figure 11 As shown in B, the polypeptides that can react with positive serum contain at least aa10-16.

[0266] In addition, for the four key segments mentioned above, we synthesized a series of variants, including natural and artificial variants. Following the method in Example 3, we tested the reactivity of these variants as coating antigens with a 1:300 dilution of nasopharyngeal carcinoma mixed serum IgG. The results are as follows... Figure 12 As shown.

[0267] (1)aa1-25( Figure 12A): The most significant decrease was observed after mutations in amino acids at positions 6, 9-11, and 16; there are three natural mutations at position 12, namely T, D, and S, and mutating alanine to these three amino acids does not significantly reduce the reactivity of aa1-25 with nasopharyngeal carcinoma serum; in addition, the effects of mutations in amino acids at positions 5, 7, 8, 13, 14, 15, 19, 22, 24, and 25 are relatively small.

[0268] (2)aa31-45( Figure 12 B): After K31, R33, D38 and R39 were mutated to alanine, the reactivity of the polypeptide with nasopharyngeal carcinoma serum decreased significantly, indicating that these amino acids are key amino acids constituting this epitope; in addition, the effects of mutations in amino acids at positions 32, 34, 35, 36, 37, 40, 41 and 42 were relatively small.

[0269] (3)aa51-65( Figure 12 C): When R53 and N54 and aa56-59 are mutated to alanine, the reactivity of the peptide with nasopharyngeal carcinoma serum is significantly reduced, while the effects of amino acid mutations at positions 52, 55, 60, and 61 are relatively small.

[0270] (4)aa81-98( Figure 12 D): When the three amino acids aa95-97 are mutated to alanine, the reactivity of the polypeptide with nasopharyngeal carcinoma serum decreases most significantly; in addition, the effects of mutations at positions 89, 91, 93, and 98 are relatively small.

[0271] The results above show that the core regions of the BNLF2b encoded protein are aa5-11, aa16-23, aa31-33, aa37-39, aa53-60, and aa89-98.

[0272] Example 11: The effect of combined use of different epitopes of the BNLF2b encoded protein on nasopharyngeal carcinoma screening performance

[0273] Based on the previously synthesized aa14-52, aa1-74, aa1-52, and aa1-25, two further polypeptides, aa11-65 (SEQ ID NO:103) and aa11-74 (SEQ ID NO:104), were synthesized and biotin-labeled at their C-termini. Following the method in Example 3, the reactivity of these polypeptides with serum samples from 84 nasopharyngeal carcinoma patients and 168 healthy controls was tested. The reactivity of all six polypeptides with nasopharyngeal carcinoma serum was significantly higher than that with the healthy control group (…). Figure 13ROC curve analysis using Medcal software showed that the areas under the curve for all six peptides were above 0.95, with sensitivity and specificity both exceeding 89% and 96%, respectively (Table 9). These results indicate that BNLF2b-encoded peptide fragments containing one or more core regions (aa5-11, aa16-23, aa31-33, aa37-39, aa53-60) possess excellent screening performance for nasopharyngeal carcinoma.

[0274] Table 9: Comparison of the performance of different peptides as capture antigens in nasopharyngeal carcinoma screening

[0275]

[0276] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

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[0312]

[35] Rao Dongping, Liu Qing, Cao Sumei. Cost-effectiveness evaluation of nasopharyngeal carcinoma screening using Markov model. Chinese Journal of Oncology. 2012; 34: 549-53. SEQUENCE LISTING <110> Xiamen University Xiamen Wantai Kerry Biotechnology Co., Ltd. <120> EB virus BNLF2b gene-encoded polypeptide and its detection applications <130> IDC190271 <160> 104 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 1, aa467-486 <400> 1 Ala Asn Ala Ala Cys Ala Asp Asp Val Thr His His Asp Arg Arg Ala 1 5 10 15 Asp Asn Ser Ala 20 <210> 2 <211> 20 <212> PRT <213> Synthetic Sequence <220> <223> EBV ORF 2, aa90 - 109 <400> 2 Gln Ala Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn Ser Leu 1 5 10 15 Gly Glu Asn Leu 20 <210> 3 <211> 20 <212> PRT <213> Synthetic Sequence <220> <223> EBV ORF 3, aa138 - 157 <400> 3 Leu Gly Ala Gln Ala Pro Glu Ser Arg Gly Gln Gly His Leu Arg Val 1 5 10 15 Pro Pro Arg Val 20 <210> 4 <211> 20 <212> PRT <213> Synthetic Sequence <220> <223> EBV ORF 15, aa1z - 31 <400> 4 Val Leu Pro Glu Pro Val Val Gly Gln Val His Ala Asn Asn Leu Leu 1 5 10 15 Note: There seems to be a potential error in the original text where "aa1z - 31" in line 62 might be a typo. It should probably be "aa12 - 31" as in the previous similar entries. The translation is done based on the provided text with this potential clarification in mind. Ser Thr Arg Ala 20 <210> 5 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 16, aa103 - 122 <400> 5 Arg Asp Ala Trp Thr Gln Glu Pro Ser Pro Leu Asp Arg Asp Pro Leu 1 5 10 15 Gly Tyr Asp Val 20 <210> 6 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 16, aa340 - 359 <400> 6 Ser Arg Gly Gln Ser Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg 1 5 10 15 Gly Lys Gly Lys 20 <210> 7 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 17, aa26 - 45 <400> 7 Arg Ser Gly Val Thr Glu Val Ala Gln Ile Ala Gly Arg Thr Pro Lys 1 5 10 15 Met Glu Asp Phe 20 <210> 8 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 18, aa110-129 <400> 8 Leu Gly Leu Ser Thr Asp Val Asp Leu Pro Lys Asn Ser Ile Ile Met 1 5 10 15 Leu Gly Gln Asp 20 <210> 9 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 19, aa394-413 <400> 9 Tyr Cys Leu Asn Phe Gly Lys Gln Thr Gly Val Gly Gly Arg Leu Asn 1 5 10 15 Ser Phe Arg Pro 20 <210> 10 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 19, aa372-391 <400> 10 His Ala Ser Arg Pro Val Ser Gly Pro Asp Tyr Pro Pro Leu Ala Val 1 5 10 15 Phe Cys Met Asp 20 <210> 11 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 20,aa63-82 <400> 11 Arg Thr Phe Gly Val Pro Arg Arg Gln Arg Ala Ile Asp Lys Arg Gln 1 5 10 15 Arg Ala Ser Val 20 <210> 12 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 20,aa101-120 <400> 12 Gln Ala Gln Ala Ala Ala Ser Ala Gly Thr Gly Ala Leu Ala Ser Ser 1 5 10 15 Ala Pro Ser Thr 20 <210> 13 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 21,aa670‑689 <400> 13 Met Ser Asp Ser Glu Glu Ala Glu Ser Asp Leu Ala Ser Asp Ile Pro 1 5 10 15 Thr Thr Glu Asp 20 <210> 14 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 22, aa330-349 <400> 14 Val Tyr Glu Ala Leu Leu Trp Asp Gln Thr Tyr Gly Val Pro Asp Ser 1 5 10 15 Val Ile Glu Ala 20 <210> 15 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 23,aa205-224 <400> 15 Thr Val Val His Gly Pro Val Val Ser Lys Ala Ile Pro Arg Ser Thr 1 5 10 15 Val Light Val Thr 20 <210> 16 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 26,aa110-129 <400> 16 Ser Leu Gln Phe Tyr Lys Arg Pro Gln Gly Gly Ser Arg Pro Glu Phe 1 5 10 15 Val Lys Leu Thr 20 <210> 17 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 27,aa256-275 <400> 17 Ala Met Ala Ser Gly Gly Leu Tyr Leu Gly His Ser Ser Ile Ile Ala 1 5 10 15 Cys Val Met Ala 20 <210> 18 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 28, aa9-28 <400> 18 Thr Asp Gly Glu Ile Ser Ser Ser Glu Glu Glu Asp Glu Asp Pro Thr 1 5 10 15 Pro Ala His Ala 20 <210> 19 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 29, aa1-20 <400> 19 Met Val Pro Ser Gln Arg Leu Ser Arg Thr Ser Ser Ser Ile Ser Ser Asn 1 5 10 15 Glu Asp Pro Ala 20 <210> 20 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 30, aa789-801 <400> 20 His Thr His Arg Ala Ser Ser Lys Asn Val Arg Val Phe Leu Val Leu 1 5 10 15 Tyr Tyr Thr Ser 20 <210> twenty one <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 31, aa185-204 <400> twenty one Ile Asn Ser Ala Phe Glu Pro Glu Val Pro Thr Pro Val Leu Ala Pro 1 5 10 15 Pro Pro Val Val 20 <210> twenty two <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 32, aa246-265 <400> twenty two Cys Thr Phe Glu Glu Val Pro Ser Leu Ala Met Gly Asp Ser Gly Leu 1 5 10 15 Ser Glu Ala Leu 20 <210> twenty three <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 33, aa26-45 <400> twenty three Gly Val Leu Pro Ala Gly Ala Ser Ser Pro Thr Asn Ala Ala Ala Ala 1 5 10 15 Ser Leu Thr Glu 20 <210> twenty four <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 34,aa134-153 <400> 24 Pro Arg Glu Ser Asn Asp Pro Asn Ala Thr Arg Arg Ala Arg Ser Arg 1 5 10 15 Ser Arg Gly Arg 20 <210> 25 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 34, aa61-80 <400> 25 Glu Pro Leu Thr Ala Arg Gln Arg Glu Val Met Ile Thr Gln Ala Thr 1 5 10 15 Gly Arg Leu Ala 20 <210> 26 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 35,aa665-684 <400> 26 Met Ser Leu Arg Pro Ser Ser Asn Pro Glu Thr Leu Ser Pro Ser Thr 1 5 10 15 See Asp Asn See 20 <210> 27 <211> 20 <212> PRT <213> artificial sequence <220> <223> EBV ORF 35,aa822-841 <400> 27 Pro Ser Thr Ser Ser Lys Leu Arg Pro Arg Trp Thr Phe Thr Ser Pro 1 5 10 15 Pro Val Thr Thr 20 <210> 28 <211> 20 <212> PRT <213> Synthetic sequence <220> <223> EBV ORF 36, aa60 - 79 <400> 28 Pro Gln Gln Val Glu Arg Pro Ile Leu Pro Pro Val Glu Ser Thr Pro 1 5 10 15 Gln Asp Met Glu 20 <210> 29 <211> 20 <212> PRT <213> Synthetic sequence <220> <223> EBV ORF 37, aa92 - 111 <400> 29 Ala Ile Leu Arg Arg Phe Pro Leu Asp Leu Arg Thr Leu Leu Gln Ala 1 5 10 15 Ile Gly Ala Ala 20 <210> 30 <211> 20 <212> PRT <213> Synthetic sequence <220> <223> EBV ORF 38, aa1 - 20 <400> 30 Val Ser Ala His Arg His Thr Ser His Arg Pro Val Phe Arg Ile Pro 1 5 10 15 Asp Phe Lys Tyr 20 <210> 31 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 39, aa105-124 <400> 31 Ala Pro Val Ile Gln Leu Val His Ala Val Tyr Asp Ser Met Leu Gln 1 5 10 15 Ser Asp Leu Arg 20 <210> 32 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 39, aa593-612 <400> 32 Ala Gln Thr Pro Trp Pro Val Val Gln Pro Ser Gln Thr Pro Asp Asp 1 5 10 15 Pro Thr Lys Gln 20 <210> 33 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 40, aa568-587 <400> 33 Ala Ala Gly Pro Pro Ala Ala Gly Pro Pro Ala Ala Gly Pro Pro Ala 1 5 10 15 Ala Gly Pro Pro 20 <210> 34 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 41, aa63-82 <400> 34 Phe Asn Lys Thr Ala Glu Gln Glu Tyr Gly Asp Lys Glu Val Lys Leu 1 5 10 15 Pro His Trp Thr 20 <210> 35 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 41, aa177-196 <400> 35 Leu Asp Gly Gly Thr Phe Lys Val Tyr Gln Ile Phe Gly Ser His Cys 1 5 10 15 Thr Tyr Val Ser 20 <210> 36 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 42, aa179-198 <400> 36 Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Cys Arg Ala Lys Phe Lys 1 5 10 15 Gln Leu Leu Gln 20 <210> 37 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 42, aa1-20 <400> 37 Met Met Asp Pro Asn Ser Thr Ser Glu Asp Val Lys Phe Thr Pro Asp 1 5 10 15 Pro Tyr Gln Val 20 <210> 38 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 43, aa444-463 <400> 38 Gln Pro Lys Arg Ile Arg Pro Phe His Pro Pro Gly Ser Pro Trp Ala 1 5 10 15 Asn Arg Pro Leu 20 <210> 39 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 44, aa126-145 <400> 39 Arg Lys Leu Arg Gln Asp Arg His His Ala Ser Val Asn Val Leu Met 1 5 10 15 Pro Gly Ser Asp 20 <210> 40 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 44, aa2-21 <400> 40 Ala Ser Ser Asn Arg Gly Asn Ala Arg Pro Leu Lys Ser Phe Leu His 1 5 10 15 Glu Leu Tyr Leu 20 <210> 41 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 45, aa495-514 <400> 41 Glu Asp Gly Ser Glu Asp Gly Glu Phe Ser Asp Leu Asp Leu Ser Asp 1 5 10 15 Ser Asp His Glu 20 <210> 42 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 45, aa10-29 <400> 42 Ile Leu Val Pro Glu His Leu Ala Gly Ala Leu Thr Lys Leu Met Ser 1 5 10 15 Asp Phe Ile Thr 20 <210> 43 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 47, aa60-79 <400> 43 Ser Leu Ala Ser Leu Asn Ser Pro Lys Asn Gly Ser Asn Gln Leu Val 1 5 10 15 Ile Ser Arg Cys 20 <210> 44 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 48, aa212-231 <400> 44 Gln His Pro Ser Pro Leu Ala Gln Asn Ser Thr Arg Lys Ser Ala Gln 1 5 10 15 Gln Lys Phe Leu 20 <210> 45 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 50, aa45-64 <400> 45 Asp Met Glu Thr Phe Ser Pro Glu Phe Asp Pro Glu Leu Ser Glu Pro 1 5 10 15 Pro Phe Leu Pro 20 <210> 46 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 52, aa211-230 <400> 46 Thr Ser Gly Lys Met Gly Cys Leu Ala Arg Ser Pro Lys Asp Tyr Cys 1 5 10 15 Ala Asp Leu Asn 20 <210> 47 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 53, aa166-185 <400> 47 Ile Met Ala Phe Leu Leu Asp Val Ile Ser Ala Leu His Pro Gly Tyr 1 5 10 15 Thr Ile Pro Met 20 <210> 48 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 53, aa1-20 <400> 48 Ala Phe Leu Gln Gly Val Lys Asp Ser Glu Asp Ala Ser Arg Leu Asp 1 5 10 15 Arg Asp Val Met 20 <210> 49 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 54, aa209-228 <400> 49 Thr Glu Thr Pro Asp Asp Pro Ser Pro Val Pro Phe Arg Asp Ile Leu 1 5 10 15 Arg Pro Val Thr 20 <210> 50 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 54, aa104-123 <400> 50 Ile Ala Met Leu Ala Asp Ala Ala Glu Lys Asp Leu Phe Asp Leu Ser 1 5 10 15 Phe Arg Thr Arg 20 <210> 51 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 55, aa357-376 <400> 51 Thr Pro Ser Pro Gly Arg Asn Arg Arg Arg Ser Ser Thr Ser Ser Ser 1 5 10 15 Ser Ser Arg Ser 20 <210> 52 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 56, aa50-69 <400> 52 Glu Thr Asn Glu Gly Leu Glu Tyr Asp Glu Asp Ser Glu Asn Asp Glu 1 5 10 15 Leu Leu Phe Leu 20 <210> 53 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 57, aa326-345 <400> 53 Met Thr Asp Pro Ser Glu Asn Ala Gly Arg Ile Gly Ile Lys Asp Arg 1 5 10 15 Val Pro Val Asn 20 <210> 54 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 59, aa55-74 <400> 54 Pro Gln Asp Asn Arg Pro Trp His Glu Ala Arg Ser Ser Gly Arg Val 1 5 10 15 Ala Glu Asp Asp 20 <210> 55 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 59, aa231-250 <400> 55 Ile Ala Pro Pro Phe Asp Val Ser Arg Leu Asn Lys Met Ala Lys Gln 1 5 10 15 Leu Cys Leu Leu 20 <210> 56 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 60, aa195-214 <400> 56 Thr Met Phe Asp Ile Phe Gln Ser Ala Phe Gly Leu Glu Glu Met Thr 1 5 10 15 Leu Glu Lys Leu 20 <210> 57 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 60, aa82-101 <400> 57 Ser Leu Pro Met Phe Gly Ala Ser Pro Ala Leu His Thr Pro Val Gln 1 5 10 15 Val Gln Met Cys 20 <210> 58 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 61, aa18-37 <400> 58 Leu Gln Gln Asp Ser Thr Thr Gln Gly Cys Leu Gly Ala Glu Thr Pro 1 5 10 15 Ser Ile Met Tyr 20 <210> 59 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 62, aa41-60 <400> 59 Val Ser Ala Val Tyr Val Pro Gln Ile Ala Gly Pro Pro Lys Thr Tyr 1 5 10 15 Met Asn Val Thr 20 <210> 60 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 63, aa280-299 <400> 60 Glu Ser Arg Ala Leu Val Arg Gly Ile Ala His Ile Phe Ser Pro His 1 5 10 15 Ala Leu Tyr Val 20 <210> 61 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 64, aa88-107 <400> 61 Arg Pro Trp Thr Glu Ile Arg Gln Asp Thr Gln Asp Gln Arg Asp Lys 1 5 10 15 Trp Glu Pro Glu 20 <210> 62 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 64, aa154-173 <400> 62 Phe Val Phe Pro Cys Cys Leu Met Leu Phe Arg Gly Ala Ser Ser Glu 1 5 10 15 Lys Val Val Asp 20 <210> 63 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 65, aa118 - 137 <400> 63 Thr Gly Thr Ser Thr Gly Val Thr Ser Asn Val Thr Thr Arg Ser Ser 1 5 10 15 Ser Thr Thr Ser 20 <210> 64 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 66, aa88 - 107 <400> 64 Gly Glu Thr Lys Thr Asn Thr Gln Asp Gln Asn Gln Asn Gln Thr Thr 1 5 10 15 Arg Thr Arg Thr 20 <210> 65 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 66, aa359 - 378 <400> 65 Lys Asp Val Leu Ala Phe Arg Pro Ser Leu Val Thr Asn Cys Thr Ala 1 5 10 15 Pro Leu Lys Thr 20 <210> 66 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 67, aa244 - 263 <400> 66 Pro Asp Glu Ile Ala Arg Ile Asp Leu Asp Asp Leu Ser Val Ala Asp 1 5 10 15 Asp Leu Ser Arg 20 <210> 67 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 68, aa120 - 139 <400> 67 Lys Asn Cys His Lys His His Ile Ser Thr Glu Met Glu Leu Ser Met 1 5 10 15 Leu Asp Leu Glu 20 <210> 68 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 69, aa17 - 36 <400> 68 Phe Ser Gly Arg Glu Ala Arg Leu Lys Phe His Phe Phe Ser Trp Ser 1 5 10 15 Thr Phe Met Leu 20 <210> 69 <211> 20 <212> PRT <213> Artificial Sequence <220> [[ID=,62]]<223> EBV ORF 70, aa171 - 190 <400> 69 [[ID=,66]]Glu Ser Val Lys Arg Val Arg Val Asp Glu Gly Ala Asn Thr Arg Arg 1 5 10 15 Thr Ile Arg Asp 20 <210> 70 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 71, aa201-220 <400> 70 Thr Lys Asp Leu Pro Asp Leu Arg Gly Pro Phe Ser Tyr Pro Ser Leu 1 5 10 15 Thr Ser Ala Gln 20 <210> 71 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 72, aa143-162 <400> 71 Pro Phe Ser Trp Phe Arg Lys Thr Ser Cys Thr Glu Gly Gly Ala Asp 1 5 10 15 Ser Thr Ser Arg 20 <210> 72 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 73, aa195-214 <400> 72 Arg Pro Arg Pro Thr Ala Gln Gly His Arg Pro Arg Thr His Val Gly 1 5 10 15 Pro Lys Pro Ser 20 <210> 73 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 73, aa154-173 <400> 73 Ser Leu Arg Thr Leu Gln Val Glu Phe Pro Ala Leu Gly Gln Lys Thr 1 5 10 15 Leu Pro Thr Ser 20 <210> 74 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 75, aa514-533 <400> 74 Ala Gln Pro Pro Pro Pro Gly Thr Gln Ala Pro Glu Ala His Cys Val 1 5 10 15 Ala Glu Ser Thr 20 <210> 75 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 75, aa432-451 <400> 75 Asp Ala Thr Leu Tyr Arg Lys Asp Ile Ala Gly Leu Ser Lys Ser Val 1 5 10 15 Asn Glu Leu Gln 20 <210> 76 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 76, aa149-168 <400> 76 Ser Arg Ser Asn Lys Arg Lys Arg Asp Pro Glu Glu Asp Glu Glu Gly 1 5 10 15 Gly Gly Leu Phe 20 <210> 77 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 77, aa180-199 <400> 77 Arg Pro Val Ser Lys Arg Pro Thr His Lys Pro Val Thr Leu Gly Pro 1 5 10 15 Phe Pro Ile Asp 20 <210> 78 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 78, aa19-38 <400> 78 Thr Ser Val Asn Ala Thr Glu Asp Ala Cys Thr Lys Ser Tyr Ser Ala 1 5 10 15 Phe Leu Ser Gly 20 <210> 79 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 79, aa323-342 <400> 79 Trp Ser Thr Gly Glu Glu Ala Gly Arg Tyr Arg Arg Ile Leu Leu Thr 1 5 10 15 Leu Gly Thr Cys 20 <210> 80 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 79, aa579 - 598 <400> 80 Pro Val Leu Val Val Asp Phe Ala Ser Leu Tyr Pro Ser Ile Ile Gln 1 5 10 15 Ala His Asn Leu 20 <210> 81 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 80, aa32 - 51 <400> 81 Ala Thr Thr Val Gln Pro Thr Ala Thr Arg Gln Gln Thr Ser Phe Pro 1 5 10 15 Phe Arg Val Cys 20 <210> 82 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 81, aa11 - 30 <400> 82 Leu Arg Pro Gly Gly Gln Arg Pro Arg Asn Pro Gly Asp His Cys Leu 1 5 10 15 Gln Arg Asp Arg 20 <210> 83 <211> 20 <212> PRT <213> Synthetic sequence <220> <223> EBV ORF 82, aa155 - 174 <400> 83 Glu Ala Phe Lys Glu Arg Leu Tyr Ser Gly Asn Leu Val Ala Ile Pro 1 5 10 15 Ser Leu Lys Gln 20 <210> 84 <211> 20 <212> PRT <213> Synthetic sequence <220> <223> EBV ORF 83, aa66 - 85 <400> 84 Lys Val Ala Glu Ser Ser Tyr Leu Met Phe Arg Ala Met Tyr Ala Val 1 5 10 15 Phe Thr Arg Asp 20 <210> 85 <211> 20 <212> PRT <213> Synthetic sequence <220> <223> EBV ORF 84, aa188 - 207 <400> 85 Asp Leu Ser Leu Pro Lys Pro Trp His Leu Pro Val Thr Cys Val Gly 1 5 10 15 Lys Asn Asp Lys 20 <210> 86 <211> 20 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa31 - 50 <400> 86 Lys Glu Arg Val Pro Val Glu Asp Arg Pro Pro Thr Pro Met Pro Thr 1 5 10 15 Ser Gln Arg Leu 20 <210> 87 <211> 20 <212> PRT <213> Artificial sequence <220>[[ID=�0]] <223> EBV ORF 86, aa19 - 38 <400> 87 Leu Pro Gly Ser Ser Thr Glu Thr Arg Pro Ser His Pro Cys Pro Glu 1 5 10 15 Asp Pro Asp Val 20 <210> 88 <211> 39 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa14 - 52 <400> 88 Leu Arg Arg Ser Phe Arg Arg Met Ser Lys Arg Ser Lys Asn Lys Ala 1 5 10 15 Lys Lys Glu Arg Val Pro Val Glu Asp Arg Pro Pro Thr Pro Met Pro 20 25 30 Thr Ser Gln Arg Leu Ile Arg 35 <210> 89 <211> 74 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa1-74 <400> 89 Met Arg Pro Gly Arg Pro Leu Ala Gly Phe Tyr Ala Thr Leu Arg Arg 1 5 10 15 Ser Phe Arg Arg Met Ser Lys Arg Ser Lys Asn Lys Ala Lys Lys Glu 20 25 30 [[ID=**23**]] 35 40 45 Arg Leu Ile Arg Arg Asn Ala Leu Gly Gly Gly Val Arg Pro Asp Ala 50 55 60 Glu Asp Cys Ile Gln Arg Phe His Pro Leu 65 70 <210> 90 <211> 61 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa14-74 <400> 90 Leu Arg Arg Ser Phe Arg Arg Met Ser Lys Arg Ser Lys Asn Lys Ala 1 5 10 15 Lys Lys Glu Arg Val Pro Val Glu Asp Arg Pro Pro Thr Pro Met Pro 20 25 30 ​Thr Ser Gln Arg Leu Ile Arg Arg Asn Ala Leu Gly Gly Gly Val Arg 35 40 45 Pro Asp Ala Glu Asp Cys Ile Gln Arg Phe His Pro Leu 50 55 60 <210> 91 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 85, aa1-52 <400> 91 Met Arg Pro Gly Arg Pro Leu Ala Gly Phe Tyr Ala Thr Leu Arg Arg 1 5 10 15 Ser Phe Arg Arg Met Ser Lys Arg Ser Lys Asn Lys Ala Lys Lys Glu 20 25 30 Arg Val Pro Val Glu Asp Arg Pro Pro Thr Pro Met Pro Thr Ser Gln 35 40 45 Arg Leu Ile Arg 50 <210> 92 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 85, aa1-15 <400> 92 Met Arg Pro Gly Arg Pro Leu Ala Gly Phe Tyr Ala Thr Leu Arg 1 5 10 15 <210> 93 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 85, aa11 - 25 <400> 93 Tyr Ala Thr Leu Arg Arg Ser Phe Arg Arg Met Ser Lys Arg Ser 1 5 10 15 <210> 94 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 85, aa21 - 35 <400> 94 Met Ser Lys Arg Ser Lys Asn Lys Ala Lys Lys Glu Arg Val Pro 1 5 10 15 <210> 95 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 85, aa31 - 45 <400> 95 Lys Glu Arg Val Pro Val Glu Asp Arg Pro Pro Thr Pro Met Pro 1 5 10 15 <210> 96 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> EBV ORF 85, aa41 - 55 <400> 96 Pro Thr Pro Met Pro Thr Ser Gln Arg Leu Ile Arg Arg Asn Ala 1 5 10 15 <210> 97 <211> 15 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa51-65 <400> 97 Ile Arg Arg Asn Ala Leu Gly Gly Gly Val Arg Pro Asp Ala Glu 1 5 10 15 <210> 98 <211> 15 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa61-75 <400> 98 Arg Pro Asp Ala Glu Asp Cys Ile Gln Arg Phe His Pro Leu Glu 1 5 10 15 <210> 99 <211> 15 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa71-85 <400> 99 Phe His Pro Leu Glu Pro Ala Leu Gly Val Ser Thr Lys Asn Phe 1 5 10 15 <210> 100 <211> 18 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa81-98 <400> 100 Ser Thr Lys Asn Phe Asp Leu Leu Ser Leu Arg Cys Glu Leu Gly Trp 1 5 10 15 Cys Gly <210> 101 <211> 98 <212> PRT <213> Artificial sequence <220> <223> The full-length protein encoded by the BNLF2b gene <400> 101 Met Arg Pro Gly Arg Pro Leu Ala Gly Phe Tyr Ala Thr Leu Arg Arg 1 5 10 15 Ser Phe Arg Arg Met Ser Lys Arg Ser Lys Asn Lys Ala Lys Lys Glu 20 25 30 Arg Val Pro Val Glu Asp Arg Pro Pro Thr Pro Met Pro Thr Ser Gln 35 40 45 Arg Leu Ile Arg Arg Asn Ala Leu Gly Gly Gly Val Arg Pro Asp Ala 50 55 60 Glu Asp Cys Ile Gln Arg Phe His Pro Leu Glu Pro Ala Leu Gly Val 65 70 75 80 Ser Thr Lys Asn Phe Asp Leu Leu Ser Leu Arg Cys Glu Leu Gly Trp 85 90 95 Cys Gly <210> 102 <211> 25 <212> PRT <213> Artificial sequence <220> <223> EBV ORF 85, aa1-25 <400> 102 Met Arg Pro Gly Arg Pro Leu Ala Gly Phe Tyr Ala Thr Leu Arg Arg 1 5 10 15 Ser Phe Arg Arg Met Ser Lys Arg Ser 20 25 <210> 103 <211> 55 <212> PRT <213> artificial sequence <220> <223> EBV ORF 85,aa11‑65 <400> 103 Tyr Ala Thr Leu Arg Arg Ser Phe Arg Arg Met Ser Lys Arg Ser Lys 1 5 10 15 Asn Lys Ala Lys Lys Glu Arg Val Pro Val Glu Asp Arg Pro Pro Thr 20 25 30 Pro Met Pro Thr Ser Gln Arg Leu Ile Arg Arg Asn Ala Leu Gly Gly 35 40 45 Gly Val Arg Pro Asp Ala Glu 50 55 <210> 104 <211> 64 <212> PRT <213> artificial sequence <220> <223> EBV ORF 85,aa11‑74 <400> 104 Tyr Ala Thr Leu Arg Arg Ser Phe Arg Arg Met Ser Lys Arg Ser Lys 1 5 10 15 Asn Lys Ala Lys Lys Glu Arg Val Pro Val Glu Asp Arg Pro Pro Thr 20 25 30 Pro Met Pro Thr Ser Gln Arg Leu Ile Arg Arg Asn Ala Leu Gly Gly 35 40 45 Gly Val Arg Pro Asp Ala Glu Asp Cys Ile Gln Arg Phe His Pro Leu 50 55 60

Claims

1. The use of reagents capable of measuring levels of antibodies specific to the protein encoded by the BNLF2b gene in the preparation of a kit for determining whether a subject has nasopharyngeal carcinoma or is at risk of developing nasopharyngeal carcinoma; wherein, The reagent capable of determining the level of an antibody specific to the protein encoded by the BNLF2b gene comprises a capture reagent selected from isolated peptides or variants thereof, wherein: (1) The isolated polypeptide consists of 39 to 74 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and contains sequences selected from the following: amino acid residues 14-52, 1-52, 11-65, 11-74, or 1-74 of the wild-type protein encoded by the BNLF2b gene; (2) The variant differs from the polypeptide from which it is derived only in the substitution of one amino acid residue and retains the activity of the polypeptide from which it is derived to be recognized and bound by the anti-EBV antibody; and the substitution of one amino acid residue is located at one of the following amino acid positions corresponding to the wild-type protein: 5, 7, 8, 12, 13, 14, 15, 19, 22, 24, 25, 32, 34, 35, 36, 37, 40, 41, 42, 52, 55, 60, 61, 89, 91, 93 or 98; The amino acid sequence of the wild-type protein encoded by the BNLF2b gene is shown in SEQ ID NO:

101.

2. The use as described in claim 1, wherein, The amino acid sequences of amino acid residues at positions 14-52 are shown in SEQ ID NO: 88; the amino acid sequences of amino acid residues at positions 1-52 are shown in SEQ ID NO: 91; the amino acid sequences of amino acid residues at positions 11-65 are shown in SEQ ID NO: 103; the amino acid sequences of amino acid residues at positions 11-74 are shown in SEQ ID NO: 104; and the amino acid sequences of amino acid residues at positions 1-74 are shown in SEQ ID NO:

89.

3. The use as described in claim 1, wherein, The isolated polypeptide consists of sequences selected from the following: amino acid residues 14-52, 1-52, 11-65, 11-74, or 1-74 of the wild-type protein encoded by the BNLF2b gene.

4. The use as described in claim 1, wherein, The variant differs from the isolated polypeptide only in one of the following amino acid substitutions: A at position 5, A at position 7, G at position 8, G, T, D, or S at position 12, A at position 13, G at position 14, A at position 15, A at position 22, A at position 24, A at position 25, A at position 32, A at position 34, and A at position 35. The amino acid at position 36 is replaced with A, the amino acid at position 37 is replaced with A, N, Q, S or R, the amino acid at position 40 is replaced with A, the amino acid at position 41 is replaced with A, the amino acid at position 42 is replaced with A, the amino acid at position 52 is replaced with K, H, A, S or D, the amino acid at position 55 is replaced with S, the amino acid at position 60 is replaced with A, the amino acid at position 61 is replaced with K, H, S or A, the amino acid at position 89 is replaced with A or T, the amino acid at position 91 is replaced with A, the amino acid at position 93 is replaced with Q, and the amino acid at position 98 is replaced with A.

5. The use as described in claim 1, wherein, The isolated polypeptide or its variants are attached to the surface of a solid support, or have modifying groups that can be attached to the solid support.

6. The use as described in claim 5, wherein, The modifying group is biotin or avidin.

7. The use as described in claim 5, wherein, The solid support is selected from magnetic beads or microtiter plates.

8. The use as described in claim 7, wherein, The microtiter plate is a microplate or an enzyme-labeled plate.

9. The use as described in claim 1, wherein, The reagent can be used to determine the level of antibodies specific to the protein encoded by the BNLF2b gene via immunological assay.

10. The use as described in claim 9, wherein, The immunological assays are selected from enzyme immunoassay, chemiluminescent immunoassay, fluorescence immunoassay or radioimmunoassay.

11. The use as described in claim 1, wherein, The kit determines whether a subject has nasopharyngeal carcinoma or is at risk of nasopharyngeal carcinoma based on the level of antibodies specific to the protein encoded by the BNLF2b gene in a sample from the subject, wherein the subject is a human.

12. The use as described in claim 1, wherein, The kit determines whether a subject has nasopharyngeal carcinoma or is at risk of nasopharyngeal carcinoma based on the level of antibodies specific to the protein encoded by the BNLF2b gene in a sample from the subject, wherein the sample is a blood sample.

13. The use as described in claim 12, wherein, The blood sample was whole blood.

14. The use as described in claim 12, wherein, The blood sample is plasma or serum.

15. The use according to any one of claims 1-14, wherein, The reagent capable of determining the level of an antibody specific to the protein encoded by the BNLF2b gene further comprises a detection reagent selected from isolated polypeptides or variants thereof with a detectable label, wherein the isolated polypeptides or variants thereof are as defined in any one of claims 1-4.

16. The use as described in claim 15, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, or biotin.

17. The use as described in claim 16, wherein, The enzyme is selected from horseradish peroxidase or alkaline phosphatase, and / or the chemiluminescent reagent is selected from acridine ester compounds.

18. The use according to any one of claims 1-14, wherein, The reagent capable of determining the level of an antibody specific to the protein encoded by the BNLF2b gene also includes a detection reagent selected from secondary antibodies with a detectable label.

19. The use as described in claim 18, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, or biotin.

20. The use as described in claim 19, wherein, The enzyme is selected from horseradish peroxidase or alkaline phosphatase, and / or the chemiluminescent reagent is selected from acridine ester compounds.

21. The use as described in claim 18, wherein, The secondary antibody is specific to the species from which the test antibody originates.

22. The use as described in claim 18, wherein, The secondary antibody is an anti-immunoglobulin antibody.

23. The use as described in claim 22, wherein, The anti-immunoglobulin antibody is selected from anti-IgG antibody, anti-IgM antibody or anti-IgA antibody.

24. A kit for determining the level of an antibody specific to the protein encoded by the BNLF2b gene in a sample from a subject and / or for determining whether the subject has nasopharyngeal carcinoma or is at risk of developing nasopharyngeal carcinoma, wherein, The kit contains a capture reagent as defined in any one of claims 1-4.

25. The kit of claim 24, wherein, The capture reagent is defined as in any one of claims 5-8.

26. The kit of claim 24, wherein, The subjects were human.

27. The kit of claim 24, further comprising a detection reagent, wherein, The detection reagent is defined as in any one of claims 15-17.

28. The kit of claim 24, further comprising a detection reagent, wherein, The detection reagent is defined as in any one of claims 18-23.

29. The kit of claim 24, wherein, The kit also includes one or more reagents or devices selected from: (i) a device for collecting or storing samples from a subject; and (ii) other reagents required for performing the assay, the other reagents being selected from buffer solutions, diluents, blocking solutions, and / or standards.

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