WO2021136082A1 - EB病毒BNLF2b基因编码多肽及其检测用途 - Google Patents

EB病毒BNLF2b基因编码多肽及其检测用途 Download PDF

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WO2021136082A1
WO2021136082A1 PCT/CN2020/139310 CN2020139310W WO2021136082A1 WO 2021136082 A1 WO2021136082 A1 WO 2021136082A1 CN 2020139310 W CN2020139310 W CN 2020139310W WO 2021136082 A1 WO2021136082 A1 WO 2021136082A1
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amino acid
acid residues
bnlf2b
gene
antibody
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PCT/CN2020/139310
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English (en)
French (fr)
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李廷栋
葛胜祥
郭小怡
洪聪明
宋浏伟
温顺华
汤家宝
张军
夏宁邵
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厦门大学
厦门万泰凯瑞生物技术有限公司
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Priority to US17/790,438 priority Critical patent/US20230324396A1/en
Priority to KR1020227025634A priority patent/KR20220119148A/ko
Priority to AU2020416358A priority patent/AU2020416358B2/en
Priority to JP2022540998A priority patent/JP2023509181A/ja
Priority to EP20909593.4A priority patent/EP4086274A4/en
Publication of WO2021136082A1 publication Critical patent/WO2021136082A1/zh
Priority to AU2024202271A priority patent/AU2024202271A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/03—Herpetoviridae, e.g. pseudorabies virus
    • C07K14/05—Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/05—Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to immunological detection, especially the field of immunological diagnosis of tumors.
  • the present invention provides a method for diagnosing nasopharyngeal carcinoma based on the level of anti-EB virus (EBV) antibodies, and a kit for the method.
  • the present invention also provides the polypeptide encoded by the Epstein-Barr virus BNLF2b gene used in the above diagnosis and its use in the diagnosis of nasopharyngeal carcinoma.
  • Nasopharyngeal carcinoma is a malignant tumor that tends to occur on the top and side walls of the nasopharyngeal cavity, and its incidence ranks first among malignant tumors of the ear, nose and throat.
  • my country is an area with a high incidence of nasopharyngeal cancer, with the highest incidence in Guangdong and Guangxi [1].
  • the occurrence of nasopharyngeal carcinoma is related to EB virus infection, genetic factors, environmental factors and other factors [2]. Since the early symptoms of nasopharyngeal carcinoma are not obvious, and the location of the onset is relatively secret, most nasopharyngeal carcinomas can not be found until the late stage [3].
  • the five-year survival rate of early nasopharyngeal carcinoma can reach more than 90%, while the prognosis of advanced nasopharyngeal carcinoma is significantly reduced [3].
  • early nasopharyngeal carcinoma can be treated with local radiotherapy, while advanced nasopharyngeal carcinoma needs chemotherapy. Therefore, early diagnosis is the key to improving the survival rate and quality of life of patients with nasopharyngeal carcinoma.
  • EBV antibody detection was mainly based on immunofluorescence, with low throughput, long time-consuming and highly subjective results, which was difficult to meet the needs of large-scale screening [13-15]; with the development of enzyme-linked immunoassay (ELISA), researchers from various countries have established a large number of EB virus antibody ELISA detection kits, which significantly improves the detection throughput and objectivity [16-23].
  • ELISA enzyme-linked immunoassay
  • researchers from various countries have established a large number of EB virus antibody ELISA detection kits, which significantly improves the detection throughput and objectivity [16-23].
  • the study also found that through the combined use of different serological markers, the sensitivity and specificity of nasopharyngeal cancer screening can be further improved, and the most widely used is the combined use of EBNA1-IgA and VCA-IgA [24-26].
  • the results of a prospective cohort study show that through screening, the early diagnosis rate of nasopharyngeal carcinoma can be increased from 10-20% to more than 60% [27-29].
  • the research group proposed a two-step screening strategy, that is, on the basis of the combined screening of EBNA1-IgA and VCA-IgA, further testing of EAD-IgA is performed on people who are at high risk in the first screening. , EAD-IgA positive patients will be diagnosed by nasopharyngoscope.
  • the two-step screening method the positive predictive value of nasopharyngeal cancer screening increased from 4.69% to 18.52% [30].
  • the positive predictive value of two-step screening is significantly improved, which can meet the needs of larger-scale nasopharyngeal cancer screening.
  • serological screening or DNA screening the two-step screening operation is more complicated and more costly.
  • the persistently positive DNA requires another blood sampling, which further increases the difficulty of screening. Therefore, although the one-step serological antibody screening is cost-effective [35], whether the two-step screening is more cost-effective remains to be evaluated.
  • nasopharyngeal cancer screening methods are only 18.52%, that is, among the high-risk screening populations, at least 71.48% of non-nasopharyngeal cancer patients need to undergo nasopharyngoscopy.
  • Nasopharyngoscopy is time-consuming, which directly limits the scale of nasopharyngeal cancer screening. At the same time, it will also cause a psychological burden to non-nasopharyngeal cancer patients who are screened positive.
  • the field still needs to develop a new nasopharyngeal cancer screening method, which has higher specificity and positive predictive value than existing methods, and at the same time, has the characteristics of high throughput and low cost to meet the needs of large-scale screening.
  • the demand for screening has enabled more people to benefit from screening.
  • the inventor of the present application unexpectedly found that: the antibody level of the subject’s serum against the protein encoded by the EBVBNLF2b gene (which can, for example, use the protein encoded by the BNLF2b gene or polypeptide fragments as the coating antigen by indirect method or dual antigen Sandwich method for detection) can efficiently distinguish nasopharyngeal carcinoma patients from healthy controls.
  • the inventor also paid a lot of creative work to obtain a BNLF2b gene encoding polypeptide fragment that is particularly suitable as a capture reagent (for example, a coating antigen in ELISA).
  • the inventors successfully established a method and platform for the diagnosis of nasopharyngeal carcinoma based on the detection of the antibody level against the protein encoded by the EBV BNLF2b gene, which can significantly improve the specificity and positive predictive value of nasopharyngeal carcinoma screening.
  • the present invention provides an isolated polypeptide or a variant thereof, wherein the polypeptide is composed of at least 7 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and contains at least 1 (for example, at least 2, at least 3, or all 4) are selected from the following sequences: amino acid residues 5-11, amino acid residues 16-23, and amino acids 31-39 of the wild-type protein encoded by the BNLF2b gene Residues, or amino acid residues at positions 53-60 (for example, amino acid residues at positions 53-61);
  • the variant differs from the polypeptide from which it is derived only in one or a few (for example, one, two, three, four, five, six, seven, eight, or nine )
  • substitution of amino acid residues for example, conservative substitution or non-conservative substitution
  • the biological function of the polypeptide from which it is derived for example, the activity of being recognized and bound by anti-EBV antibodies.
  • the wild-type protein encoded by the BNLF2b gene has a sequence as shown in SEQ ID NO: 101.
  • the biological function of the polypeptide of the present invention or its variants includes, but is not limited to, the activity of being recognized and bound as an epitope peptide by an anti-EBV antibody (for example, an antibody specific to the protein encoded by the BNLF2b gene).
  • an anti-EBV antibody for example, an antibody specific to the protein encoded by the BNLF2b gene.
  • the isolated polypeptide comprises: amino acid residues 53-60 (for example, amino acid residues 53-61) and amino acid residues 5-23 of the wild-type protein encoded by the BNLF2b gene , Amino acid residues 16-39, amino acid residues 31-60 (e.g. amino acid residues 31-61), amino acid residues 5-39, amino acid residues 16-60 (e.g. Amino acid residues 16-61), or amino acid residues 5-60 (for example, amino acid residues 5-61).
  • the isolated polypeptide is at least 8 (e.g., at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14) of the wild-type protein encoded by the BNLF2b gene. It is composed of at least 15 consecutive amino acid residues and includes amino acid residues 53-60 of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 53-61 of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 51-65 of the wild-type protein encoded by the BNLF2b gene.
  • the 51-65 amino acid residues have the sequence shown in SEQ ID NO: 97.
  • the polypeptide consists of at least 15 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide is at least 19 (e.g., at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25) of the wild-type protein encoded by the BNLF2b gene.
  • Consecutive amino acid residues and include amino acid residues 5-23 of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 1-25 of the wild-type protein encoded by the BNLF2b gene.
  • amino acid residues 1-25 have the sequence shown in SEQ ID NO: 102.
  • the polypeptide consists of at least 25 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide is 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) contiguous amino acid residues, and include wild-type amino acid residues encoded by the BNLF2b gene Amino acid residues 16-39 of type protein.
  • the polypeptide comprises amino acid residues 14-52 of the wild-type protein encoded by the BNLF2b gene.
  • the 14-52 amino acid residues have the sequence shown in SEQ ID NO: 88.
  • the polypeptide consists of at least 39 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide is composed of at least 30 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene, and comprises amino acid residues 31-60 of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide comprises amino acid residues 31-61 of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide is 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) continuous It is composed of amino acid residues and includes amino acid residues 5-39 of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 1-52 of the wild-type protein encoded by the BNLF2b gene.
  • amino acid residues 1-52 have the sequence shown in SEQ ID NO:91.
  • the polypeptide consists of at least 53 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide is 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, and include amino acid residues 16-60 of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 16-61 of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 14-74 of the wild-type protein encoded by the BNLF2b gene (for example, the sequence shown in SEQ ID NO: 90). In certain embodiments, the polypeptide consists of at least 61 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 11-65 of the wild-type protein encoded by the BNLF2b gene (for example, the sequence shown in SEQ ID NO: 103). In certain embodiments, the polypeptide consists of at least 55 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 11-74 of the wild-type protein encoded by the BNLF2b gene (for example, the sequence shown in SEQ ID NO: 104). In certain embodiments, the polypeptide consists of at least 64 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide is 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) continuous It is composed of amino acid residues and includes amino acid residues 5-60 of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 5-61 of the wild-type protein encoded by the BNLF2b gene.
  • the polypeptide comprises amino acid residues 1-74 of the wild-type protein encoded by the BNLF2b gene.
  • amino acid residues 1-74 have the sequence shown in SEQ ID NO:89.
  • the polypeptide consists of at least 74 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide consists of a sequence selected from the group consisting of amino acid residues 51-56, amino acid residues 1-25, and 14 of the wild-type protein encoded by the BNLF2b gene. -52 amino acid residues, amino acid residues 1-52, amino acid residues 14-74, amino acid residues 11-65, amino acid residues 11-74, or amino acid residues 1-74 Residues. In certain exemplary embodiments, the isolated polypeptide consists of a sequence selected from the following: SEQ ID NOs: 88-91, 97, 102-104.
  • the variant of the present invention differs from the polypeptide from which it is derived only in the substitution of 1, 2, 3, or 4 amino acid residues (e.g., conservative substitutions or non-conservative substitutions), and It retains the biological function of the polypeptide from which it is derived (for example, the activity of being recognized and bound by anti-EBV antibodies).
  • the variant does not include amino acid substitutions in amino acid positions corresponding to the following positions, which refer 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.
  • the variant comprises amino acid substitutions at one or more of the amino acid positions (for example, 1, 2, 3, or 4) corresponding to the following positions, the following position references are The amino acid positions shown in the wild-type protein encoded by the BNLF2b gene: 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 variant comprises amino acid substitutions at one or more of the amino acid positions (for example, 1, 2, 3, or 4) corresponding to the following positions, the following position references are 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.
  • the variant comprises one or several (for example, one, two, three, or four) amino acid substitutions selected from the group consisting of: an amino acid substitution at a position corresponding to position 5 Is A, the amino acid at the position corresponding to position 7 is replaced with A, the amino acid at the position corresponding to position 8 is replaced with G, and the amino acid at the position corresponding to position 12 is replaced with G, T, D or S ,
  • the amino acid at the position corresponding to position 13 is replaced with A
  • the amino acid at the position corresponding to position 14 is replaced with G
  • the amino acid at the position corresponding to position 15 is replaced with A
  • the amino acid at the position is replaced with A
  • the amino acid at the position corresponding to position 24 is replaced with A
  • the amino acid at the position corresponding to position 25 is replaced with A
  • the amino acid at the position corresponding to position 32 is replaced with A
  • in The amino acid at the position corresponding to position 34 is replaced with A
  • the amino acid is replaced with A, N, Q, S or R, the amino acid at the position corresponding to position 40 is replaced with A, the amino acid at the position corresponding to position 41 is replaced with A, and the amino acid at the position corresponding to position 42 is replaced with A.
  • the amino acid is replaced with A, the amino acid at the position corresponding to position 52 is replaced with K, H, A, S, or D, the amino acid at the position corresponding to position 55 is replaced with S, and the amino acid at the position corresponding to position 60 is replaced with S.
  • the amino acid is replaced with A, the amino acid at the position corresponding to position 61 is replaced with K, H, S, or A, the amino acid at the position corresponding to position 89 is replaced with A or T, and the amino acid at the position corresponding to position 91 is replaced with A or T.
  • the amino acid is substituted with A, the amino acid at the position corresponding to position 93 is substituted with Q, and the amino acid at the position corresponding to position 98 is substituted with A.
  • the variant comprises one or several (for example, one, two, three, or four) amino acid substitutions selected from the group consisting of: an amino acid substitution at a position corresponding to position 5 Is A, the amino acid at the position corresponding to position 7 is replaced with A, the amino acid at the position corresponding to position 8 is replaced with G, and the amino acid at the position corresponding to position 12 is replaced with G, T, D or S ,
  • the amino acid at the position corresponding to position 13 is replaced with A
  • the amino acid at the position corresponding to position 14 is replaced with G
  • the amino acid at the position corresponding to position 22 is replaced with A
  • the amino acid at the position corresponding to position 25 is replaced with A.
  • the amino acid at position is replaced with A, the amino acid at the position corresponding to position 32 is replaced with A, the amino acid at the position corresponding to position 34 is replaced with A, and the amino acid at the position corresponding to position 35 is replaced with A,
  • the amino acid at the position corresponding to position 36 is replaced with A
  • the amino acid at the position corresponding to position 40 is replaced with A
  • the amino acid at the position corresponding to position 41 is replaced with A
  • the amino acid of is replaced with A
  • the amino acid at the position corresponding to position 52 is replaced with K, H, A, S, or D
  • the amino acid at the position corresponding to position 55 is replaced with S, at the position corresponding to position 60
  • the amino acid of is replaced with A
  • the amino acid at the position corresponding to position 61 is replaced with K, H, S, or A
  • the amino acid at the position corresponding to position 89 is replaced with A or T, at the position corresponding to position 91
  • the amino acid of is replaced
  • the isolated polypeptide has no more than 97 wild-type proteins encoded by the BNLF2b gene (e.g., no more than 96, no more than 95, no more than 94, no more than 93, not more than 92, not more than 91, not more than 90, not more than 89, not more than 88, not more than 87, not more than 86, not more than 85 , No more than 84, no more than 83, no more than 82, no more than 81, no more than 80, no more than 79, no more than 78, no more than 76, no More than 75, or not more than 74) contiguous amino acid residues.
  • BNLF2b gene e.g., no more than 96, no more than 95, no more than 94, no more than 93, not more than 92, not more than 91, not more than 90, not more than 89, not more than 88, not more than 87, not more than 86, not more than 85 , No more
  • the isolated polypeptide consists of 15-80 (eg, 15-74) consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene. In certain embodiments, the isolated polypeptide consists of 39-74 consecutive amino acid residues of the wild-type protein encoded by the BNLF2b gene.
  • the isolated polypeptide or a variant thereof is attached to the surface of a solid-phase carrier, or has a modification group that can be attached to a solid-phase carrier.
  • the C-terminus of the isolated polypeptide or variant thereof is attached to the surface of a solid-phase carrier, or has a modification group that can be attached to a solid-phase carrier.
  • the modifying group is biotin or avidin.
  • the solid phase carrier is selected from magnetic beads or microtiter plates (for example, microtiter plates or microtiter plates).
  • the isolated polypeptide or variant thereof bears a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • polypeptide of the present invention or its variant is not limited by its production method.
  • it can be produced by genetic engineering methods (recombinant technology), or can be produced by chemical synthesis methods.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of the invention or a variant thereof.
  • the present invention also provides a vector comprising the isolated nucleic acid molecule as described above.
  • the vector of the present invention can be a cloning vector or an expression vector.
  • the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a cosmid and the like.
  • the present invention also provides a host cell comprising the isolated nucleic acid molecule or vector of the present invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. 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.
  • the present invention also provides a method for preparing the polypeptide of the present invention or a variant thereof, which comprises culturing the host cell of the present invention under conditions that allow the expression of the polypeptide or a variant thereof, and obtaining The polypeptide or variants thereof are recovered from the host cell culture.
  • the present invention provides a kit comprising a capture reagent selected from the isolated polypeptide described in the first aspect or a variant thereof.
  • the kit further includes instructions for using the isolated polypeptide or a variant thereof as a capture reagent to detect antibodies specific to the protein encoded by the BNLF2b gene in the sample, or using the isolated polypeptide
  • the polypeptide or its variant is used as a capture reagent to determine the level of antibodies specific to the protein encoded by the BNLF2b gene in a sample from a subject, so as to determine whether the subject has or is suffering from nasopharyngeal carcinoma The description of the risk.
  • the subject is a mammal, such as a human.
  • the capture reagent is attached to the surface of a solid support.
  • the C-terminus of the capture reagent is connected to a solid support.
  • the solid phase carrier includes a well plate, a test tube, and beads (such as latex particles) made or coated with a polymer material (such as polyvinyl chloride, polystyrene, polyacrylamide, or cellulose). Or thin films (such as nitrocellulose membrane), or magnetic beads pre-coated with functional groups (such as amino, carboxyl, biotin or avidin).
  • the solid phase carrier is selected from magnetic beads or microtiter plates (for example, microtiter plates or microtiter plates).
  • the capture reagent has a modification group that can be attached to a solid support.
  • the C-terminus of the capture reagent has a modification group that can be connected to a solid phase carrier.
  • the kit may further include a coating reagent for coating the capture reagent on the solid support, such as a coating buffer (e.g., carbonate buffer, phosphoric acid). Salt buffer, Tris-HCL buffer or borate buffer).
  • a coating buffer e.g., carbonate buffer, phosphoric acid
  • Salt buffer Tris-HCL buffer or borate buffer
  • modifying group is biotin or avidin
  • the surface of the solid support has a corresponding linking group.
  • the kit includes at least a solid phase carrier coated with avidin or streptavidin in a separate container or in a separate compartment of a single container unit, and the above Capture reagent.
  • the method of determining the level of anti-EB virus (EBV) antibodies in a sample from a subject to determine whether the subject has or is at risk of nasopharyngeal cancer is Double antigen sandwich form. It is well known to those skilled in the art to determine the level of antibodies in a sample by the double antigen sandwich method. In this type of assay, “capture antigen” and “detect antigen” make the sample antibody form a bridge between two specific antigens. Therefore, the two antigens are usually the same, or have the same core epitope, or have immunity. Cross-reactivity allows one antibody to bind two antigens.
  • EBV anti-EB virus
  • the kit further comprises a detection reagent selected from the isolated polypeptide described in the first aspect or a variant thereof.
  • the detection reagent bears a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • the detectable label is selected from an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).
  • the polypeptide sequence contained in the detection reagent and the capture reagent are the same or substantially the same.
  • the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent comprise the same core fragment, and the core fragment is selected from the wild-type protein encoded by the BNLF2b gene. Amino acid residues 5-11, amino acid residues 16-23, amino acid residues 31-39, and/or amino acid residues 53-60 (for example, amino acid residues 53-61).
  • the 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) of the wild-type protein encoded by the BNLF2b gene. 53-61 amino acid residues), 5-23 amino acid residues, 16-39 amino acid residues, 5-39 amino acid residues, 16-60 amino acid residues (e.g., 16-61 Amino acid residues at positions), or amino acid residues at positions 5-60 (for example, amino acid residues at positions 5-61).
  • amino acid residues 53-60 e.g., amino acid residues
  • the capture reagent simultaneously contain amino acid residues 53-60 (e.g., amino acid residues) of the wild-type protein encoded by the BNLF2b gene. 53-61 amino acid residues), 5-23 amino acid residues, 16-39 amino acid residues, 5-39 amino acid residues, 16-60 amino acid residues (e.g., 16-61 Amino acid residues at positions), or amino
  • the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 51-56 and the first amino acid residues of the wild-type protein encoded by the BNLF2b gene. -25 amino acid residues, 14-52 amino acid residues, 1-52 amino acid residues, 14-74 amino acid residues, 11-65 amino acid residues, 11-74 amino acid residues Group, or amino acid residues 1-74.
  • the method for determining the level of anti-EB virus (EBV) antibodies in a sample from a subject to determine whether the subject has or is at risk of nasopharyngeal cancer In the form of indirect method.
  • the indirect method to determine the level of antibodies in a sample is well known to those skilled in the art.
  • the "capture antigen” first forms an immune complex with the antibody in the sample, and then the captured antibody is detected by a secondary antibody (e.g., anti-immunoglobulin antibody).
  • the kit further comprises a detection reagent selected from a secondary antibody with a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • the detectable label is selected from an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).
  • the secondary antibody is specific for antibodies of the species (e.g., human) from which the antibody to be tested is derived.
  • the secondary antibody is an anti-immunoglobulin antibody.
  • the kit is used to detect anti-EBV IgG antibodies.
  • the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.
  • the kit is used to detect anti-EBV IgM antibodies.
  • the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.
  • the kit is used to detect anti-EBV IgA antibodies.
  • the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.
  • the kit of the present invention may also include one or more reagents or devices selected from: (i) a device for collecting or storing a sample from a subject (for example, a blood sampling device); (ii) Other reagents (such as buffers, diluents, blocking solutions, and/or standards) required for performing the assay.
  • a device for collecting or storing a sample from a subject for example, a blood sampling device
  • Other reagents such as buffers, diluents, blocking solutions, and/or standards
  • the present invention provides a method for detecting antibodies specific to the protein encoded by the BNLF2b gene in a sample, which includes the following steps:
  • the capture reagent is selected from the isolated polypeptide described in the first aspect or a variant thereof;
  • step (2) the amount of the immune complex is determined by immunological detection.
  • the immunological detection is selected from an enzyme immunoassay (e.g., ELISA), a chemiluminescence immunoassay, a fluorescence immunoassay, or a radioimmunoassay.
  • an enzyme immunoassay e.g., ELISA
  • chemiluminescence immunoassay e.g., a chemiluminescence immunoassay
  • fluorescence immunoassay e.g., fluorescence immunoassay
  • radioimmunoassay e.g., radioimmunoassay.
  • the capture reagent is attached to the surface of a solid support.
  • the capture reagent has a modification group that can be attached to a solid support.
  • the method before step (1), further comprises a step of coating the capture reagent on the surface of the solid support.
  • the C-terminus of the capture reagent is attached to the surface of the solid-phase support, or has a modification group that can be attached to the solid-phase support.
  • the assay is performed in a dual antigen sandwich format. It is well known to those skilled in the art to determine the level of antibodies in a sample by the double antigen sandwich method. In this type of assay, “capture antigen” and “detect antigen” make the sample antibody form a bridge between two specific antigens. Therefore, the two antigens are usually the same, or have the same core epitope, or have immunity. Cross-reactivity allows one antibody to bind two antigens.
  • a detection reagent is used to detect the amount of the immune complex, and the detection reagent is selected from the isolated polypeptide described in the first aspect or a variant thereof.
  • the detection reagent is selected from the isolated polypeptide or variant thereof described in the first aspect, and the isolated polypeptide or variant thereof bears a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • the detectable label is selected from an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).
  • the polypeptide sequence contained in the detection reagent and the capture reagent are the same or substantially the same.
  • the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent comprise the same core fragment, and the core fragment is selected from the wild-type protein encoded by the BNLF2b gene. Amino acid residues 5-11, amino acid residues 16-23, amino acid residues 31-39, and/or amino acid residues 53-60 (for example, amino acid residues 53-61).
  • the 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) of the wild-type protein encoded by the BNLF2b gene. 53-61 amino acid residues), 5-23 amino acid residues, 16-39 amino acid residues, 5-39 amino acid residues, 16-60 amino acid residues (e.g., 16-61 Amino acid residues at positions), or amino acid residues at positions 5-60 (for example, amino acid residues at positions 5-61).
  • amino acid residues 53-60 e.g., amino acid residues
  • the capture reagent simultaneously contain amino acid residues 53-60 (e.g., amino acid residues) of the wild-type protein encoded by the BNLF2b gene. 53-61 amino acid residues), 5-23 amino acid residues, 16-39 amino acid residues, 5-39 amino acid residues, 16-60 amino acid residues (e.g., 16-61 Amino acid residues at positions), or amino
  • the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 51-56 and the first amino acid residues of the wild-type protein encoded by the BNLF2b gene. -25 amino acid residues, 14-52 amino acid residues, 1-52 amino acid residues, 14-74 amino acid residues, 11-65 amino acid residues, 11-74 amino acid residues Group, or amino acid residues 1-74.
  • the determination is performed in the form of an indirect method.
  • the indirect method to determine the level of antibodies in a sample is well known to those skilled in the art.
  • the "capture antigen” first forms an immune complex with the antibody in the sample, and then the captured antibody is detected by a secondary antibody (for example, an anti-immunoglobulin antibody).
  • a detection reagent is used to detect the amount of the immune complex, and the detection reagent is selected from a secondary antibody with a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent reagent such as acridine ester compound
  • a fluorescent dye such as acridine ester compound
  • biotin biotin
  • the secondary antibody is specific for antibodies of the species (e.g., human) from which the antibody to be tested is derived.
  • the secondary antibody is an anti-immunoglobulin antibody.
  • the antibody being detected is an IgG antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.
  • the antibody being tested is an IgM antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.
  • the antibody being tested is an IgA antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.
  • the present invention also relates to the use of the isolated polypeptide described in the first aspect or a variant thereof in the preparation of a kit for detecting antibodies specific to the protein encoded by the BNLF2b gene in a sample .
  • the kit detects antibodies specific to the protein encoded by the BNLF2b gene in the sample by the method described in the third aspect.
  • the present invention provides a method for determining whether a subject has or is at risk of nasopharyngeal cancer, which includes:
  • the level is higher than a reference value, it is determined whether the subject has nasopharyngeal cancer or is at risk of nasopharyngeal cancer.
  • the reference value is a value derived from a subject who does not suffer from nasopharyngeal cancer or a healthy person (for example, a subject who has no detectable disease and has no history of cancer or nasopharyngeal cancer), or indicates that there is no nasopharyngeal cancer.
  • the sample is a blood sample, such as whole blood, plasma, or serum.
  • the subject is a mammal, such as a human.
  • the level of antibodies specific to the protein encoded by the BNLF2b gene in the sample is determined by immunological assay.
  • the immunological assay is selected from an enzyme immunoassay (e.g., ELISA), a chemiluminescence immunoassay, a fluorescence immunoassay, or a radioimmunoassay.
  • the assay includes using the isolated polypeptide of the first aspect or a variant thereof as a capture reagent.
  • step (1) includes the following steps:
  • the capture reagent is selected from the isolated polypeptide described in the first aspect or a variant thereof;
  • the amount of the immune complex is determined by immunological detection.
  • the immunological detection is selected from an enzyme immunoassay (e.g., ELISA), a chemiluminescence immunoassay, a fluorescence immunoassay, or a radioimmunoassay.
  • the capture reagent is attached to the surface of a solid support.
  • the capture reagent has a modification group that can be attached to a solid support.
  • the method before step (1), further comprises a step of coating the capture reagent on the surface of the solid support.
  • the C-terminus of the capture reagent is attached to the surface of the solid-phase support, or has a modification group that can be attached to the solid-phase support.
  • the assay is performed in a dual antigen sandwich format. It is well known to those skilled in the art to determine the level of antibodies in a sample by the double antigen sandwich method. In this type of assay, “capture antigen” and “detect antigen” make the sample antibody form a bridge between two specific antigens. Therefore, the two antigens are usually the same, or have the same core epitope, or have immunity. Cross-reactivity allows one antibody to bind two antigens.
  • a detection reagent is used to detect the amount of the immune complex, and the detection reagent is selected from the isolated polypeptide described in the first aspect or a variant thereof.
  • the detection reagent is selected from the isolated polypeptide or variant thereof described in the first aspect, and the isolated polypeptide or variant thereof bears a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • the detectable label is selected from an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).
  • the polypeptide sequence contained in the detection reagent and the capture reagent are the same or substantially the same.
  • the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent comprise the same core fragment, and the core fragment is selected from the wild-type protein encoded by the BNLF2b gene. Amino acid residues 5-11, amino acid residues 16-23, amino acid residues 31-39, and/or amino acid residues 53-60 (for example, amino acid residues 53-61).
  • the 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) of the wild-type protein encoded by the BNLF2b gene. 53-61 amino acid residues), 5-23 amino acid residues, 16-39 amino acid residues, 5-39 amino acid residues, 16-60 amino acid residues (e.g., 16-61 Amino acid residues at positions), or amino acid residues at positions 5-60 (for example, amino acid residues at positions 5-61).
  • amino acid residues 53-60 e.g., amino acid residues
  • the capture reagent simultaneously contain amino acid residues 53-60 (e.g., amino acid residues) of the wild-type protein encoded by the BNLF2b gene. 53-61 amino acid residues), 5-23 amino acid residues, 16-39 amino acid residues, 5-39 amino acid residues, 16-60 amino acid residues (e.g., 16-61 Amino acid residues at positions), or amino
  • the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 51-56 and the first amino acid residues of the wild-type protein encoded by the BNLF2b gene. -25 amino acid residues, 14-52 amino acid residues, 1-52 amino acid residues, 14-74 amino acid residues, 11-65 amino acid residues, 11-74 amino acid residues Group, or amino acid residues 1-74.
  • the determination is performed in the form of an indirect method.
  • the indirect method to determine the level of antibodies in a sample is well known to those skilled in the art.
  • the "capture antigen” first forms an immune complex with the antibody in the sample, and then the captured antibody is detected by a secondary antibody (for example, an anti-immunoglobulin antibody).
  • a detection reagent is used to detect the amount of the immune complex, and the detection reagent is selected from a secondary antibody with a detectable label.
  • the detectable label is selected from enzymes (e.g. horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g. acridinium ester compounds), fluorescent dyes or biotin.
  • enzymes e.g. horseradish peroxidase or alkaline phosphatase
  • chemiluminescent reagents e.g. acridinium ester compounds
  • fluorescent dyes e.g. acridinium ester compounds
  • the secondary antibody is specific for antibodies of the species (e.g., human) from which the antibody to be tested is derived.
  • the secondary antibody is an anti-immunoglobulin antibody.
  • the antibody being detected is an IgG antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.
  • the antibody being tested is an IgM antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.
  • the antibody being tested is an IgA antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.
  • the method further includes: before step (1), providing a sample from the subject.
  • the method further comprises: after step (2), administering a therapeutically effective amount of a treatable nasopharyngeal carcinoma to a subject who is judged to have or is at risk of nasopharyngeal carcinoma
  • Anti-tumor therapy e.g., chemotherapy, radiation therapy and/or immunotherapy.
  • the anti-tumor therapy that can treat nasopharyngeal carcinoma is selected from surgical treatment, radiotherapy (such as external radiation therapy EBRT, brachytherapy), or chemotherapy (such as carboplatin, paclitaxel, docetaxel) Cy, gemcitabine, doxorubicin, epirubicin, bleomycin, methotrexate), targeted therapy (such as cetuximab), immunotherapy (such as PD-1 monoclonal antibody) and various methods Combination therapy (such as radiotherapy + chemotherapy, radiotherapy + surgery).
  • radiotherapy such as external radiation therapy EBRT, brachytherapy
  • chemotherapy such as carboplatin, paclitaxel, docetaxel
  • Cy gemcitabine
  • doxorubicin epirubicin
  • bleomycin bleomycin
  • methotrexate methotrexate
  • targeted therapy such as cetuximab
  • immunotherapy such as PD-1 monoclonal antibody
  • Combination therapy such as radiotherapy + chemotherapy, radiotherapy +
  • the present invention also relates to the use of a reagent capable of determining the level of an antibody specific to the protein encoded by the BNLF2b gene in the preparation of a kit for determining whether a subject has nasopharyngeal carcinoma or Are you at risk of nasopharyngeal cancer.
  • the reagent can determine the level of antibodies specific to the protein encoded by the BNLF2b gene by immunological assay.
  • the immunological assay is selected from an enzyme immunoassay (e.g., ELISA), a chemiluminescence immunoassay, a fluorescence immunoassay, or a radioimmunoassay.
  • the subject is a mammal, such as a human.
  • the sample is a blood sample, such as whole blood, plasma, or serum.
  • the kit determines whether the subject has or is at risk of nasopharyngeal cancer by the following method:
  • the level is higher than a reference value, it is determined whether the subject has nasopharyngeal cancer or is at risk of nasopharyngeal cancer.
  • the reagent capable of measuring the level of the antibody specific to the protein encoded by the BNLF2b gene comprises a capture reagent selected from the isolated polypeptide described in the first aspect or a variant thereof.
  • the above step (1) includes the following steps:
  • the capture reagent is selected from the isolated polypeptide described in the first aspect or a variant thereof;
  • the amount of the immune complex is determined by immunological detection.
  • the immunological detection is selected from an enzyme immunoassay (e.g., ELISA), a chemiluminescence immunoassay, a fluorescence immunoassay, or a radioimmunoassay.
  • the capture reagent is attached to the surface of a solid support.
  • the capture reagent has a modification group that can be attached to a solid support.
  • the method before the above step (1), the method further comprises the step of coating the capture reagent on the surface of the solid support.
  • the C-terminus of the capture reagent is attached to the surface of the solid-phase support, or has a modification group that can be attached to the solid-phase support.
  • the reagent capable of measuring the level of the antibody specific to the protein encoded by the BNLF2b gene further comprises a detection reagent selected from the isolated polypeptide described in the first aspect or a variant thereof.
  • the detection reagent is used to detect the amount of the immune complex.
  • the detection reagent is selected from the isolated polypeptide or variant thereof described in the first aspect, and the isolated polypeptide or variant thereof bears a detectable label.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • the detectable label is selected from an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).
  • the polypeptide sequence contained in the detection reagent and the capture reagent are the same or substantially the same.
  • the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent comprise the same core fragment, and the core fragment is selected from the wild-type protein encoded by the BNLF2b gene. Amino acid residues 5-11, amino acid residues 16-23, amino acid residues 31-39, and/or amino acid residues 53-60 (for example, amino acid residues 53-61).
  • the 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) of the wild-type protein encoded by the BNLF2b gene. 53-61 amino acid residues), 5-23 amino acid residues, 16-39 amino acid residues, 5-39 amino acid residues, 16-60 amino acid residues (e.g., 16-61 Amino acid residues at positions), or amino acid residues at positions 5-60 (for example, amino acid residues at positions 5-61).
  • amino acid residues 53-60 e.g., amino acid residues
  • the capture reagent simultaneously contain amino acid residues 53-60 (e.g., amino acid residues) of the wild-type protein encoded by the BNLF2b gene. 53-61 amino acid residues), 5-23 amino acid residues, 16-39 amino acid residues, 5-39 amino acid residues, 16-60 amino acid residues (e.g., 16-61 Amino acid residues at positions), or amino
  • the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent simultaneously contain amino acid residues 51-56 and the first amino acid residues of the wild-type protein encoded by the BNLF2b gene. -25 amino acid residues, 14-52 amino acid residues, 1-52 amino acid residues, 14-74 amino acid residues, 11-65 amino acid residues, 11-74 amino acid residues Group, or amino acid residues 1-74.
  • the reagent capable of measuring the level of the antibody specific to the protein encoded by the BNLF2b gene further comprises a detection reagent, and the detection reagent is selected from a secondary antibody with a detectable label.
  • the detection reagent is used to detect the amount of the immune complex.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent reagent such as acridine ester compound
  • a fluorescent dye such as acridine ester compound
  • biotin biotin
  • the secondary antibody is specific for antibodies of the species (e.g., human) from which the antibody to be tested is derived.
  • the secondary antibody is an anti-immunoglobulin antibody.
  • the antibody being detected is an IgG antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.
  • the antibody being tested is an IgM antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.
  • the antibody being tested is an IgA antibody.
  • the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.
  • 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).
  • EBV Epstein-Barr virus
  • the full-length protein encoded by the BNLF2b gene contains 98 amino acids, and its sequence is shown in SEQ ID NO: 101.
  • the full-length protein encoded by the BNLF2b gene may also be referred to as the wild-type protein encoded by the BNLF2b gene or the protein encoded by the BNLF2b gene.
  • amino acid sequence of the protein encoded by the BNLF2b gene when referring to the amino acid sequence of the protein encoded by the BNLF2b gene, it uses the sequence shown in SEQ ID NO: 101 for description.
  • 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.
  • mutations or variations can be naturally generated or artificially introduced without affecting its biological function.
  • the term "BNLF2b gene-encoded protein” and similar expressions shall include all such sequences, including, for example, the sequence shown in SEQ ID NO: 101 and its natural or artificial variants. Moreover, when describing the 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 corresponding fragments in variants (natural or artificial).
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percent identity, the sequences being compared are located in the same position Fragments.
  • K D value refers to the ratio of kd (specific binding molecule-target molecule interaction dissociation rate; also called koff) to ka (specific binding molecule-target molecule interaction rate of association; also called kon)
  • M molar concentration
  • an antibody that specifically binds to a certain antigen means that the antibody has a concentration of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, The affinity (K D ) of 10 -8 M, 10 -9 M, or 10 -10 M or less binds the antigen.
  • the K D value can be determined by a method well known in the art, for example, measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • immunological assay refers to an assay that utilizes the specific interaction/binding affinity between antigen and antibody. It can generally be used to detect the presence or presence of a specific antigen or antibody in a sample. Level. Such immunological tests are well known to those skilled in the art, and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA) , Western blotting, immunoturbidimetry, surface plasmon resonance, etc.
  • EIA enzyme immunoassay
  • CLIA chemiluminescence immunoassay
  • RIA radioimmunoassay
  • FFA fluorescence immunoassay
  • the immunological detection is an enzyme immunoassay (EIA), such as an ELISA detection method, an Elispot detection method, or a CLEIA detection method.
  • EIA enzyme immunoassay
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also called “antigenic determinant” in the art.
  • an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which can be "linear ⁇ " or “conformational”.
  • linear epitope all interaction points between a protein and an interacting molecule (for example, an antibody) exist linearly along the protein's primary amino acid sequence.
  • conformational epitopes points of interaction exist across protein amino acid residues that are separated from each other.
  • the term "detectable label” can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. It is particularly preferable that such a label can be applied to immunological detection (for example, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).
  • Such labels include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Red protein (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridinium ester compounds), magnetic beads (E.g, ), calorimetric markers such as colloidal gold or colored glass or plastic (for example, polystyrene, polypropylene, latex, etc.) beads, and avidin (for
  • Patents teaching the use of this marker include, but are not limited to, U.S. Patent Nos. 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 by the present invention can be detected by methods known in the art. For example, a radioactive label can be detected using photographic film or a scintillation calculator, and a fluorescent label can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing colored markers.
  • the detectable label as described above can be attached to the detection antibody or antigen via linkers of different lengths to reduce potential steric hindrance.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL. Constant domains are not directly involved in the binding of antibodies and antigens, but exhibit a variety of effector functions, such as mediating immunoglobulins and host tissues or factors, including various cells of the immune system (for example, effector cells) and classical complement The combination of the first component (C1q) of the system.
  • VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form an antigen binding site.
  • isolated refers to those obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolation a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly 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.
  • prokaryotic cells such as Escherichia coli or subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 fruit fly cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • the molecules are the same at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by 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 similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent bonds or hydrogen bonds, etc.) is replaced by residues. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine Acid, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods of identifying conservative substitutions of amino acids 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 is incorporated herein by reference).
  • subject includes, but is not limited to, various animals, particularly mammals, such as humans.
  • the present invention finds for the first time that the antibody level against the protein encoded by the Epstein Barr virus BNLF2b gene can be used to diagnose nasopharyngeal carcinoma or assess the risk of nasopharyngeal carcinoma, and its diagnostic effect is significantly better than existing markers, which can significantly improve the screening of nasopharyngeal carcinoma Specificity and positive predictive value.
  • the present invention also provides for the first time a nasopharyngeal carcinoma serological screening kit based on the EB virus BNLF2b gene encoding protein or polypeptide fragment.
  • the technical solution of the present invention can achieve detection sensitivity equivalent to that of known combined detection methods for EB virus antibodies, and even better detection specificity, and can achieve rapid and high-throughput detection, and has important clinical features. Value.
  • Figure 1 shows the reactivity of IgA and IgG antibodies in 1 nasopharyngeal carcinoma mixed serum (NPC) and 3 healthy control serums (H1-H3) in Examples 2 and 3 with 87 EBV polypeptides (number 1-87) .
  • the left picture is IgA antibody, and the right picture is IgG antibody.
  • the horizontal axis represents different serum samples, and the vertical axis represents different EBV peptides. The darker the color, the higher the reactivity.
  • Figure 2 shows the reactivity of antibodies in the serum of nasopharyngeal carcinoma patients and healthy controls in Examples 2 and 3 with different EBV polypeptides.
  • the abscissa represents the EBV polypeptide
  • the ordinate represents the OD value (logarithmic scale) of the reaction.
  • Figure 3 shows the reactivity of IgG antibodies in the serum of nasopharyngeal cancer patients and healthy controls in Example 4 with the protein aa14-52 encoded by BNLF2b.
  • Fig. 3A is the detected OD value (logarithmic scale) of different samples
  • Fig. 3B is the result of ROC curve analysis, the abscissa represents 100-specificity%, and the ordinate represents sensitivity%.
  • Figure 4 shows the reactivity of IgA, IgM and IgG antibodies in the serum of nasopharyngeal carcinoma patients and healthy controls with the protein aa1-74 encoded by BNLF2b in Example 4.
  • Figure A is the detection OD value
  • the abscissa represents the serum classification
  • the ordinate represents the OD value (logarithmic scale)
  • Figure B is the result of ROC curve analysis, the abscissa represents 100-specificity%, and the ordinate represents sensitivity%
  • Panel C shows the reactivity of IgA and IgM antibodies in healthy control serum with the protein aa1-74 encoded by BNLF2b.
  • the abscissa represents the type of antibody
  • the ordinate represents the OD value (logarithmic scale).
  • Figure 5 shows the results of 50 nasopharyngeal carcinoma sera and 500 healthy control sera detected by the BNLF2b antibody double antigen sandwich method in Example 5.
  • Figure 5A is the detected OD value
  • the abscissa is the serum classification
  • the ordinate is the OD value (logarithmic scale)
  • Figure 5B is the ROC curve analysis result
  • the abscissa is 100-specificity%
  • the ordinate is sensitivity%.
  • Figure 6 shows the reactivity of 74 nasopharyngeal carcinoma sera and 250 healthy control sera in Example 6 with EBNA1/IgA and VCA/IgA, and the calculated risk rate of nasopharyngeal carcinoma based on the combined detection of the two.
  • the abscissa represents serum classification
  • the ordinate in Fig. 6A and Fig. 6B is the detected OD value (logarithmic scale)
  • the ordinate in Fig. 6C is the risk of nasopharyngeal cancer.
  • Figure 7 shows the results of the BNLF2b antibody double antigen sandwich method in Example 6 to detect the serum of nasopharyngeal carcinoma patients and healthy control serum.
  • the abscissa represents the serum classification
  • the ordinate represents the OD value of the reaction.
  • Figure 8 shows the ROC curve of the BNLF2b antibody double antigen sandwich method and the combined detection of EBNA1/IgA+VCA/IgA in Example 6. Among them, the abscissa is 100-specificity%, and the ordinate is sensitivity%.
  • Figure 9 shows the reactivity of the IgG antibody in the serum of nasopharyngeal carcinoma in Example 10 with different peptides encoded by BNLF2b.
  • the abscissa represents different BNLF2b polypeptides
  • the ordinate represents the number of nasopharyngeal carcinoma mixed serum. The darker the color, the higher the reactivity.
  • Figure 10 shows the reactivity of the IgG antibody in the two nasopharyngeal carcinoma mixed sera in Example 10 with different peptides of the BNLF2b encoded protein.
  • AD stands for nasopharyngeal carcinoma serum and truncated peptide in aa1-25 segment, truncated peptide in aa31-45 segment, truncated peptide in aa51-65 segment and truncated peptide in aa81-98 segment, respectively
  • the abscissa represents the position of the polypeptide (for example, 1-25 represents the amino acid position 1-25), and the ordinate represents the ratio of the detected OD value to the detected OD value of the untruncated peptide.
  • Figure 11 shows the reactivity of the nasopharyngeal carcinoma serum in Example 10 with different truncated polypeptides in the aa1-25 segment.
  • Figure 11A shows the reactivity of serum from different nasopharyngeal cancer patients with aa1-25, aa1-15 and aa11-25
  • Figure 11B shows the reactivity of these sera with aa1-25 and its truncated polypeptide.
  • the abscissa represents the amino acid position of the synthetic peptide
  • the ordinate represents the nasopharyngeal carcinoma serum number. The darker the color, the higher the reactivity.
  • Fig. 12 shows the reactivity of IgG antibodies with BNLF2b-encoded protein mutants in patients with nasopharyngeal carcinoma in Example 10.
  • AD represents the reactivity of the nasopharyngeal carcinoma serum with the mutant in the aa1-25 segment, the mutant in the aa31-45 segment, the mutant in the aa51-65 segment, and the mutant in the aa81-98 segment, respectively.
  • the coordinate represents the mutation position, and the ordinate represents the ratio of the detected OD value to the detected OD value of the corresponding unmutated peptide.
  • Figure 13 shows the reactivity of IgG antibodies in the serum of nasopharyngeal carcinoma patients and healthy controls in Example 11 with different BNLF2b-encoded polypeptides. Among them, the abscissa represents the polypeptide fragment, and the ordinate represents the detected OD value.
  • Table 1 The sequence information involved in this application is described in the following table.
  • EBV ORF 23 15 EBV ORF 23, aa205-224 67 EBV ORF 68,aa120-139 16 EBV ORF 26, aa110-129 68 EBV ORF 69,aa17-36 17 EBV ORF 27, a256-275 69 EBV ORF 70,aa171-190 18 EBV ORF 28, aa9-28 70 EBV ORF 71,aa201-220 19 EBV ORF 29, aa1-20 71 EBV ORF 72,aa143-162 20 EBV ORF 30, aa789-801 72 EBV ORF 73,aa195-214 twenty one EBV ORF 31, aa185-204 73 EBV ORF 73,aa154-173 twenty two EBV ORF 32, aa246-265 74 EBV ORF 75,aa514-533 twenty three EBV ORF 33, aa26-45 75 EBV ORF 75,aa432-451 twenty four EBV ORF 34, aa134-153 76 E
  • Example 3 Evaluation of the reactivity between Epstein-Barr virus polypeptides and serum IgG antibodies
  • Example 2 The reactivity of the 87 polypeptides (1-87) synthesized in Example 1 with serum IgG was tested according to the method in Example 2. Among them, HRP-labeled mouse anti-human IgG diluted 1:5000 (Milan, Beijing) ) Instead of goat anti-human IgA. The results are shown in Figure 1. Among these peptides, the reactivity of peptides encoded by 5 genes including BZLF1 and BRLF1 with nasopharyngeal carcinoma mixed serum is higher than 0.1 (Table 3-1). Among them are BNLF1 (ZTA) and BRLF1 ( RTA) and BILF2 (gp78) have been reported.
  • Example 4 Establishment of the indirect method of anti-BNLF2b antibody and preliminary evaluation of its performance
  • the polypeptide aa14-52 was diluted with carbonate buffer (pH 9.6) to 125ng/ml, and each well was coated with 100 ⁇ L. According to the method in Example 3, 86 samples of nasopharyngeal carcinoma patients and 195 samples of healthy human sera were tested . The results are shown in Figure 3A. When aa14-52 was used as the coating antigen, 57 out of 86 sera from nasopharyngeal carcinoma patients had OD values higher than 0.1, and 195 healthy human sera had OD values lower than 0.025.
  • polypeptide aa1-74 as the coating antigen, 63 healthy human sera and 221 nasopharyngeal carcinoma sera were tested in the same way as 4.1. The results are shown in Figure 4A, 55 out of 63 sera from patients with nasopharyngeal carcinoma had an OD exceeding 0.1, while only 10 out of 221 healthy controls had an OD exceeding 0.1.
  • the ROC curve analysis was further performed on the above indirect method based on the polypeptide aa1-74, and the result is shown in Figure 4B. This method can efficiently distinguish nasopharyngeal cancer patients from healthy people, 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).
  • the same polypeptide aa1-74 was used as the coating antigen to detect the levels of IgA and IgM antibodies in 221 healthy control sera by indirect methods.
  • the IgA antibody detection uses a 1:20000 dilution of HRP-labeled goat anti-human IgA (KPL). , Gaithersburg, MD)
  • IgM antibody detection uses 1:50000 dilution of HRP-labeled goat anti-human IgM (Wanyumeilan, Beijing).
  • KPL horse-labeled goat anti-human IgA
  • Anti-BNLF2b IgA antibodies and anti-BNLF2b IgM antibodies have good specificity. Only 4 and 7 sera have a detection value of more than 0.1, respectively. The specificity is 98.19% (217/221). ) And 96.83% (214/221).
  • Table 4 shows the performance of the above methods to distinguish between nasopharyngeal cancer and non-nasopharyngeal cancer. The results show that whether it is the indirect method of IgG antibody with polypeptide aa14-52 as the coating antigen, or the polypeptide aa1-74 as the coating antigen.
  • the indirect methods of IgG, IgA and IgM antibodies can effectively distinguish nasopharyngeal carcinoma from healthy controls, and have good detection sensitivity and specificity.
  • Example 5 Establishment of double antigen sandwich method for anti-BNLF2b antibody
  • Example 4 Since the results of Example 4 show that the three antibodies of IgA, IgM and IgG all have good specificity in predicting the risk of nasopharyngeal cancer, the total antibody against BNLF2b can be used to predict the risk of nasopharyngeal cancer. Based on this, this example established a double antigen sandwich method for detecting anti-BNLF2b antibodies to evaluate its performance in screening for nasopharyngeal carcinoma.
  • the cut-off value is 0.1
  • the sensitivity of the three peptides is above 85%, indicating that aa1-52, aa14-74 Or when aa1-74 is the coating antigen, this method has high detection sensitivity and specificity.
  • Example 6 Comparison of anti-BNLF2b antibody double antigen sandwich method and existing nasopharyngeal cancer screening reagents
  • the results of the combined detection of EBNA1/IgA+VCA/IgA are shown in Figure 6C.
  • 69 and 8 have risk rates exceeding 0.98, respectively.
  • the sensitivity and specificity are 93.64, respectively. % And 96.80%.
  • the sensitivity of anti-BNLF2b antibody detection is 93.55% (116/124), and the specificity is 99.73% (748/750).
  • the positive predictive value of anti-BNLF2b antibody detection is 33.2%, which is compared with the positive prediction of EBNA1/IgA+VCA/IgA combined detection
  • the value (4.4%, 38/862) increased significantly (P ⁇ 0.0001).
  • the above results show that the anti-BNLF2b antibody detection can significantly improve the specificity and positive predictive value of nasopharyngeal cancer screening compared with the existing EBNA1/IgA+VCA/IgA combined screening method.
  • Example 7 Application of anti-BNLF2b antibody double antigen sandwich method in the screening of nasopharyngeal carcinoma
  • Example 8 Combined use of anti-BNLF2b antibody double antigen sandwich method and existing nasopharyngeal cancer screening reagents
  • Example 7 Combining the data in Example 6 and Example 7, a total of 79 cases of nasopharyngeal carcinoma and 1,570 cases of non-nasopharyngeal carcinoma.
  • the test results of 79 nasopharyngeal carcinoma specimens are shown in Table 7-1.
  • the anti-BNLF2b antibody test and EBNA1/IgA, VCA/IgA, and risk factors were positive at the same time in 70, 67, and 72 samples, respectively.
  • the test results of 1570 non-nasopharyngeal cancer specimens are shown in Table 7-2. There were only 5, 5, and 4 specimens that were positive for the anti-BNLF2b antibody test and EBNA/IgA, VCA/IgA, and risk factor at the same time, respectively.
  • Table 7-1 Test results of 79 specimens of nasopharyngeal carcinoma
  • Example 2 high-risk specimens from the pre-screening cohort of Zhongshan People's Hospital that were determined to be high-risk by EBNA1/IgA+VCA/IgA combined detection (sample 2), of which 8 were diagnosed as nasopharyngeal carcinoma by nasopharyngoscopy.
  • the results are shown in Table 8.
  • the sensitivity and positive predictive value of the combined detection are 87.50% and 29.17%, respectively.
  • sample 2 Combine sample 2 with sample 1, where sample 1 includes 8 cases with a risk coefficient> 0.98 in the 250 healthy controls in Example 6, and 126 cases with a risk coefficient> 0.98 in the screening cohort of 1325 people in Example 7 (5 An example is nasopharyngeal carcinoma).
  • sample 1 includes 8 cases with a risk coefficient> 0.98 in the 250 healthy controls in Example 6, and 126 cases with a risk coefficient> 0.98 in the screening cohort of 1325 people in Example 7
  • An example is nasopharyngeal carcinoma.
  • the positive predictive value of the combined use was increased to 36.36% (Table 8 ). Therefore, the combined use of anti-BNLF2b antibody detection and EBNA1/IgA+VCA/IgA can further improve the specificity and positive predictive value of nasopharyngeal cancer screening. In order to reduce the workload, you can first detect the anti-BNLF2b antibody, and then further detect EBNA1/IgA and VCA/IgA.
  • Example 9 Application of anti-BNLF2b antibody double antigen sandwich method in auxiliary diagnosis of nasopharyngeal carcinoma
  • nasopharyngeal carcinoma The clinical symptoms of nasopharyngeal carcinoma are not specific enough to be distinguished from other head and neck diseases. Clinically, suspected cases are diagnosed by nasopharyngoscope and pathological examination. We collected 63 suspected cases of nasopharyngeal carcinoma, 31 of which were finally diagnosed as nasopharyngeal carcinoma. We tested these 63 specimens by the double antigen sandwich method according to the method in Example 6, and the results showed that with 0.1 as the cut-off value, 30 of the 31 nasopharyngeal carcinoma cases tested positive for the BNLF2b antibody. , And in 32 cases of non-nasopharyngeal carcinoma, only 2 cases were positive for BNLF2b antibody.
  • the sensitivity of BNLF2b antibody detection was 96.78% (30/31), the specificity was 93.75% (30/32), and the positive predictive value was 93.75% (30/32).
  • the above results show that in outpatient cases, the BNLF2b antibody test can significantly reduce the number of unnecessary nasopharyngoscopes and reduce the economic burden and physical burden of patients.
  • Example 10 Study on the immunodominant epitope of the protein encoded by BNLF2b
  • the BNLF2b gene encodes a total of 98 amino acids (SEQ ID NO: 101).
  • SEQ ID NO: 101 The BNLF2b gene encodes a total of 98 amino acids.
  • 9 peptides by walking method. Each polypeptide has 15 amino acids (the last polypeptide is 18 amino acids), and the overlap between two adjacent polypeptides is 5 amino acids, and biotin is labeled at the C-terminus for subsequent detection (SEQ ID NOs: 92-100).
  • the above-mentioned polypeptide fragments were used as coating antigens to detect the IgG in the serum of nasopharyngeal carcinoma patients diluted 1:300.
  • the results are shown in Figure 9, the BNLF2b-encoded protein epitopes are mainly located in the four segments aa1-25, aa31-45, aa51-65 and aa81-98.
  • Example 3 We further followed the method in Example 3 and preliminarily evaluated aa1-25 (SEQ ID NO: 102), aa31-45 (SEQ ID NO: 95), aa51-65 (SEQ ID NO: 97), aa81-98 (SEQ ID NO: 100) the sensitivity of four segment peptides in nasopharyngeal cancer screening, the results found that the detection sensitivity of aa51-65 was 76.7% (33/ 43), aa1-25 is 72.1% (31/43), and the sensitivity of the other two peptides is 55.8% (24/43).
  • the sensitivities of aa14-52 and aa1-74 were 90.7% (39/43) and 93.0% (40/43), and the three missed serum fragment 4 peptides of aa1-74 were all negative, while aa14-52 was negative, aa1
  • the OD value of -74 positive serum reacted with aa51-65 was 0.393.
  • aa1-15 and aa11-25 contain two independent epitopes, and the key amino acids are aa5-7 and aa16-18, respectively.
  • aa1-25 segment contains epitopes spanning aa1-15 and aa11-25
  • aa1-15, aa11-25 and aa1-25 as the coating antigens
  • 43 nasopharyngeal carcinoma sera IgG The results are shown in Figure 11A.
  • the OD values of 14 serums for aa1-25 are higher than the sum of aa1-15 and aa11-25.
  • the 14 sera were tested with different truncated polypeptides in the aa1-25 segment as coating antigens.
  • the polypeptides capable of reacting with the positive sera contained at least aa10-16.
  • aa1-25 ( Figure 12A): the 6th, 9-11 and 16th amino acid mutations are most significantly reduced; there are 3 natural mutations at the 12th position, namely T, D and S. Amino acid mutation into these three amino acids does not significantly reduce the reactivity of aa1-25 with nasopharyngeal carcinoma serum; in addition, amino acid mutations at positions 5, 7, 8, 13, 14, 15, 19, 22, 24, and 25 The impact is relatively small.
  • Example 11 The effect of the combined use of different epitopes of the BNLF2b encoded protein on the screening performance of nasopharyngeal carcinoma. Based on the previously synthesized aa14-52, aa1-74, aa1-52, aa1-25, aa11- 65 (SEQ ID NO: 103) and aa11-74 (SEQ ID NO: 104) two polypeptides, and biotin is labeled at the C terminal. According to the method in Example 3, the reaction of these polypeptides with 84 sera of nasopharyngeal carcinoma patients and 168 sera of healthy people was detected respectively.

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Abstract

提供了基于抗EB病毒(EBV)抗体水平来诊断鼻咽癌的方法，以及用于所述方法的试剂盒。还提供了用于上述诊断的EB病毒BNLF2b基因编码的多肽及其用于诊断鼻咽癌的用途。

Description

EB病毒BNLF2b基因编码多肽及其检测用途 技术领域
本发明涉及免疫学检测，特别是肿瘤的免疫学诊断领域。具体而言，本发明提供了基于抗EB病毒(EBV)抗体水平来诊断鼻咽癌的方法，以及用于所述方法的试剂盒。本发明还提供了用于上述诊断的EB病毒BNLF2b基因编码的多肽及其用于诊断鼻咽癌的用途。
背景技术
鼻咽癌是一种好发于鼻咽腔顶部和侧壁的恶性肿瘤，其发病率居耳鼻喉恶性肿瘤之首。我国是鼻咽癌高发区，以广东、广西地区发病率最高[1]。鼻咽癌的发生与EB病毒感染、遗传因素、环境因素等多种因素有关[2]。由于鼻咽癌早期症状不明显，而且发病位置较为隐秘，大部分的鼻咽癌到了晚期才能发现[3]。但是，早期鼻咽癌的五年生存率可以达到90％以上，而晚期鼻咽癌的预后显著降低[3]。同时，早期鼻咽癌可以通过局部放疗进行治疗，而晚期鼻咽癌则要进行化疗。因此，早期诊断是提高鼻咽癌患者生存率和生存质量的关键。
早在1976年，Werner Henle等就发现，鼻咽癌患者血清中EBV IgA抗体显著升高[4]。随后，研究发现，鼻咽癌患者血清中针对EBNA1、EA-D、VCA等多种蛋白的抗体水平均显著升高[5-12]。早期EBV抗体检测以免疫荧光法为主，通量低、耗时长且结果主观性较强，难以满足大规模筛查的需求[13-15]；随着酶联免疫法(ELISA)的发展，各国科研人员建立了大量的EB病毒抗体ELISA检测试剂盒，使得检测通量和客观性显著提高[16-23]。此外，研究还发现，通过联合使用不同的血清学标志物，可进一步提高鼻咽癌筛查的灵敏度和特异性，其中应用最多的是EBNA1-IgA和VCA-IgA联合使用[24-26]。前瞻性队列研究的结果表明，通过筛查，鼻咽癌的早期诊断率可以由10-20％提高到60％以上[27-29]。在此基础上，本课题组提出了两步法筛查的策略，即：在EBNA1-IgA与VCA-IgA联合筛查的基础上，对第一步筛查为高危的人群进一步检测EAD-IgA，EAD-IgA阳性者再进行鼻咽镜确诊。通过两步法筛查，鼻咽癌筛查的阳性预测值由原来的4.69％提高到18.52％[30]。
除EBV抗体水平外，鼻咽癌患者血液中EBV DNA和micro RNA中的水平也显著升高[31-33]。Allan Chan等在香港的一项前瞻性研究的结果表明，以持续性EBV DNA 阳性做为标准进行鼻咽癌筛查，可以显著提高鼻咽癌筛查的特异性，在初筛EBV DNA阳性的1112人(5.5％)中，仅309人EBV DNA持续阳性，其中34人最终确诊鼻咽癌[34]。以持续性EBV阳性做为筛查标准，灵敏度和特异性分别达到97.1％和98.6％，阳性预测值由3.06％提高到11％，通过筛查鼻咽癌的早期诊断率由20％提高到71％[34]。
相比一步法筛查，两步法筛查的阳性预测值显著提高，可以满足更大规模的鼻咽癌筛查的需求。但是，无论是血清学筛查，还是DNA筛查，两步法筛查操作均更为复杂，成本也更高。此外，DNA持续性阳性还需要再次采血，进一步增加了筛查的难度。因此，尽管一步法血清学抗体筛查具有显著的成本效益[35]，但两步法筛查是否具有更高的成本效益还有待进一步评估。此外，目前报导的鼻咽癌筛查方法阳性预测值最高仅为18.52％，即：在筛查高危的人群中，至少有71.48％的非鼻咽癌患者要进行鼻咽镜检查。而鼻咽镜检查耗时长，这就直接限制了鼻咽癌筛查的规模，同时，也会给筛查阳性的非鼻咽癌患者造成心理负担。
因此，本领域仍然需要开发新的鼻咽癌筛查方法，其相比现有方法具有更高的特异性和阳性预测值，同时，具有高通量、成本低的特点，以满足大规模筛查的需求，使更多人能够从筛查中受益。
发明内容
本申请的发明人经过大量的研究后出人意料地发现：受试者血清中针对EBVBNLF2b基因编码蛋白的抗体水平(其可以例如，使用BNLF2b基因编码蛋白或多肽片段作为包被抗原通过间接法或双抗原夹心法来进行检测)可高效区分鼻咽癌患者与健康对照。此外，发明人还付出了大量的创造性劳动，获得了特别适用于作为捕获试剂(例如，ELISA中的包被抗原)的BNLF2b基因编码多肽片段。由此，发明人基于针对EBV BNLF2b基因编码蛋白的抗体水平的检测，成功地建立了用于诊断鼻咽癌的方法和平台，其能够显著提高鼻咽癌筛查的特异性和阳性预测值。
分离的多肽或其变体
在第一方面，本发明提供了一种分离的多肽或其变体，其中，所述多肽由BNLF2b基因编码的野生型蛋白的至少7个连续氨基酸残基组成，且包含至少1个(例如至少2个、至少3个、或全部4个)选自下列的序列：BNLF2b基因编码的野生型蛋 白的第5-11位氨基酸残基、第16-23位氨基酸残基、第31-39位氨基酸残基、或第53-60位氨基酸残基(例如第53-61位氨基酸残基)；
其中，所述变体与其所源自的多肽相异仅在于1个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸残基的置换(例如，保守置换或非保守置换)，且保留了其所源自的多肽的生物学功能(例如被抗EBV抗体识别并结合的活性)。
在某些实施方案中，所述BNLF2b基因编码的野生型蛋白具有如SEQ ID NO:101所示的序列。
在本文中，本发明的多肽或其变体的生物学功能包括但不限于作为表位肽被抗EBV抗体(例如对BNLF2b基因编码的蛋白特异性的抗体)识别并结合的活性。
在某些实施方案中，所述分离的多肽包含：BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基(例如第53-61位氨基酸残基)、第5-23位氨基酸残基、第16-39位氨基酸残基、第31-60位氨基酸残基(例如第31-61位氨基酸残基)、第5-39位氨基酸残基、第16-60位氨基酸残基(例如第16-61位氨基酸残基)、或第5-60位氨基酸残基(例如第5-61位氨基酸残基)。
在一个实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少8个(例如，至少9个、至少10个、至少11个、至少12个、至少13个、至少14个或至少15个)连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第53-61位氨基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第51-65位氨基酸残基。
在某些实施方案中，所述第51-65位氨基酸残基具有SEQ ID NO:97所示的序列。
在某些实施方案中，所述多肽由BNLF2b基因编码的野生型蛋白的至少15个连续氨基酸残基组成。
在一个实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少19 个(例如，至少20个、至少21个、至少22个、至少23个、至少24个、或至少25个)连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第5-23位氨基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第1-25位氨基酸残基。
在某些实施方案中，所述第1-25位氨基酸残基具有SEQ ID NO:102所示的序列。
在某些实施方案中，所述多肽由BNLF2b基因编码的野生型蛋白的至少25个连续氨基酸残基组成。
在一个实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少24个(例如，至少25个、至少26个、至少27个、至少28个、至少29个、至少30个、至少31个、至少32个、至少33个、至少34个、至少35个、至少36个、至少37个、至少38个、或至少39个)连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第16-39位氨基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第14-52位氨基酸残基。
在某些实施方案中，所述第14-52位氨基酸残基具有SEQ ID NO:88所示的序列。
在某些实施方案中，所述多肽由BNLF2b基因编码的野生型蛋白的至少39个连续氨基酸残基组成。
在一个实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少30个连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第31-60位氨基酸残基。
在某些实施方案中，所述分离的多肽包含BNLF2b基因编码的野生型蛋白的第31-61位氨基酸残基。
在一个实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少35个(例如，至少36个、至少37个、至少38个、至少39个、至少40个、至少41个、至少42个、至少43个、至少44个、至少45个、至少46个、至少47个、至少48个、至少49个、至少50个、至少51个、至少52个、或至少53个)连续氨基酸残基组成， 且包含BNLF2b基因编码的野生型蛋白的第5-39位氨基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第1-52位氨基酸残基。
在某些实施方案中，所述第1-52位氨基酸残基具有SEQ ID NO:91所示的序列。
在某些实施方案中，所述多肽由BNLF2b基因编码的野生型蛋白的至少53个连续氨基酸残基组成。
在一个实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少45个(例如，至少46个、至少47个、至少48个、至少49个、至少50个、至少51个、至少52个、至少53个、至少54个、至少55个、至少56个、至少57个、至少58个、至少59个、至少60个、至少61个、至少62个、至少63个、或至少64个)连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第16-60位氨基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第16-61位氨基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第14-74位氨基酸残基(例如，SEQ ID NO:90所示的序列)。在某些实施方案中，所述多肽由BNLF2b基因编码的野生型蛋白的至少61个连续氨基酸残基组成。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第11-65位氨基酸残基(例如，SEQ ID NO:103所示的序列)。在某些实施方案中，所述多肽由BNLF2b基因编码的野生型蛋白的至少55个连续氨基酸残基组成。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第11-74位氨基酸残基(例如，SEQ ID NO:104所示的序列)。在某些实施方案中，所述多肽由BNLF2b基因编码的野生型蛋白的至少64个连续氨基酸残基组成。
在一个实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少56个(例如，至少57个、至少58个、至少59个、至少60个、至少61个、至少62个、至少63个、至少64个、至少65个、至少66个、至少67个、至少68个、至少69个、至少70个、至少71个、至少72个、至少73个、或至少74个)连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第5-60位氨基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第5-61位氨 基酸残基。
在某些实施方案中，所述多肽包含BNLF2b基因编码的野生型蛋白的第1-74位氨基酸残基。
在某些实施方案中，所述第1-74位氨基酸残基具有SEQ ID NO:89所示的序列。
在某些实施方案中，所述多肽由BNLF2b基因编码的野生型蛋白的至少74个连续氨基酸残基组成。
在某些示例性实施方案中，所述分离的多肽由选自下列的序列组成：BNLF2b基因编码的野生型蛋白的第51-56位氨基酸残基、第1-25位氨基酸残基、第14-52位氨基酸残基、第1-52位氨基酸残基、第14-74位氨基酸残基、第11-65位氨基酸残基、第11-74位氨基酸残基、或第1-74位氨基酸残基。在某些示例性实施方案中，所述分离的多肽由选自下列的序列组成：SEQ ID NOs:88-91、97、102-104。
在某些实施方案中，本发明的变体与其所源自的多肽相异仅在于1个、2个、3个或4个氨基酸残基的置换(例如，保守置换或非保守置换)，且保留了其所源自的多肽的生物学功能(例如被抗EBV抗体识别并结合的活性)。
在某些实施方案中，所述变体不包括在相应于如下位置的氨基酸位置中的氨基酸置换，所述如下位置参考由BNLF2b基因编码的野生型蛋白所示的氨基酸位置：6、9、10、11、16、31、33、38、39、53、54、56、57、58、59、95、96、97。
在某些实施方案中，所述变体在相应于如下位置的氨基酸位置中的一处或多处(例如1处，2处，3处或4处)包含氨基酸置换，所述如下位置参考由BNLF2b基因编码的野生型蛋白所示氨基酸位置：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或98。
在某些实施方案中，所述变体在相应于如下位置的氨基酸位置中的一处或多处(例如1处，2处，3处或4处)包含氨基酸置换，所述如下位置参考由BNLF2b基因编码的野生型蛋白所示氨基酸位置：5、7、8、12、13、14、22、25、32、34、35、36、40、41、42、52、55、60、61、89、91、93或98。
在某些实施方案中，所述变体包含选自下列的1个或几个(例如，1个、2个、3个或4个)氨基酸置换：在相应于位置5的位置上的氨基酸置换为A，在相应于位置7的 位置上的氨基酸置换为A，在相应于位置8的位置上的氨基酸置换为G，在相应于位置12的位置上的氨基酸置换为G、T、D或S，在相应于位置13的位置上的氨基酸置换为A，在相应于位置14的位置上的氨基酸置换为G，在相应于位置15的位置上的氨基酸置换为A，在相应于位置22的位置上的氨基酸置换为A，在相应于位置24的位置上的氨基酸置换为A，在相应于位置25的位置上的氨基酸置换为A，在相应于位置32的位置上的氨基酸置换为A，在相应于位置34的位置上的氨基酸置换为A，在相应于位置35的位置上的氨基酸置换为A，在相应于位置36的位置上的氨基酸置换为A，在相应于位置37的位置上的氨基酸置换为A、N、Q、S或R，在相应于位置40的位置上的氨基酸置换为A，在相应于位置41的位置上的氨基酸置换为A，在相应于位置42的位置上的氨基酸置换为A，在相应于位置52的位置上的氨基酸置换为K、H、A、S或D，在相应于位置55的位置上的氨基酸置换为S，在相应于位置60的位置上的氨基酸置换为A，在相应于位置61的位置上的氨基酸置换为K、H、S或A，在相应于位置89的位置上的氨基酸置换为A或T，在相应于位置91的位置上的氨基酸置换为A，在相应于位置93的位置上的氨基酸置换为Q，在相应于位置98的位置上的氨基酸置换为A。
在某些实施方案中，所述变体包含选自下列的1个或几个(例如，1个、2个、3个或4个)氨基酸置换：在相应于位置5的位置上的氨基酸置换为A，在相应于位置7的位置上的氨基酸置换为A，在相应于位置8的位置上的氨基酸置换为G，在相应于位置12的位置上的氨基酸置换为G、T、D或S，在相应于位置13的位置上的氨基酸置换为A，在相应于位置14的位置上的氨基酸置换为G，在在相应于位置22的位置上的氨基酸置换为A，在相应于位置25的位置上的氨基酸置换为A，在相应于位置32的位置上的氨基酸置换为A，在相应于位置34的位置上的氨基酸置换为A，在相应于位置35的位置上的氨基酸置换为A，在相应于位置36的位置上的氨基酸置换为A，在相应于位置40的位置上的氨基酸置换为A，在相应于位置41的位置上的氨基酸置换为A，在相应于位置42的位置上的氨基酸置换为A，在相应于位置52的位置上的氨基酸置换为K、H、A、S或D，在相应于位置55的位置上的氨基酸置换为S，在相应于位置60的位置上的氨基酸置换为A，在相应于位置61的位置上的氨基酸置换为K、H、S或A，在相应于位置89的位置上的氨基酸置换为A或T，在相应于位置91的位置上的氨基酸置换为A，在相应于位置93的位置上的氨基酸置换为Q，在相应于位置98的位置上的氨基酸置换为A。
在某些实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的不多于97个(例如，不多于96个，不多于95个，不多于94个，不多于93个，不多于92个，不多于91个，不多于90个，不多于89个，不多于88个，不多于87个，不多于86个，不多于85个，不多于84个，不多于83个，不多于82个，不多于81个，不多于80个，不多于79个，不多于78个，不多于76个，不多于75个，或不多于74个)的连续氨基酸残基组成。
在某些实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的15～80个(例如15～74个)连续氨基酸残基组成。在某些实施方案中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的39～74个连续氨基酸残基组成。
在一个实施方案中，所述分离的多肽或其变体连接于固相载体表面，或者带有可与固相载体连接的修饰基团。在某些实施方案中，所述分离的多肽或其变体的C端连接于固相载体表面，或者带有可与固相载体连接的修饰基团。在某些实施方案中，所述修饰基团是生物素或亲和素。在某些实施方案中，所述固相载体选自磁珠或微量滴定板(例如微孔板或酶标板)。
在另一个实施方案中，所述分离的多肽或其变体带有可检测的标记。在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
多肽的制备
本发明的多肽或其变体不受其产生方式的限定，例如，其可以通过基因工程方法(重组技术)产生，也可以通过化学合成方法产生。
在另一个方面，本发明提供了一种分离的核酸分子，其包含编码本发明的多肽或其变体的核苷酸序列。
在另一个方面，本发明还提供了一种载体，其包含如上所述的分离的核酸分子。本发明的载体可以是克隆载体，也可以是表达载体。在一个优选实施方案中，本发明的载体是例如质粒，粘粒，噬菌体，柯斯质粒等等。
在另一个方面，本发明还提供了包含本发明的分离的核酸分子或载体的宿主细胞。 此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如293T细胞。
在另一个方面，本发明还提供了制备本发明的多肽或其变体的方法，其包括，在允许所述多肽或其变体表达的条件下，培养本发明的宿主细胞，和从培养的宿主细胞培养物中回收所述多肽或其变体。
检测试剂盒
在第二方面，本发明提供了一种试剂盒，其包含捕获试剂，所述捕获试剂选自第一方面所述的分离的多肽或其变体。在某些实施方案中，所述试剂盒还包含使用所述分离的多肽或其变体作为捕获试剂来检测样品中的对BNLF2b基因编码的蛋白特异性的抗体的说明书，或者使用所述分离的多肽或其变体作为捕获试剂来测定来自受试者的样品中对BNLF2b基因编码的蛋白特异性的抗体的水平，从而确定所述受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中的说明书。
在某些实施方案中，所述受试者是哺乳动物，例如人。
在一些实施方案中，所述捕获试剂连接于固相载体表面。在某些实施方案中，所述捕获试剂的C端与固相载体连接。在本文中，所述的固相载体包括由聚合物材料(例如聚氯乙烯、聚苯乙烯、聚丙酰胺或纤维素)制成或包被的凹孔平板、试管、珠粒(例如乳胶颗粒)或薄膜(例如硝酸纤维素膜)，或由功能基团(例如氨基、羧基、生物素或亲和素)预包被的磁珠。在某些实施方案中，所述固相载体选自磁珠或微量滴定板(例如微孔板或酶标板)。
在另一些实施方案中，所述捕获试剂带有可与固相载体连接的修饰基团。在某些实施方案中，所述捕获试剂的C端带有可与固相载体连接的修饰基团。在此类实施方案中，所述试剂盒可进一步包含用于将所述捕获试剂包被于所述固相载体上的包被试剂，例如包被缓冲液(例如，碳酸盐缓冲液、磷酸盐缓冲液、Tris-HCL缓冲液或硼酸盐缓冲液)。将蛋白或多肽包被于固相载体上的方法是本领域熟知的，例如物理吸附、通过氨基化或羧基化表面实现的共价偶联或通过亲和素-生物素系统、聚赖氨酸预包被表面、蛋白A或蛋白G预包被表面实现的介导结合。在某些实施方案中，所述修饰基团是生物素或亲和素，所述固相载体表面带有相应的连接基团。
在某些实施方案中，所述试剂盒在单独的容器中或在单个容器单元的分开的隔室 中至少包含用抗生物素蛋白或链霉抗生物素蛋白包被的固相载体，以及上述捕获试剂。
在一些实施方案中，所述测定来自受试者的样品中抗EB病毒(EBV)抗体的水平从而确定所述受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中的方法以双抗原夹心形式进行。通过双抗原夹心法来测定样品中的抗体水平是本领域技术人员熟知的。在此类测定中，“捕获抗原”、“检测抗原”使得样品抗体在两种特异性抗原之间形成桥，因此，两种抗原通常是相同的、或具有相同的核心表位、或具有免疫交叉反应性，使得一种抗体能够结合两种抗原。
因此，在某些实施方案中，所述试剂盒还包含检测试剂，所述检测试剂选自第一方面所述的分离的多肽或其变体。
在某些实施方案中，所述检测试剂带有可检测的标记。在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。在某些示例性实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)。
在某些实施方案中，所述检测试剂和捕获试剂中所包含的多肽序列相同或基本相同。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列包含相同的核心片段，所述核心片段选自BNLF2b基因编码的野生型蛋白的第5-11位氨基酸残基、第16-23位氨基酸残基、第31-39位氨基酸残基、和/或第53-60位氨基酸残基(例如第53-61位氨基酸残基)。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列同时包含BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基(例如第53-61位氨基酸残基)、第5-23位氨基酸残基、第16-39位氨基酸残基、第5-39位氨基酸残基、第16-60位氨基酸残基(例如第16-61位氨基酸残基)、或第5-60位氨基酸残基(例如第5-61位氨基酸残基)。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列同时包含BNLF2b基因编码的野生型蛋白的第51-56位氨基酸残基、第1-25位氨基酸残基、第14-52位氨基酸残基、第1-52位氨基酸残基、第14-74位氨基酸残基、第11-65位氨基酸残基、第11-74位氨基酸残基、或第1-74位氨基酸残基。
在另一些实施方案中，所述测定来自受试者的样品中抗EB病毒(EBV)抗体的水平从而确定所述受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中的方法以间接法形式进行。通过间接法来测定样品中的抗体水平是本领域技术人员熟知的。在此类测定 中，“捕获抗原”首先与样品中的抗体形成免疫复合物，随后通过二级抗体(例如，抗-免疫球蛋白抗体)对被捕获的抗体进行检测。
因此，在某些实施方案中，所述试剂盒还包含检测试剂，所述检测试剂选自带有可检测的标记的二级抗体。在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。在某些示例性实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)。
在某些实施方案中，所述二级抗体对待测抗体所来自的物种(例如人)的抗体是特异的。
在某些实施方案中，所述二级抗体是抗-免疫球蛋白抗体。
在某些示例性实施方案中，所述试剂盒用于检测抗EBV IgG抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgG抗体，例如抗人IgG抗体。
在某些示例性实施方案中，所述试剂盒用于检测抗EBV IgM抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgM抗体，例如抗人IgM抗体。
在某些示例性实施方案中，所述试剂盒用于检测抗EBV IgA抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgA抗体，例如抗人IgA抗体。
在某些实施方案中，本发明的试剂盒还可以包含一种或多种选自下列的试剂或装置：(i)用于收集或贮存来自受试者的样品的装置(例如采血装置)；(ii)用于进行所述测定所需的其他试剂(例如缓冲液，稀释液，封闭液，和/或标准品)。
检测用途及方法
在第三方面，本发明提供了用于检测样品中的对BNLF2b基因编码的蛋白特异性的抗体的方法，其包括以下步骤：
(1)将所述样品与捕获试剂接触，以获得抗原-抗体免疫复合物；所述捕获试剂选自第一方面所述的分离的多肽或其变体；
(2)测定步骤(1)获得的抗原-抗体免疫复合物的量。
在某些实施方案中，在步骤(2)中，通过免疫学检测来测定所述免疫复合物的量。
在某些实施方案中，所述免疫学检测选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在一些实施方案中，所述捕获试剂连接于固相载体表面。
在另一些实施方案中，所述捕获试剂带有可与固相载体连接的修饰基团。在此类实施方案中，在步骤(1)之前，所述方法还包含将所述捕获试剂包被于固相载体表面的步骤。
在某些实施方案中，所述捕获试剂的C端连接于固相载体表面，或者带有可与固相载体连接的修饰基团。
在一些实施方案中，所述测定以双抗原夹心形式进行。通过双抗原夹心法来测定样品中的抗体水平是本领域技术人员熟知的。在此类测定中，“捕获抗原”、“检测抗原”使得样品抗体在两种特异性抗原之间形成桥，因此，两种抗原通常是相同的、或具有相同的核心表位、或具有免疫交叉反应性，使得一种抗体能够结合两种抗原。
因此，在某些实施方案中，在步骤(2)中，使用检测试剂来检测所述免疫复合物的量，所述检测试剂选自第一方面所述的分离的多肽或其变体。
在某些实施方案中，所述检测试剂选自第一方面所述的分离的多肽或其变体，所述分离的多肽或其变体带有可检测的标记。在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。在某些示例性实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)。
在某些实施方案中，所述检测试剂和捕获试剂中所包含的多肽序列相同或基本相同。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列包含相同的核心片段，所述核心片段选自BNLF2b基因编码的野生型蛋白的第5-11位氨基酸残基、第16-23位氨基酸残基、第31-39位氨基酸残基、和/或第53-60位氨基酸残基(例如第53-61位氨基酸残基)。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列同时包含BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基(例如第53-61位氨基酸残基)、第5-23位氨基酸残基、第16-39位氨基酸残基、第5-39位氨基酸残基、第16-60位氨基酸残基(例如第16-61位氨基酸残基)、或第5-60位氨基酸残基(例如第5-61位氨基酸残基)。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列同时包含BNLF2b基因编码的野生型蛋白的第51-56位氨基酸残基、第1-25位氨基酸残基、第14-52位氨基酸残基、第1-52位氨基酸残基、第14-74位氨基酸残基、第 11-65位氨基酸残基、第11-74位氨基酸残基、或第1-74位氨基酸残基。
在另一些实施方案中，所述测定以间接法形式进行。通过间接法来测定样品中的抗体水平是本领域技术人员熟知的。在此类测定中，“捕获抗原”首先与样品中的抗体形成免疫复合物，随后通过二级抗体(例如，抗-免疫球蛋白抗体)对被捕获的抗体进行检测。
因此，在某些实施方案中，在步骤(2)中，使用检测试剂来检测所述免疫复合物的量，所述检测试剂选自带有可检测的标记的二级抗体。
在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
在某些实施方案中，所述二级抗体对待测抗体所来自的物种(例如人)的抗体是特异的。
在某些实施方案中，所述二级抗体是抗-免疫球蛋白抗体。
在某些示例性实施方案中，被检测的抗体是IgG抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgG抗体，例如抗人IgG抗体。
在某些示例性实施方案中，被检测的抗体是IgM抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgM抗体，例如抗人IgM抗体。
在某些示例性实施方案中，被检测的抗体是IgA抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgA抗体，例如抗人IgA抗体。
在另一方面，本发明还涉及第一方面所述的分离的多肽或其变体在制备试剂盒中的用途，所述试剂盒用于检测样品中的对BNLF2b基因编码的蛋白特异性的抗体。在某些实施方案中，所述试剂盒通过第三方面所述的方法检测样品中的对BNLF2b基因编码的蛋白特异性的抗体。
诊断用途及方法
在第四方面，本发明提供了用于确定受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中的方法，其包括：
(1)测定来自所述受试者的样品中对BNLF2b基因编码的蛋白特异性的抗体的水平；和，
(2)将所述水平与参考值比较。
在某些实施方案中，如果所述水平高于参考值的话，则判断所述受试者患有鼻咽癌或是否处于鼻咽癌的风险中。
在本文中，所述参考值是来源于未患鼻咽癌的对象或健康人(例如，没有可检测到的疾病、并且没有癌症或鼻咽癌历史的对象)的值，或者指示未患鼻咽癌的对象或健康人的相应样品中的对BNLF2b基因编码的蛋白特异性的抗体水平。
在某些实施方案中，所述样品是血液样品，例如全血、血浆或血清。
在某些实施方案中，所述受试者是哺乳动物，例如人。
在某些实施方案中，通过免疫学测定来测定所述样品中对BNLF2b基因编码的蛋白特异性的抗体的水平。在某些实施方案中，所述免疫学测定选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在某些实施方案中，所述测定包括使用第一方面所述的分离的多肽或其变体作为捕获试剂。
在某些实施方案中，步骤(1)包括以下步骤：
(1a)将来自所述受试者的样品与捕获试剂接触，以获得抗原-抗体免疫复合物；所述捕获试剂选自第一方面所述的分离的多肽或其变体；
(1b)测定步骤(1a)获得的抗原-抗体免疫复合物的量。
在某些实施方案中，在步骤(1b)中，通过免疫学检测来测定所述免疫复合物的量。在某些实施方案中，所述免疫学检测选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在一些实施方案中，所述捕获试剂连接于固相载体表面。
在另一些实施方案中，所述捕获试剂带有可与固相载体连接的修饰基团。在此类实施方案中，在步骤(1)之前，所述方法还包含将所述捕获试剂包被于固相载体表面的步骤。
在某些实施方案中，所述捕获试剂的C端连接于固相载体表面，或者带有可与固相载体连接的修饰基团。
在一些实施方案中，所述测定以双抗原夹心形式进行。通过双抗原夹心法来测定样品中的抗体水平是本领域技术人员熟知的。在此类测定中，“捕获抗原”、“检测抗原” 使得样品抗体在两种特异性抗原之间形成桥，因此，两种抗原通常是相同的、或具有相同的核心表位、或具有免疫交叉反应性，使得一种抗体能够结合两种抗原。
因此，在某些实施方案中，在步骤(1b)中，使用检测试剂来检测所述免疫复合物的量，所述检测试剂选自第一方面所述的分离的多肽或其变体。
在某些实施方案中，所述检测试剂选自第一方面所述的分离的多肽或其变体，所述分离的多肽或其变体带有可检测的标记。在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。在某些示例性实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)。
在某些实施方案中，所述检测试剂和捕获试剂中所包含的多肽序列相同或基本相同。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列包含相同的核心片段，所述核心片段选自BNLF2b基因编码的野生型蛋白的第5-11位氨基酸残基、第16-23位氨基酸残基、第31-39位氨基酸残基、和/或第53-60位氨基酸残基(例如第53-61位氨基酸残基)。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列同时包含BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基(例如第53-61位氨基酸残基)、第5-23位氨基酸残基、第16-39位氨基酸残基、第5-39位氨基酸残基、第16-60位氨基酸残基(例如第16-61位氨基酸残基)、或第5-60位氨基酸残基(例如第5-61位氨基酸残基)。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列同时包含BNLF2b基因编码的野生型蛋白的第51-56位氨基酸残基、第1-25位氨基酸残基、第14-52位氨基酸残基、第1-52位氨基酸残基、第14-74位氨基酸残基、第11-65位氨基酸残基、第11-74位氨基酸残基、或第1-74位氨基酸残基。
在另一些实施方案中，所述测定以间接法形式进行。通过间接法来测定样品中的抗体水平是本领域技术人员熟知的。在此类测定中，“捕获抗原”首先与样品中的抗体形成免疫复合物，随后通过二级抗体(例如，抗-免疫球蛋白抗体)对被捕获的抗体进行检测。
因此，在某些实施方案中，在步骤(1b)中，使用检测试剂来检测所述免疫复合物的量，所述检测试剂选自带有可检测的标记的二级抗体。
在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸 酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
在某些实施方案中，所述二级抗体对待测抗体所来自的物种(例如人)的抗体是特异的。
在某些实施方案中，所述二级抗体是抗-免疫球蛋白抗体。
在某些示例性实施方案中，被检测的抗体是IgG抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgG抗体，例如抗人IgG抗体。
在某些示例性实施方案中，被检测的抗体是IgM抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgM抗体，例如抗人IgM抗体。
在某些示例性实施方案中，被检测的抗体是IgA抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgA抗体，例如抗人IgA抗体。
在某些实施方案中，所述方法还包括：在步骤(1)之前，提供来自所述受试者的样品。
在某些实施方案中，所述方法还包括：在步骤(2)之后，给被判断为患有鼻咽癌或处于鼻咽癌的风险中的受试者施用治疗有效量的可治疗鼻咽癌的抗肿瘤疗法(例如，化学疗法、放射疗法和/或免疫疗法)。
在某些实施方案中，所述可治疗鼻咽癌的抗肿瘤疗法选自手术治疗，放射疗法(如体外放射线治疗EBRT，近距离放疗)，或化学疗法(如卡铂，紫杉醇，多西他赛，吉西他滨，阿霉素，表阿霉素，博莱霉素，甲氨蝶呤)，靶向治疗(如西妥昔单抗)，免疫治疗(如PD-1单抗)以及多种方法联合治疗(如放疗+化疗，放疗+手术)。
在另一方面，本发明还涉及能够测定对BNLF2b基因编码的蛋白特异性的抗体的水平的试剂在制备试剂盒中的用途，所述试剂盒用于确定受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中。
在某些实施方案中，所述试剂能够通过免疫学测定来测定对BNLF2b基因编码的蛋白特异性的抗体的水平。在某些实施方案中，所述免疫学测定选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在某些实施方案中，所述受试者是哺乳动物，例如人。
在某些实施方案中，所述样品是血液样品，例如全血、血浆或血清。
在某些实施方案中，所述试剂盒通过下述方法来确定受试者是否患有鼻咽癌或是 否处于鼻咽癌的风险中：
(1)测定来自所述受试者的样品中对BNLF2b基因编码的蛋白特异性的抗体的水平；和，
(2)将所述水平与参考值比较。
在某些实施方案中，如果所述水平高于参考值的话，则判断所述受试者患有鼻咽癌或是否处于鼻咽癌的风险中。
在某些实施方案中，所述能够测定对BNLF2b基因编码的蛋白特异性的抗体的水平的试剂包含捕获试剂，所述捕获试剂选自第一方面所述的分离的多肽或其变体。
在某些实施方案中，上述步骤(1)包括以下步骤：
(1a)将来自所述受试者的样品与捕获试剂接触，以获得抗原-抗体免疫复合物；所述捕获试剂选自第一方面所述的分离的多肽或其变体；
(1b)测定步骤(1a)获得的抗原-抗体免疫复合物的量。
在某些实施方案中，在步骤(1b)中，通过免疫学检测来测定所述免疫复合物的量。在某些实施方案中，所述免疫学检测选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在一些实施方案中，所述捕获试剂连接于固相载体表面。
在另一些实施方案中，所述捕获试剂带有可与固相载体连接的修饰基团。在此类实施方案中，在上述步骤(1)之前，所述方法还包含将所述捕获试剂包被于固相载体表面的步骤。
在某些实施方案中，所述捕获试剂的C端连接于固相载体表面，或者带有可与固相载体连接的修饰基团。
在一些实施方案中，所述能够测定对BNLF2b基因编码的蛋白特异性的抗体的水平的试剂还包含检测试剂，所述检测试剂选自第一方面所述的分离的多肽或其变体。
在某些实施方案中，在上述步骤(1b)中，使用所述检测试剂来检测所述免疫复合物的量。
在某些实施方案中，所述检测试剂选自第一方面所述的分离的多肽或其变体，所述分离的多肽或其变体带有可检测的标记。在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、 荧光染料或生物素。在某些示例性实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)。
在某些实施方案中，所述检测试剂和捕获试剂中所包含的多肽序列相同或基本相同。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列包含相同的核心片段，所述核心片段选自BNLF2b基因编码的野生型蛋白的第5-11位氨基酸残基、第16-23位氨基酸残基、第31-39位氨基酸残基、和/或第53-60位氨基酸残基(例如第53-61位氨基酸残基)。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列同时包含BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基(例如第53-61位氨基酸残基)、第5-23位氨基酸残基、第16-39位氨基酸残基、第5-39位氨基酸残基、第16-60位氨基酸残基(例如第16-61位氨基酸残基)、或第5-60位氨基酸残基(例如第5-61位氨基酸残基)。在某些实施方案中，所述基本相同是指，所述检测试剂和捕获试剂中所包含的两个多肽序列同时包含BNLF2b基因编码的野生型蛋白的第51-56位氨基酸残基、第1-25位氨基酸残基、第14-52位氨基酸残基、第1-52位氨基酸残基、第14-74位氨基酸残基、第11-65位氨基酸残基、第11-74位氨基酸残基、或第1-74位氨基酸残基。
在另一些实施方案中，所述能够测定对BNLF2b基因编码的蛋白特异性的抗体的水平的试剂还包含检测试剂，所述检测试剂选自带有可检测的标记的二级抗体。
在某些实施方案中，在上述步骤(1b)中，使用所述检测试剂来检测所述免疫复合物的量。
在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
在某些实施方案中，所述二级抗体对待测抗体所来自的物种(例如人)的抗体是特异的。
在某些实施方案中，所述二级抗体是抗-免疫球蛋白抗体。
在某些示例性实施方案中，被检测的抗体是IgG抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgG抗体，例如抗人IgG抗体。
在某些示例性实施方案中，被检测的抗体是IgM抗体。在此类实施方案中，所述抗-免疫球蛋白抗体选自抗IgM抗体，例如抗人IgM抗体。
在某些示例性实施方案中，被检测的抗体是IgA抗体。在此类实施方案中，所述 抗-免疫球蛋白抗体选自抗IgA抗体，例如抗人IgA抗体。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“BNLF2b”是指，EB病毒(EBV)的BNLF2b基因，其是本领域技术人员公知的(参见，例如NCBI GENBANK数据库登录号：CAA24811.1)。由BNLF2b基因编码的全长蛋白包含98个氨基酸，其序列如SEQ ID NO:101所示。在本文中，由BNLF2b基因编码的全长蛋白也可简称为BNLF2b基因编码的野生型蛋白、或BNLF2b基因编码蛋白。
如本文中所使用的，当提及BNLF2b基因编码蛋白的氨基酸序列时，其使用SEQ ID NO：101所示的序列来进行描述。例如，表述“BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基”是指，SEQ ID NO：101所示的多肽的第53-60位氨基酸残基。然而，本领域技术人员理解，在BNLF2b基因编码蛋白的氨基酸序列中，可天然产生或人工引入突变或变异，而不影响其生物学功能。因此，在本发明中，术语“BNLF2b基因编码蛋白”及其类似表述应包括所有此类序列，包括例如SEQ ID NO：101所示的序列以及其天然或人工的变体。并且，当描述BNLF2b基因编码蛋白的序列片段时，其不仅包括SEQ ID NO：101的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“BNLF2b基因编码蛋白的第53-60位氨基酸残基”包括，SEQ ID NO：101的第53-60位氨基酸残基，以及其变体(天然或人工)中的相应片段。根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
如本文中所使用的，术语“特异性结合”是指，两分子(即结合分子与靶分子)之间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。两分子之间的结合亲和力可用K D值描述。K D值是指由kd(特定的结合分子-靶分子相互作用的解离速率；亦称为koff)与ka(特定结合分子-靶分子相互作用的缔合速率；亦称为kon)之比得到的解离常数，或者指表示为摩尔浓度(M)的kd/ka。K D值越小，两分子结合越紧密，亲和力越高。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异 性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。K D值可通过本领域熟知的方法确定，例如使用表面等离子体共振术(SPR)在BIACORE仪中测定。
如本文中所使用的，术语“免疫学测定”是指，利用抗原-抗体之间的特异性相互作用/结合亲和力来进行的测定，其一般可用于检测特定抗原或者抗体在样品中的存在或水平。此类免疫学检测是本领域技术人员公知的，包括但不限于，酶免疫测定法(EIA)、化学发光免疫分析法(CLIA)、放射免疫测定法(RIA)、荧光免疫测定法(FIA)、Western印迹法、免疫比浊法、表面等离子共振法等。在某些实施方式中，所述免疫学检测为酶免疫测定法(EIA)，例如ELISA检测法、Elispot检测法或CLEIA检测法。关于免疫学检测的详细描述，可参见例如，Fundamental Immunology,Ch.7 Paul,W.,ed.，第2版，Raven Press,N.Y.(1989)。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“可检测的标记”可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟知的，包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如， 3H、 125I、 35S、 14C或 32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物)、磁珠(例如，
Figure PCTCN2020139310-appb-000001
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。本发明中涵盖的标记物可通过本领域已知的方 法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，可通过不同长度的接头将如上所述的可检测的标记连接至检测抗体或抗原，以降低潜在的位阻。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动 物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基 进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“受试者”包括但不限于各种动物，特别是哺乳动物，例如人。
有益效果
本发明首次发现，针对EB病毒BNLF2b基因编码蛋白的抗体水平能够用于诊断鼻咽癌或评估鼻咽癌风险，且其诊断效果显著优于现有标志物，可显著提升鼻咽癌筛查的特异性和阳性预测值。本发明还首次提供了基于EB病毒BNLF2b基因编码蛋白或多肽片段的鼻咽癌血清学筛查试剂盒。
与现有技术相比，本发明的技术方案能够达到与已知的EB病毒抗体联合检测方法相当的检测灵敏度，甚至更优的检测特异性，且可以实现快速、高通量检测，具备重大临床应用价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技 术人员来说将变得显然。
附图说明
图1显示了实施例2和3中1份鼻咽癌混合血清(NPC)和3份健康对照血清(H1-H3)中IgA和IgG抗体与87条EBV多肽(编号1-87)的反应性。左图为IgA抗体，右图为IgG抗体，横轴代表不同的血清样本，纵轴代表不同的EBV多肽，颜色越深，代表反应性越高。
图2显示了实施例2和3中鼻咽癌患者和健康对照血清中抗体与不同EBV多肽的反应性。其中，横坐标代表EBV多肽，纵坐标为反应的OD值(对数刻度)。
图3显示了实施例4中鼻咽癌患者和健康对照血清中IgG抗体与BNLF2b编码蛋白aa14-52的反应性。其中，图3A为不同样本的检测OD值(对数刻度)；图3B为ROC曲线分析的结果，横坐标代表100-特异性％，纵坐标代表灵敏度％。
图4显示了实施例4中鼻咽癌患者和健康对照者血清中IgA、IgM和IgG抗体与BNLF2b编码蛋白aa1-74的反应性。其中，图A为检测OD值，横坐标代表血清分类，纵坐标代表OD值(对数刻度)；图B为ROC曲线分析的结果，横坐标代表100-特异性％，纵坐标代表灵敏度％；图C为健康对照血清中IgA和IgM抗体与BNLF2b编码蛋白aa1-74的反应性，横坐标代表抗体类型，纵坐标代表OD值(对数刻度)。
图5显示了实施例5中BNLF2b抗体双抗原夹心法检测50份鼻咽癌血清和500份健康对照血清的结果。其中，图5A为检测的OD值，横坐标为血清分类，纵坐标为OD值(对数刻度)；图5B为ROC曲线分析结果，横坐标为100-特异性％，纵坐标为灵敏度％。
图6显示了实施例6中74份鼻咽癌血清和250份健康对照血清与EBNA1/IgA、VCA/IgA的反应性以及二者联合检测计算出的发生鼻咽癌的风险率。其中，横坐标代表血清分类，图6A和图6B中纵坐标为检测OD值(对数刻度)，图6C纵坐标为发生鼻咽癌的风险率。
图7显示了实施例6中BNLF2b抗体双抗原夹心法检测鼻咽癌患者血清和健康对照血清的结果。其中，横坐标代表血清分类，纵坐标代表反应的OD值。
图8显示了实施例6中BNLF2b抗体双抗原夹心法和EBNA1/IgA+VCA/IgA联合检测的ROC曲线图。其中，横坐标为100-特异性％，纵坐标为灵敏度％。
图9显示了实施例10中鼻咽癌血清中IgG抗体与BNLF2b编码蛋白不同肽段的反 应性。其中，横坐标代表不同的BNLF2b多肽，纵坐标代表鼻咽癌混合血清编号，颜色越深，代表反应性越高。
图10显示了实施例10中两份鼻咽癌混合血清中IgG抗体与BNLF2b编码蛋白不同肽段的反应性。其中，A-D分别代表鼻咽癌血清与aa1-25区段内截短肽，aa31-45区段内截短肽，aa51-65区段内截短肽和aa81-98区段内截短肽的反应性，横坐标代表多肽位置(例如1-25表示第1-25位氨基酸)，纵坐标代表检测OD值与未截短的肽段的检测OD值的比值。
图11显示了实施例10中鼻咽癌血清与aa1-25区段内不同截短多肽的反应性。其中，图11A显示不同鼻咽癌患者血清与aa1-25，aa1-15和aa11-25的反应性，图11B显示这些血清与aa1-25及其截短多肽的反应性。横坐标代表合成肽的氨基酸位置，纵坐标代表鼻咽癌血清编号，颜色越深，代表反应性越高。
图12显示了实施例10中鼻咽癌患者中IgG抗体与BNLF2b编码蛋白突变体的反应性。其中，A-D分别代表鼻咽癌血清与aa1-25区段内突变体，aa31-45区段内突变体，aa51-65区段内突变体和aa81-98区段内突变体的反应性，横坐标代表突变位置，纵坐标代表检测OD值与相应未突变的肽段的检测OD值的比值。
图13显示了实施例11中鼻咽癌患者和健康对照血清中IgG抗体与不同的BNLF2b编码多肽的反应性。其中，横坐标代表多肽片段，纵坐标代表检测OD值。
序列信息
表1：本申请涉及的序列的信息描述于下面的表中。
SEQ ID NO: 描述 SEQ ID NO: 描述
1 EBV ORF 1，aa467-486 53 EBV ORF 57,aa326-345
2 EBV ORF 2，aa90-109 54 EBV ORF 59,aa55-74
3 EBV ORF 3，aa138-157 55 EBV ORF 59,aa231-250
4 EBV ORF 15，aa12-31 56 EBV ORF 60,aa195-214
5 EBV ORF 16，aa103-122 57 EBV ORF 60,aa82-101
6 EBV ORF 16，aa340-359 58 EBV ORF 61,aa18-37
7 EBV ORF 17，aa26-45 59 EBV ORF 62,aa41-60
8 EBV ORF 18，aa110-129 60 EBV ORF 63,aa280-299
9 EBV ORF 19，aa394-413 61 EBV ORF 64,aa88-107
10 EBV ORF 19，aa372-391 62 EBV ORF 64,aa154-173
11 EBV ORF 20，aa63-82 63 EBV ORF 65,aa118-137
12 EBV ORF 20，aa101-120 64 EBV ORF 66,aa88-107
13 EBV ORF 21，aa670-689 65 EBV ORF 66,aa359-378
14 EBV ORF 22，aa330-349 66 EBV ORF 67,aa244-263
15 EBV ORF 23，aa205-224 67 EBV ORF 68,aa120-139
16 EBV ORF 26，aa110-129 68 EBV ORF 69,aa17-36
17 EBV ORF 27，aa256-275 69 EBV ORF 70,aa171-190
18 EBV ORF 28，aa9-28 70 EBV ORF 71,aa201-220
19 EBV ORF 29，aa1-20 71 EBV ORF 72,aa143-162
20 EBV ORF 30，aa789-801 72 EBV ORF 73,aa195-214
21 EBV ORF 31，aa185-204 73 EBV ORF 73,aa154-173
22 EBV ORF 32，aa246-265 74 EBV ORF 75,aa514-533
23 EBV ORF 33，aa26-45 75 EBV ORF 75,aa432-451
24 EBV ORF 34，aa134-153 76 EBV ORF 76,aa149-168
25 EBV ORF 34,aa61-80 77 EBV ORF 77,aa180-199
26 EBV ORF 35，aa665-684 78 EBV ORF 78,aa19-38
27 EBV ORF 35，aa822-841 79 EBV ORF 79,aa323-342
28 EBV ORF 36，aa60-79 80 EBV ORF 79,aa579-598
29 EBV ORF 37，aa92-111 81 EBV ORF 80,aa32-51
30 EBV ORF 38，aa1-20 82 EBV ORF 81,aa11-30
31 EBV ORF 39，aa105-124 83 EBV ORF 82,aa155-174
32 EBV ORF 39，aa593-612 84 EBV ORF 83,aa66-85
33 EBV ORF 40，aa568-587 85 EBV ORF 84,aa188-207
34 EBV ORF 41，aa63-82 86 EBV ORF 85，aa31-50
35 EBV ORF 41，aa177-196 87 EBV ORF 86，aa19-38
36 EBV ORF 42，aa179-198 88 EBV ORF 85，aa14-52
37 EBV ORF 42,aa1-20 89 EBV ORF 85，aa1-74
38 EBV ORF 43,aa444-463 90 EBV ORF 85，aa14-74
39 EBV ORF 44,aa126-145 91 EBV ORF 85，aa1-52
40 EBV ORF 44,aa2-21 92 EBV ORF 85，aa1-15
41 EBV ORF 45,aa495-514 93 EBV ORF 85，aa11-25
42 EBV ORF 45,aa10-29 94 EBV ORF 85，aa21-35
43 EBV ORF 47,aa60-79 95 EBV ORF 85，aa31-45
44 EBV ORF 48,aa212-231 96 EBV ORF 85，aa41-55
45 EBV ORF 50,aa45-64 97 EBV ORF 85，aa51-65
46 EBV ORF 52,aa211-230 98 EBV ORF 85，aa61-75
47 EBV ORF 53,aa166-185 99 EBV ORF 85，aa71-85
48 EBV ORF 53,aa1-20 100 EBV ORF 85，aa81-98
49 EBV ORF 54,aa209-228 101 BNLF2b基因编码的全长蛋白
50 EBV ORF 54,aa104-123 102 EBV ORF 85，aa1-25
51 EBV ORF 55,aa357-376 103 EBV ORF 85，aa11-65
52 EBV ORF 56,aa50-69 104 EBV ORF 85，aa11-74
实施例1：EB病毒基因编码多肽的合成
根据GenBank中EBV B95-8毒株的86个开放阅读框(ORF)编码蛋白的氨基酸序列信息(GenBank ID：V01555.2)，通过生物信息学工具在线预测每个蛋白的B细 胞表位，根据预测结果，每个蛋白选取1-2条可能的B细胞表位多肽委托厦门精聚生物科技有限公司进行合成。在合成时，在多肽N末端偶联生物素，以便于后续实验。最终成功合成87条EB病毒基因编码多肽(SEQ ID NO:1-87)，这些多肽来自68个ORF，其具体信息如表1所示。
实施例2：EB病毒多肽与血清IgA抗体的反应性评价
将实施例1中获得的生物素标记的多肽(编号1-87)分别稀释至500ng/ml，按每孔100μL的标准加入链亲和素包被的96孔微孔板中，37摄氏度反应2小时。反应结束后，用PBST洗涤2次，每孔加入200μL封闭液，37摄氏度封闭2小时。封闭结束后，倒掉封闭液，每孔分别加入100μL 1:20稀释的鼻咽癌患者混合血清或阴性对照血清，37摄氏度反应30分钟。反应结束后，用PBST洗涤5次，并加入1:20000稀释的HRP标记的羊抗人IgA(KPL，Gaithersburg,MD)，37摄氏度继续反应30分钟。PBST洗涤5次后，每孔加入100μL TMB显色液，37摄氏度温育15分钟后，每孔加入50μL终止液，混匀后通过酶标仪测定450和630nm处的吸光度。
结果如图1所示，在87条多肽中，有8条多肽对鼻咽癌混合血清反应的OD值在0.3以上(表2-1)。在这8条多肽中，有BSRL1、BLLF1b、BGLF3、BDLF2和BVRF2基因编码的5条多肽在之前的研究中未被报导用于鼻咽癌的辅助诊断。
我们进一步通过36份阴性血清评价了这5条多肽的特异性，结果如图2及表2-2所示，BSRF1、BGLF3和BVRF2基因编码的3条多肽的特异性相对较好。同时，我们通过12份鼻咽癌血清对上述3条多肽的检测灵敏度进行评价，结果如图2所示，这3条多肽的反应性均较低，其中，BSRF1和BVRF2编码多肽与6份血清的反应性高于0.1，而BGLF3编码多肽仅与3份血清的反应性高于0.1。
表2-1：EB病毒多肽与鼻咽癌患者血清IgA抗体的反应性
Figure PCTCN2020139310-appb-000002
表2-2：EB病毒多肽与阴性对照血清IgA抗体的反应性
Figure PCTCN2020139310-appb-000003
实施例3：EB病毒多肽与血清IgG抗体的反应性评价
按照实施例2中的方法检测实施例1中合成的87种多肽(1-87)与血清IgG的反应性，其中用1:5000稀释的HRP标记的鼠抗人IgG(万域美澜，北京)代替羊抗人IgA。结果如图1所示，在这些多肽中，有BZLF1，BRLF1等5条基因编码多肽与鼻咽癌混合血清的反应性高于0.1(表3-1)，其中有BNLF1(ZTA)，BRLF1(RTA)和BILF2(gp78)已有报导。
我们进一步通过36份阴性血清评价了BVRF2和BNLF2b编码多肽的特异性，结果如图2及表3-2所示，BNLF2b基因编码多肽与36份阴性血清反应的OD值均低于0.034，具有良好的特异性。同时，我们通过12份鼻咽癌血清对BNLF2b基因编码多肽的检测灵敏度进行评价，结果如图2所示，12份鼻咽癌患者血清中，有6份的反应OD值高于0.1。
表3-1：EB病毒多肽与阳性血清IgG抗体的反应性
Figure PCTCN2020139310-appb-000004
表3-2：EB病毒多肽与阴性血清IgG抗体的反应性
Figure PCTCN2020139310-appb-000005
Figure PCTCN2020139310-appb-000006
实施例4：抗BNLF2b抗体间接法的建立及性能初步评价
我们进一步通过DNASTAR软件包中Protean软件对BNLF2b编码蛋白(SEQ ID NO:101)的亲疏水性以及抗原性进行分析，合成了两段多肽aa14-52(SEQ ID NO:88)和aa1-74(SEQ ID NO:89)，将其分别作为包被抗原用于间接法检测。
4.1基于多肽aa14-52的间接法检测
将多肽aa14-52用碳酸盐缓冲液(pH9.6)稀释至125ng/ml，每孔包被100μL，按照实施例3中的方法对86份鼻咽癌患者和195份健康人血清进行检测。结果如图3A所示，当以aa14-52作为包被抗原时，86份鼻咽癌患者血清中有57份的OD值高于0.1，195份健康人血清的OD值均低于0.025。同时，使用相同的方法对122位EBNA1/IgA+VCA/IgA联合筛查(该联合检测方法可参见实施例6)为高危但最终确诊为非鼻咽癌者的受试者血清进行了检测，结果如图3A所示，仅3例OD值超过0.1。对上述基于多肽aa14-52的间接法检测数据通过MedCalc 16.2.1软件(MedCalc Software，Ostend,Belgium)进行ROC曲线分析，结果如图3B所示，曲线下面积AUC为0.942，当cut-off值为0.025时，约登指数为0.80，特异性为100％，灵敏度为80.23％(69/86)。
4.2基于多肽aa1-74的间接法检测
以多肽aa1-74作为包被抗原，按照与4.1相同的方法对63份健康人血清和221份鼻咽癌血清进行检测。结果如图4A所示，63份鼻咽癌患者血清中55份检测OD值超过0.1，而221份健康对照者血清中仅10份检测OD超过0.1。进一步对对上述基于多肽aa1-74的间接法进行ROC曲线分析，结果如图4B所示，该方法能够高效的区分鼻咽癌患者和健康人，AUC为0.950。当cut-off值设为0.1时，灵敏度为87.30％(55/63)，特异性为95.48％(211/221)。
此外，同样以多肽aa1-74为包被抗原，通过间接法检测221份健康对照血清中IgA和IgM抗体的水平，其中，IgA抗体检测使用1:20000稀释的HRP标记的羊抗人IgA(KPL，Gaithersburg,MD)，IgM抗体检测使用1:50000稀释的HRP标记的羊抗人IgM(万域美澜，北京)。结果如图4C所示，抗BNLF2b IgA抗体和抗BNLF2b IgM抗体检测均具有较好的特异性，分别仅有4份和7份血清的检测值超过0.1，特异性分别为98.19％(217/221)和96.83％(214/221)。
表4显示了上述各方法区分鼻咽癌和非鼻咽癌的性能，结果表明，无论是以多肽aa14-52为包被抗原的IgG抗体间接法，还是以多肽aa1-74为包被抗原的IgG、IgA和IgM抗体间接法均能够高效区分鼻咽癌与健康对照，具有良好的检测灵敏度和特异性。
表4：抗BNLF2b抗体间接法区分鼻咽癌和非鼻咽癌的性能
Figure PCTCN2020139310-appb-000007
实施例5：抗BNLF2b抗体双抗原夹心法的建立
由于实施例4的结果显示IgA、IgM和IgG三种抗体在鼻咽癌风险预测中均具有较好的特异性，因此，可通过检测针对BNLF2b的总抗体进行鼻咽癌风险预测。基于此，本实施例建立了检测抗BNLF2b抗体的双抗原夹心法，以评价其筛查鼻咽癌的性能。
用碳酸盐缓冲液(pH 9.6)将BNLF2b编码多肽aa1-74稀释成100ng/mL，按每孔100μL的标准96孔微孔板中，37摄氏度反应2小时。反应结束后，用PBST洗涤2次，每孔加入200μL封闭液，37摄氏度封闭2小时。封闭结束后，倒掉封闭液，每孔分别加入50μL含有67ng/ml生物素化的多肽aa1-74的稀释液和50μL鼻咽癌患者血清或阴性对照血清，37摄氏度反应60分钟。反应结束后，用PBST洗涤5次，并加入1：5000稀释的HRP标记的链霉亲和素和1:15000稀释的HRP标记的aa1-74，37摄氏度继续反应30分钟。PBST洗涤5次后，每孔加入100μL TMB显色液，37摄氏度温育15分钟后，每孔加入50μL终止液，混匀后通过酶标仪测定450nm和630nm处的吸光度。用该方法对50份鼻咽癌患者血清和500份健康人血清进行检测，结果如图5A所示，在50份鼻咽癌患者血清中，有46份的反应OD值高于0.1，而与500份阴性血清中，仅1份OD值超过0.1。进一步对上述结果进行ROC曲线分析，结果如图5B所示，曲线下面积为0.977，当阈值为0.1时，该试剂的灵敏度和特异性分别为92.0％和99.8％，约登指数为0.91。
此外，为了比较BNLF2b不同肽段在鼻咽癌诊断中的性能，除aa1-74外，我们还合成了aa1-52(SEQ ID NO:91)和aa14-74(SEQ ID NO:90)两个肽段，并且均在C端标记了生物素。取另外175份鼻咽癌患者血清，按照相同的方法进行检测，结果如下表所示，以0.1作为临界值时，三段多肽的灵敏度均在85％以上，表明以aa1-52、aa14-74或aa1-74为包被抗原时，该方法均具备较高的检测灵敏度和特异性。
表5：不同BNLF2b多肽用于标记抗原时检测灵敏度的比较
Figure PCTCN2020139310-appb-000008
实施例6：抗BNLF2b抗体双抗原夹心法与现有鼻咽癌筛查试剂的比较
选取74份鼻咽癌患者血清和250份健康体检人群血清，用中山生物EBNA1/IgA检测试剂盒、欧盟VCA/IgA检测试剂盒(货号：EI 2791-9601A)以及实施例5的双抗原夹心法(aa1-74)进行平行检测。EBNA1/IgA的检测结果如图6A所示，VCA/IgA的检测结果如图6B所示。EBNA1/IgA与VCA/IgA通常需要联合作为筛查指标，当两者联合检测时，鼻咽癌发生的风险率计算公式为：LogitP＝-3.934+2.203×VCA/IgA+4.797×EBNA1/IgA(Liu,Z.,et al.2013，Am J Epidemiol)，阈值为0.98。EBNA1/IgA+VCA/IgA联合检测的结果如图6C所示，在74例鼻咽癌患者和250份健康体检人群中分别有69份和8份风险率超过0.98，灵敏度和特异性分别为93.64％和96.80％。
抗BNLF2b抗体检测结果如图7所示，在鼻咽癌患者和健康体检人群血清中分别有70份和1份检测OD值超过0.1，以0.1做为cut-off值，抗BNLF2b抗体检测的灵敏度和特异性分别为94.59％和99.60％。进一步通过SPSS软件进行卡方检验发现，抗BNLF2b抗体检测与EBNA1/IgA+VCA/IgA联合检测的灵敏度无显著差异(P＝1.00)，但抗BNLF2b抗体检测的特异性显著高于EBNA1/IgA+VCA/IgA联合检测(P＝0.037)。
对上述抗BNLF2b抗体检测和EBNA1/IgA+VCA/IgA联合检测的结果分别进行ROC曲线分析，结果如图8所示，抗BNLF2b抗体检测和EBNA1/IgA+VCA/IgA联合检测的AUC分别为0.97和0.99，整体上无显著差异(P＝0.316)，但是在灵敏度在94.59％或以下时，抗BNLF2b抗体检测的特异性均高于联合检测。此外，74例鼻咽癌患者中有28份为I/II期，在这28例患者中，BNLF2b和EBNA1/IgA+VCA/IgA联合 检测均有一份漏检，灵敏度为96.43％。
将实施例5与实施例6中的数据合并计算，抗BNLF2b抗体检测的灵敏度为93.55％(116/124)，特异性为99.73％(748/750)。按照文献(Liu,Z.,et al.2013，Am J Epidemiol)中的发病率计算公式，抗BNLF2b抗体检测的阳性预测值为33.2％，相比EBNA1/IgA+VCA/IgA联合检测的阳性预测值(4.4％，38/862)显著提高(P<0.0001)。以上结果说明，抗BNLF2b抗体检测相比现有EBNA1/IgA+VCA/IgA联合筛查方法可显著提升鼻咽癌筛查的特异性和阳性预测值。
实施例7：抗BNLF2b抗体双抗原夹心法在鼻咽癌筛查中的应用
在鼻咽癌高发区广东省中山市阜沙和南朗两个镇开展人群筛查，其中阜沙入组496人，南朗入组829人，合计1325人。根据实施例6中的方法(以下简称为抗BNLF2b抗体检测)，对这些标本同时检测EBNA1/IgA、VCA/IgA和抗BNLF2b抗体，根据EBNA1/IgA和VCA/IgA联合检测公式(LogitP＝-3.934+2.203×VCA/IgA+4.797×EBNA1/IgA)计算发生鼻咽癌的风险系数。结果如下表所示，在1325人中，EBNA1/IgA、VCA/IgA和抗BNLF2b抗体阳性分别为163人，218人和32人。在风险系数超过0.98的126人中，最终5人确诊为鼻咽癌，而风险系数不超过0.98的人中，在肿瘤登记系统中未发现有提前确诊的鼻咽癌。在该5份确诊为鼻咽癌的标本中，VCA/IgA仅能检出1份，其余方法能够100％检出。而在1320例非鼻咽癌标本中，抗BNLF2b抗体检测的特异性和阳性预测值分别为97.95％和15.63％，均高于EBNA1/IgA、VCA/IgA以及二者联合检测。
表6：抗BNLF2b抗体与现有标志物在人群筛查中的性能比较
Figure PCTCN2020139310-appb-000009
实施例8：抗BNLF2b抗体双抗原夹心法与现有鼻咽癌筛查试剂联合使用
将实施例6和实施例7中的数据进行合并，共79例鼻咽癌和1570例非鼻咽癌。79 例鼻咽癌标本的检测结果如表7-1所示，抗BNLF2b抗体检测与EBNA1/IgA、VCA/IgA和风险系数同时为阳性的样本分别有70、67和72例。1570例非鼻咽癌标本的检测结果如表7-2所示，抗BNLF2b抗体检测与EBNA/IgA、VCA/IgA和风险系数同时为阳性的标本分别仅为5、5和4例。抗BNLF2b抗体检测与EBNA/IgA、VCA/IgA、EBNA/IgA+VCA/IgA的进一步联合检测的结果如表7-3所示，与EBNA/IgA联合检测后，阳性预测值从15.15％提高至50％，与风险系数联合检测后，阳性预测值从15.15％提高55.56％。
表7-1：对79例鼻咽癌标本的检测结果
Figure PCTCN2020139310-appb-000010
表7-2：1570例非鼻咽癌标本的检测结果
Figure PCTCN2020139310-appb-000011
表7-3：抗BNLF2b抗体检测以及与现有标志物联合检测用于鼻咽癌筛查的性能
Figure PCTCN2020139310-appb-000012
我们进一步选取中山市人民医院在前期筛查队列中经EBNA1/IgA+VCA/IgA联合检测确定为高危的标本227份(样本2)，其中8份经鼻咽镜检查确诊为鼻咽癌。结果如表8所示，抗BNLF2b抗体阳性有24份，其中7份为鼻咽癌，联合检测的灵敏度和阳性预测值分别为87.50％和29.17％。
将样本2与样本1合并，其中样本1包括实施例6中250人健康对照中风险系数>0.98的8例、以及实施例7中1325人的筛查队列中风险系数>0.98的126例(5例为鼻咽癌)。将两个样本合并后，合计13份鼻咽癌，348份非鼻咽癌， EBNA1/IgA+VCA/IgA联合检测的阳性预测值为3.60％。在此基础上，经抗BNLF2b抗体检测后，13例鼻咽癌中12例为阳性，而348例非鼻咽癌中仅21例为阳性，联合使用的阳性预测值提高到36.36％(表8)。因此，抗BNLF2b抗体检测与EBNA1/IgA+VCA/IgA的联合使用能够进一步提高鼻咽癌筛查的特异性和阳性预测值。为了降低工作量，可以先检测抗BNLF2b抗体，再进一步检测EBNA1/IgA和VCA/IgA。
表8：抗BNLF2b抗体检测在高风险系数人群中的性能
Figure PCTCN2020139310-appb-000013
实施例9：抗BNLF2b抗体双抗原夹心法在鼻咽癌辅助诊断中的应用
鼻咽癌的临床症状不够特异，难以与其它头颈部疾病区分，临床上，对于疑似病例主要依靠鼻咽镜以及病理检查确诊。我们收集了63份疑似鼻咽癌病例，其中31份最终确诊为鼻咽癌。我们按照实施例6中的方法通过双抗原夹心法对这63份标本进行检测，结果显示，以0.1为cut-off值，在31份鼻咽癌病例中，有30份BNLF2b抗体检测结果为阳性，而在32例非鼻咽癌病例中，仅2例为BNLF2b抗体阳性。BNLF2b抗体检测的灵敏度为96.78％(30/31)，特异性为93.75％(30/32)，阳性预测值为93.75％(30/32)。以上结果说明，在门诊病例中，通过BNLF2b抗体检测可以显著降低不必要的鼻咽镜检查的数量，减轻患者的经济负担和身体负担。
实施例10：BNLF2b编码蛋白的免疫优势表位的研究
BNLF2b基因共编码98个氨基酸(SEQ ID NO:101)，为了分析BNLF2b编码蛋白的免疫优势表位，我们通过步移法设计了9条多肽。每条多肽15个氨基酸(最后一条多肽为18个氨基酸)，相邻两条多肽间重叠5个氨基酸，并在C端标记生物素以便于后续检测(SEQ ID NOs:92-100)。按照实施例3中的方法以上述多肽片段为包被抗原，对1:300稀释的鼻咽癌患者血清中IgG进行检测。结果如图9所示，BNLF2b编码蛋白表位主要位于aa1-25，aa31-45，aa51-65以及aa81-98四个区段。
我们进一步按照实施例3中的方法，通过43份鼻咽癌血清初步评价了aa1-25 (SEQ ID NO:102)、aa31-45(SEQ ID NO:95)、aa51-65(SEQ ID NO:97)、aa81-98(SEQ ID NO:100)四个区段多肽在鼻咽癌筛查中的灵敏度，结果发现，以0.1做为临界值，aa51-65的检测灵敏度为76.7％(33/43)，aa1-25为72.1％(31/43)，另外两条多肽的灵敏度均为55.8％(24/43)。aa14-52和aa1-74的灵敏度为90.7％(39/43)和93.0％(40/43)，且aa1-74漏检的3份血清4段多肽均为阴性，而aa14-52阴性，aa1-74阳性的血清与aa51-65反应的OD值为0.393。
为了进一步确定构成这些表位的关键氨基酸，我们在aa1-25，aa31-45，aa51-65和aa81-98的基础上进一步进行N端和C端截短，合成了一系列多肽。本次实验将40份鼻咽癌血清混合形成两份混合血清，1:300稀释后按照实施例3中的方法进行IgG检测。结果如图10所示。
(1)aa1-25区段(图10A)：固定C端，当N端截短至16位时仍能够与鼻咽癌血清中IgG结合，而截短至19位时则完全丧失与鼻咽癌血清IgG的反应性；类似的，固定N端，当C端截短至第7位时，多肽仍能与鼻咽癌血清IgG反应，而当C端截短至第4位时，则完全丧失与鼻咽癌血清中IgG的反应性。因此，aa1-15和aa11-25含有两个独立的表位，关键氨基酸分别为aa5-7和aa16-18。
(2)aa31-45区段(图10B)：无论是N端截短还是C端截短，不含37-39位氨基酸EDR时，多肽将完全丧失与鼻咽癌血清中IgG的反应性，因此，KER(aa31-33)和EDR(aa37-39)为构成该表位的关键氨基酸。
(3)aa51-65区段(图10C)：N端不含aa54-56或者C端不含aa60时，多肽完全丧失与鼻咽癌血清的反应性完全丧失。
(4)aa81-98区段(图10D)：N端不含aa87-89或者C端不含aa96-98时，多肽完全丧失与鼻咽癌血清的反应性完全丧失。
为了分析aa1-25区段是否含有跨aa1-15和aa11-25两段的表位，我们分别以aa1-15、aa11-25和aa1-25为包被抗原，检测了43份鼻咽癌血清IgG。结果如图11A所示，14份血清对aa1-25反应的OD值高于aa1-15和aa11-25之和。进一步以aa1-25区段内不同截短的多肽为包被抗原对这14份血清进行检测，结果如图11B所示，能够与阳性血清反应的多肽至少包含了aa10-16。
除此之外，对于上述四个关键区段，我们还合成了一系列变体，包括自然变体和人工变体。按照实施例3中的方法，以这些变体为包被抗原检测其与1:300稀释的鼻咽癌混合血清IgG的反应性。结果如图12所示。
(1)aa1-25(图12A)：第6位、第9-11位以及第16位氨基酸突变后降低最为明显；第12位存在3个自然突变，分别为T、D和S，将丙氨酸突变为这三个氨基酸并不会显著降低aa1-25与鼻咽癌血清的反应性；此外，第5、7、8、13、14、15、19、22、24、25位氨基酸突变的影响相对较小。
(2)aa31-45(图12B)：K31、R33以及D38和R39突变为丙氨酸后，多肽与鼻咽癌血清的反应性出现明显下降，说明这些氨基酸是构成该表位的关键氨基酸；此外，第32、34、35、36、37、40、41、42位氨基酸突变的影响相对较小。
(3)aa51-65(图12C)：R53和N54以及aa56-59突变为丙氨酸时，多肽与鼻咽癌血清的反应性出现显著降低，而52、55、60和61位氨基酸突变的影响相对较小。
(4)aa81-98(图12D)：aa95-97三个氨基酸突变为丙氨酸时，多肽与鼻咽癌血清的反应性下降最为明显；此外，第89、91、93、98位氨基酸突变的影响相对较小。
以上结果显示，BNLF2b编码蛋白的aa5-11、aa16-23、aa31-33、aa37-39、aa53-60和aa89-98为核心区段。
实施例11：BNLF2b编码蛋白不同表位联合使用对鼻咽癌筛查性能的影响在之前已经合成的aa14-52、aa1-74、aa1-52、aa1-25的基础上，进一步合成了aa11-65(SEQ ID NO:103)和aa11-74(SEQ ID NO:104)两条多肽，并在C端标记生物素。按照实施例3中的方法，分别检测这些多肽与84份鼻咽癌患者血清和168份健康人血清进行反应。6段多肽与鼻咽癌血清的反应性均显著高于健康对照人群(图13)。通过Medcal软件进行ROC曲线分析的结果显示，6段多肽的曲线下面积均在0.95以上，灵敏度和特异性分别均在89％和96％以上(表9)。以上结果表明，包含一个或多个核心区段(aa5-11、aa16-23、aa31-33、aa37-39、aa53-60)的BNLF2b编码多肽片段均具备优良的筛查鼻咽癌的性能。
表9：不同多肽作为捕获抗原在鼻咽癌筛查中的性能比较
Figure PCTCN2020139310-appb-000014
Figure PCTCN2020139310-appb-000015
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。
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Claims (39)

  1. 一种分离的多肽或其变体，其中，所述多肽由BNLF2b基因编码的野生型蛋白的至少7个连续氨基酸残基组成，且包含至少1个(例如至少2个、至少3个、或全部4个)选自下列的序列：BNLF2b基因编码的野生型蛋白的第5-11位氨基酸残基、第16-23位氨基酸残基、第31-39位氨基酸残基、或第53-60位氨基酸残基；
    其中，所述变体与其所源自的多肽相异仅在于1个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸残基的置换(例如，保守置换或非保守置换)，且保留了其所源自的多肽的生物学功能(例如被抗EBV抗体识别并结合的活性)；
    优选地，所述BNLF2b基因编码的野生型蛋白具有如SEQ ID NO:101所示的序列。
  2. 权利要求1所述的分离的多肽或其变体，其中，所述分离的多肽包含：BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基、第5-23位氨基酸残基、第16-39位氨基酸残基、第31-60位氨基酸残基、第5-39位氨基酸残基、第16-60位氨基酸残基、或第5-60位氨基酸残基。
  3. 权利要求1或2所述的分离的多肽或其变体，其中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少8个连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第53-60位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第53-61位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第51-65位氨基酸残基；
    优选地，所述第51-65位氨基酸残基具有SEQ ID NO:97所示的序列；
    优选地，所述多肽由BNLF2b基因编码的野生型蛋白的至少15个连续氨基酸残基组成。
  4. 权利要求1或2所述的分离的多肽或其变体，其中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少19个连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第5-23位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第1-25位氨基酸残基；
    优选地，所述第1-25位氨基酸残基具有SEQ ID NO:102所示的序列；
    优选地，所述多肽由BNLF2b基因编码的野生型蛋白的至少25个连续氨基酸残基组成。
  5. 权利要求1或2所述的分离的多肽或其变体，其中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少24个连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第16-39位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第14-52位氨基酸残基；
    优选地，所述第14-52位氨基酸残基具有SEQ ID NO:88所示的序列；
    优选地，所述多肽由BNLF2b基因编码的野生型蛋白的至少39个连续氨基酸残基组成。
  6. 权利要求1或2所述的分离的多肽或其变体，其中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少30个连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第31-60位氨基酸残基；
    优选地，所述分离的多肽包含BNLF2b基因编码的野生型蛋白的第31-61位氨基酸残基。
  7. 权利要求1或2所述的分离的多肽或其变体，其中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少35个连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第5-39位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第1-52位氨基酸残基；
    优选地，所述第1-52位氨基酸残基具有SEQ ID NO:91所示的序列；
    优选地，所述多肽由BNLF2b基因编码的野生型蛋白的至少53个连续氨基酸残基组成。
  8. 权利要求1或2所述的分离的多肽或其变体，其中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少45个连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第16-60位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第16-61位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第14-74位氨基酸残基、第 11-65位氨基酸残基、或第11-74位氨基酸残基；
    优选地，所述第14-74位氨基酸残基具有SEQ ID NO:90所示的序列；
    优选地，所述第11-65位氨基酸残基具有SEQ ID NO:103所示的序列；
    优选地，所述第11-74位氨基酸残基具有SEQ ID NO:104所示的序列；
    优选地，所述多肽由BNLF2b基因编码的野生型蛋白的至少61个、至少55个或至少64个连续氨基酸残基组成。
  9. 权利要求1或2所述的分离的多肽或其变体，其中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的至少56个连续氨基酸残基组成，且包含BNLF2b基因编码的野生型蛋白的第5-60位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第5-61位氨基酸残基；
    优选地，所述多肽包含BNLF2b基因编码的野生型蛋白的第1-74位氨基酸残基；
    优选地，所述第1-74位氨基酸残基具有SEQ ID NO:89所示的序列；
    优选地，所述多肽由BNLF2b基因编码的野生型蛋白的至少74个连续氨基酸残基组成。
  10. 权利要求1-9任一项所述的分离的多肽或其变体，其中，所述分离的多肽由选自下列的序列组成：BNLF2b基因编码的野生型蛋白的第51-56位氨基酸残基、第1-25位氨基酸残基、第14-52位氨基酸残基、第1-52位氨基酸残基、第14-74位氨基酸残基、第11-65位氨基酸残基、第11-74位氨基酸残基、或第1-74位氨基酸残基。
  11. 权利要求1-10任一项所述的分离的多肽或其变体，其中，所述变体不包括在相应于如下位置的氨基酸位置中的氨基酸置换，所述如下位置参考由BNLF2b基因编码的野生型蛋白所示的氨基酸位置：6、9、10、11、16、31、33、38、39、53、54、56、57、58、59、95、96、97。
  12. 权利要求1-11任一项所述的分离的多肽或其变体，其中，所述变体在相应于如下位置的氨基酸位置中的一处或多处(例如1处，2处，3处或4处)包含氨基酸置换，所述如下位置参考由BNLF2b基因编码的野生型蛋白所示氨基酸位置：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或98；
    优选地，所述变体在相应于如下位置的氨基酸位置中的一处或多处(例如1处，2处，3处或4处)包含氨基酸置换，所述如下位置参考由BNLF2b基因编码的野生型蛋白所示氨基酸位置：5、7、8、12、13、14、22、25、32、34、35、36、40、41、42、52、55、60、61、89、91、93或98。
  13. 权利要求1-11任一项所述的分离的多肽或其变体，其中，所述变体包含选自下列的1个或几个(例如，1个、2个、3个或4个)氨基酸置换：在相应于位置5的位置上的氨基酸置换为A，在相应于位置7的位置上的氨基酸置换为A，在相应于位置8的位置上的氨基酸置换为G，在相应于位置12的位置上的氨基酸置换为G、T、D或S，在相应于位置13的位置上的氨基酸置换为A，在相应于位置14的位置上的氨基酸置换为G，在相应于位置15的位置上的氨基酸置换为A，在相应于位置22的位置上的氨基酸置换为A，在相应于位置24的位置上的氨基酸置换为A，在相应于位置25的位置上的氨基酸置换为A，在相应于位置32的位置上的氨基酸置换为A，在相应于位置34的位置上的氨基酸置换为A，在相应于位置35的位置上的氨基酸置换为A，在相应于位置36的位置上的氨基酸置换为A，在相应于位置37的位置上的氨基酸置换为A、N、Q、S或R，在相应于位置40的位置上的氨基酸置换为A，在相应于位置41的位置上的氨基酸置换为A，在相应于位置42的位置上的氨基酸置换为A，在相应于位置52的位置上的氨基酸置换为K、H、A、S或D，在相应于位置55的位置上的氨基酸置换为S，在相应于位置60的位置上的氨基酸置换为A，在相应于位置61的位置上的氨基酸置换为K、H、S或A，在相应于位置89的位置上的氨基酸置换为A或T，在相应于位置91的位置上的氨基酸置换为A，在相应于位置93的位置上的氨基酸置换为Q，在相应于位置98的位置上的氨基酸置换为A；
    优选地，所述变体包含选自下列的1个或几个(例如，1个、2个、3个或4个)氨基酸置换：在相应于位置5的位置上的氨基酸置换为A，在相应于位置7的位置上的氨基酸置换为A，在相应于位置8的位置上的氨基酸置换为G，在相应于位置12的位置上的氨基酸置换为G、T、D或S，在相应于位置13的位置上的氨基酸置换为A，在相应于位置14的位置上的氨基酸置换为G，在在相应于位置22的位置上的氨基酸置换为A，在相应于位置25的位置上的氨基酸置换为A，在相应于位置32的位置上的氨基酸置换为A，在相应于位置34的位置上的氨基酸置换为A，在相应于位置35的位置上的氨基酸置换为 A，在相应于位置36的位置上的氨基酸置换为A，在相应于位置40的位置上的氨基酸置换为A，在相应于位置41的位置上的氨基酸置换为A，在相应于位置42的位置上的氨基酸置换为A，在相应于位置52的位置上的氨基酸置换为K、H、A、S或D，在相应于位置55的位置上的氨基酸置换为S，在相应于位置60的位置上的氨基酸置换为A，在相应于位置61的位置上的氨基酸置换为K、H、S或A，在相应于位置89的位置上的氨基酸置换为A或T，在相应于位置91的位置上的氨基酸置换为A，在相应于位置93的位置上的氨基酸置换为Q，在相应于位置98的位置上的氨基酸置换为A。
  14. 权利要求1-13任一项所述的分离的多肽或其变体，其中，所述分离的多肽由BNLF2b基因编码的野生型蛋白的不多于97个(例如，不多于96个，不多于95个，不多于94个，不多于93个，不多于92个，不多于91个，不多于90个，不多于89个，不多于88个，不多于87个，不多于86个，不多于85个，不多于84个，不多于83个，不多于82个，不多于81个，不多于80个，不多于79个，不多于78个，不多于76个，不多于75个，或不多于74个)的连续氨基酸残基组成。
  15. 权利要求1-14任一项所述的分离的多肽或其变体，其中，所述分离的多肽或其变体连接于固相载体表面，或者带有可与固相载体连接的修饰基团；
    优选地，所述修饰基团是生物素或亲和素；
    优选地，所述固相载体选自磁珠或微量滴定板(例如微孔板或酶标板)。
  16. 权利要求1-14任一项所述的分离的多肽或其变体，其中，所述分离的多肽或其变体带有可检测的标记；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
  17. 一种分离的核酸分子，其包含编码权利要求1-14任一项所述的分离的多肽或其变体的核苷酸序列。
  18. 一种载体，其包含权利要求17所述的分离的核酸分子。
  19. 一种宿主细胞，其包含权利要求17所述的分离的核酸分子或权利要求18所述的载体。
  20. 一种试剂盒，其包含捕获试剂，所述捕获试剂选自权利要求1-14任一项所述的分离的多肽或其变体；
    优选地，所述试剂盒还包含使用所述分离的多肽或其变体作为捕获试剂来测定来自受试者的样品中对BNLF2b基因编码的蛋白特异性的抗体的水平，并任选地确定所述受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中的说明书；
    优选地，所述捕获试剂选自权利要求15所述的分离的多肽或其变体；
    优选地，所述受试者是哺乳动物，例如人。
  21. 权利要求20所述的试剂盒，其还包含检测试剂，其中，所述检测试剂选自权利要求16所述的分离的多肽或其变体。
  22. 权利要求20所述的试剂盒，其还包含检测试剂，其中，所述检测试剂选自带有可检测的标记的二级抗体；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素；
    优选地，所述二级抗体对待测抗体所来自的物种(例如人)的抗体是特异的；
    优选地，所述二级抗体是抗-免疫球蛋白抗体；
    优选地，所述抗-免疫球蛋白抗体选自抗IgG抗体、抗IgM抗体、抗IgA抗体。
  23. 权利要求20-22任一项所述的试剂盒，其中，所述试剂盒还包含一种或多种选自下列的试剂或装置：(i)用于收集或贮存来自受试者的样品的装置(例如采血装置)；(ii)用于进行所述测定所需的其他试剂(例如缓冲液，稀释液，封闭液，和/或标准品)。
  24. 用于确定受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中的方法，其包括：
    (1)测定来自所述受试者的样品中对BNLF2b基因编码的蛋白特异性的抗体的水平；和，
    (2)将所述水平与参考值比较；
    优选地，如果所述水平高于参考值的话，则判断所述受试者患有鼻咽癌或是否处于鼻咽癌的风险中；
    优选地，通过免疫学测定来测定所述样品中对BNLF2b基因编码的蛋白特异性的抗体的水平；
    优选地，所述免疫学测定选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
  25. 权利要求24所述的方法，其中，所述测定包括使用权利要求1-14任一项所述的分离的多肽或其变体作为捕获试剂；
    优选地，所述捕获试剂选自权利要求15所述的分离的多肽或其变体。
  26. 权利要求25所述的方法，其中，步骤(1)包括以下步骤：
    (1a)将来自所述受试者的样品与捕获试剂接触，以获得抗原-抗体免疫复合物；所述捕获试剂选自权利要求1-14任一项所述的分离的多肽或其变体；
    (1b)测定步骤(1a)获得的抗原-抗体免疫复合物的量；
    优选地，在步骤(1b)中，通过免疫学检测来测定所述免疫复合物的量；
    优选地，所述免疫学检测选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法；
    优选地，所述捕获试剂选自权利要求15所述的分离的多肽或其变体。
  27. 权利要求26所述的方法，其中，在步骤(1b)中，使用检测试剂来检测所述免疫复合物的量，所述检测试剂选自权利要求16所述的分离的多肽或其变体。
  28. 权利要求26所述的方法，其中，在步骤(1b)中，使用检测试剂来检测所述免疫复合物的量，所述检测试剂选自带有可检测的标记的二级抗体；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素；
    优选地，所述二级抗体对待测抗体所来自的物种(例如人)的抗体是特异的；
    优选地，所述二级抗体是抗-免疫球蛋白抗体；
    优选地，所述抗-免疫球蛋白抗体选自抗IgG抗体、抗IgM抗体或抗IgA抗体；优选 地，所述抗-免疫球蛋白抗体是抗IgG抗体。
  29. 权利要求24-28任一项所述的方法，其中，所述样品是血液样品，例如全血、血浆或血清。
  30. 权利要求24-29任一项所述的方法，其中，所述受试者是人。
  31. 权利要求24-30任一项所述的方法，其还包括：
    在步骤(1)之前，提供来自所述受试者的样品；和/或
    在步骤(2)之后，给被判断为患有鼻咽癌或处于鼻咽癌的风险中的受试者施用治疗有效量的可治疗鼻咽癌的抗肿瘤疗法(例如，化学疗法、放射疗法和/或免疫疗法)。
  32. 能够测定对BNLF2b基因编码的蛋白特异性的抗体的水平的试剂在制备试剂盒中的用途，所述试剂盒用于确定受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中；
    优选地，所述试剂能够通过免疫学测定来测定对BNLF2b基因编码的蛋白特异性的抗体的水平；优选地，所述免疫学测定选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法；
    优选地，所述受试者是人；
    优选地，所述样品是血液样品，例如全血、血浆或血清；
    优选地，所述试剂盒通过权利要求24所述的方法来确定受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中。
  33. 权利要求32所述的用途，其中，所述能够测定对BNLF2b基因编码的蛋白特异性的抗体的水平的试剂包含捕获试剂，所述捕获试剂选自权利要求1-14任一项所述的分离的多肽或其变体；
    优选地，所述捕获试剂选自权利要求15所述的分离的多肽或其变体；
    优选地，所述试剂盒通过权利要求25或26所述的方法来确定受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中。
  34. 权利要求33所述的用途，其中，所述能够测定对BNLF2b基因编码的蛋白特异 性的抗体的水平的试剂还包含检测试剂，所述检测试剂选自权利要求16所述的分离的多肽或其变体；
    优选地，所述试剂盒通过权利要求27所述的方法来确定受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中。
  35. 权利要求33所述的用途，其中，所述能够测定对BNLF2b基因编码的蛋白特异性的抗体的水平的试剂还包含检测试剂，所述检测试剂选自带有可检测的标记的二级抗体；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素；
    优选地，所述二级抗体对待测抗体所来自的物种(例如人)的抗体是特异的；
    优选地，所述二级抗体是抗-免疫球蛋白抗体；
    优选地，所述抗-免疫球蛋白抗体选自抗IgG抗体、抗IgM抗体或抗IgA抗体；优选地，所述抗-免疫球蛋白抗体是抗IgG抗体；
    优选地，所述试剂盒通过权利要求28所述的方法来确定受试者是否患有鼻咽癌或是否处于鼻咽癌的风险中。
  36. 用于检测样品中的对BNLF2b基因编码的蛋白特异性的抗体的方法，其包括以下步骤：
    (1)将所述样品与捕获试剂接触，以获得抗原-抗体免疫复合物；所述捕获试剂选自权利要求1-14任一项所述的分离的多肽或其变体；
    (2)测定步骤(1)获得的抗原-抗体免疫复合物的量；
    优选地，在步骤(2)中，通过免疫学检测来测定所述免疫复合物的量；
    优选地，所述免疫学检测选自酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法；
    优选地，所述捕获试剂选自权利要求15所述的分离的多肽或其变体。
  37. 权利要求36所述的方法，其中，在步骤(2)中，使用检测试剂来检测所述免疫复合物的量，所述检测试剂选自权利要求16所述的分离的多肽或其变体。
  38. 权利要求36所述的方法，其中，在步骤(2)中，使用检测试剂来检测所述免疫复合物的量，所述检测试剂选自带有可检测的标记的二级抗体；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素；
    优选地，所述二级抗体对待测抗体所来自的物种(例如人)的抗体是特异的；
    优选地，所述二级抗体是抗-免疫球蛋白抗体；
    优选地，所述抗-免疫球蛋白抗体选自抗IgG抗体、抗IgM抗体或抗IgA抗体；优选地，所述抗-免疫球蛋白抗体是抗IgG抗体。
  39. 权利要求1-16任一项所述的分离的多肽或其变体在制备试剂盒中的用途，所述试剂盒用于检测样品中的对BNLF2b基因编码的蛋白特异性的抗体。
PCT/CN2020/139310 2020-01-02 2020-12-25 EB病毒BNLF2b基因编码多肽及其检测用途 WO2021136082A1 (zh)

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