An EBV-related gastric cancer diagnostic marker and its application

Abstract

The present invention discloses an EBV-related gastric cancer diagnostic marker and its application, wherein the diagnostic marker is selected from at least one of LF2 protein and BFRF3 protein. The present invention is the first to screen and obtain two diagnostic markers for EBV-related gastric cancer (EBVaGC) based on proteome chips and phage display technology - LF2 protein and BFRF3 protein, which can efficiently and accurately diagnose EBVaGC. In particular, the AUC of the combined diagnosis of these two proteins can reach 0.911, which is significantly better than the AUC of the individual diagnoses. And when diagnosing EBVaGC based on these two proteins, no tissue sample is required, only a serum sample is required, and it can be detected during a routine blood test. Therefore, compared with existing diagnostic methods, the present invention has the characteristics of high accuracy, small limitations, and low cost, reduces the cost of medical treatment for patients, is conducive to the realization of universal screening of the population, simplifies the diagnosis process of EBVaGC, and significantly improves the treatment efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an EBV-related gastric cancer diagnostic marker and application thereof. Background Art

[0002] Epstein-Barr virus (EBV) is a human herpesvirus first discovered in Burkitt lymphoma in 1964 by Epstein and Barr's team. EBV infection rates in the natural population exceed 90% and are highly infectious. According to the International Agency for Research on Cancer's classification criteria for carcinogens, EBV is classified as a Group 1 oncogen and is closely associated with the development and progression of gastric cancer, nasopharyngeal carcinoma, and lymphoma. Globally, approximately 200,000 cancers and 140,000 deaths are caused by EBV infection each year. EBV-associated gastric cancer (EBVaGC) is one of the main types of gastric cancer, accounting for approximately 10% of all gastric cancers. Compared with other gastric cancer types, EBVaGC has a high degree of genomic methylation and high expression of proteins such as the immunotherapy target PD1, making it highly responsive to tumor immunotherapy. Therefore, detecting and confirming EBVaGC can facilitate the precise treatment of gastric cancer.

[0003] The detection of EBV-related gastric cancer still faces bottlenecks. Compared with other types of gastric cancer, EBVaGC has no specific clinical manifestations and pathological features. Currently, the diagnosis of EBVaGC relies on EBER in situ hybridization testing, but this method has major shortcomings: EBER is the gold standard for confirming EBV infection, but this method requires sampling through gastroscopic biopsy, and its accuracy is related to the doctor's operating experience and correct sampling; and EBV is heterogeneously distributed in gastric cancer tissue, so its detection has a high false negative rate. In addition, EBER in situ hybridization technology is complex to operate and has a high detection cost (300-500 yuan / person), which not only increases the patient's medical costs but also makes it difficult to achieve universal screening. Therefore, the development of simple, accurate, and economical EBV detection methods is crucial for the diagnosis of EBVaGC. Summary of the Invention

[0004] In response to existing technical problems, the purpose of the present invention is to provide an EBV-related gastric cancer diagnostic marker and its application, aiming to use proteome chips and phage display technology to globally screen EBV gastric cancer-related proteins, and to identify EBVaGC protein markers using only serum samples, providing a simple, accurate and economical new method for the diagnosis of EBVaGC.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides an EBV-related gastric cancer diagnostic marker, wherein the diagnostic marker is selected from at least one of LF2 protein and BFRF3 protein.

[0007] In a second aspect, the present invention provides a use of a diagnostic marker in preparing a product for diagnosing EBV-related gastric cancer, wherein the diagnostic marker is selected from at least one of LF2 protein and BFRF3 protein.

[0008] Preferably, the amino acid sequence of the LF2 protein is shown in SEQ ID NO.1;

[0009] The amino acid sequence of the BFRF3 protein is shown in SEQ ID NO.2.

[0010] Preferably, the diagnostic marker is a combination of LF2 protein and BFRF3 protein.

[0011] Preferably, the antibodies related to the LF2 protein and BFRF3 protein are highly expressed in patients with EBV-related gastric cancer.

[0012] Preferably, the product is a detection kit comprising a reagent for detecting the level of IgG antibodies against LF2 protein and / or BFRF3 protein in a sample to be tested.

[0013] Preferably, when the IgG antibody unit value of the anti-Omp20 protein is ≥1.5 and the IgG antibody unit value of the anti-HcpA protein is ≥1.3, the patient can be preliminarily diagnosed as having EBV-related gastric cancer.

[0014] Preferably, the sample to be tested is any one of whole blood, plasma, and serum.

[0015] Preferably, the reagents include a standard, an antigen coating solution, a blocking solution, a sample diluent, a stop solution, an enzyme-labeled reagent, a color development reagent, and a washing solution.

[0016] Preferably, the standard is an IgG antibody.

[0017] Preferably, the antigen coating solution is TBST.

[0018] Preferably, the blocking solution is TBST containing 3% BSA.

[0019] Preferably, the sample diluent includes a protein diluent and a serum diluent; the protein diluent is PBS containing 5% glycerol; and the serum diluent is TBST containing 10% BSA.

[0020] Preferably, the stop solution is 1M H2SO4 solution.

[0021] Preferably, the enzyme-labeled reagent contains PBST containing enzyme-labeled antibodies.

[0022] Preferably, the color developing reagent is TMB.

[0023] Preferably, the washing solution is PBST.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. This study, based on proteomic microarrays and phage display technology, screened and identified two diagnostic markers for EBV-associated gastric cancer (EBVaGC)—LF2 and BFRF3—for the first time. These proteins are highly effective and accurate in diagnosing EBVaGC. The combined diagnostic area under the correlation coefficient (AUC) of these two proteins reached 0.911, significantly exceeding the AUC of each protein alone.

[0026] 2. The present invention does not require tissue samples for diagnosing EBVaGC, only serum samples are needed, and the diagnosis can be performed during a routine blood test.

[0027] 3. Compared with existing diagnostic methods, the present invention has the characteristics of high accuracy, small limitations and low cost, which reduces the medical costs of patients, facilitates the universal screening of the population, simplifies the diagnosis process of EBVaGC, and significantly improves treatment efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0029] Figure 1 The results of Example 1 using the EBV proteome chip and EBVaGC patient serum reaction; Figure 1 A is the result of EBV-positive gastric cancer chip. Figure 1 B is the EBV-negative gastric cancer chip result;

[0030] Figure 2 The AUC results of the ROC curve analysis of the diagnostic marker LF2 protein of the present invention for diagnosing EBVaGC alone in Example 2 are shown;

[0031] Figure 3 The AUC results of the ROC curve analysis of the diagnostic marker BFRF3 protein of the present invention for diagnosing EBVaGC alone in Example 2 are shown;

[0032] Figure 4 The AUC results of the combined diagnosis of EBVaGC using the diagnostic markers LF2 protein and BFRF3 protein of the present invention were analyzed using the ROC curve in Example 2. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0034] EBV infection-induced gastric cancer and related deaths are a major public health issue that needs to be addressed in our province and across my country. The large number of people infected, the widespread nature of the disease, and the risk of recurrent infection present significant challenges in clinical prevention and treatment: 1) Existing diagnostic methods are significantly limited and fail to guide medication, making personalized, precision treatment difficult to achieve; 2) Existing diagnostic methods are costly and difficult to perform, making universal population screening difficult; and 3) There is no effective vaccine to prevent EBV infection. Currently, the diagnosis of EBVaGC relies primarily on the EBER in situ hybridization assay, but this method has significant limitations: 1) During intestinal mucosal biopsy, the virus is unevenly distributed throughout the mucosal tissue, with viral loads significantly higher in ulcerated than non-ulcerated mucosa. Endoscopic biopsy sampling is dependent on the physician's experience and accurate sampling, resulting in significant uncertainty. 2) Interpretation of the EBER in situ hybridization assay requires optimal staining of the control tissue section before the test tissue section can be used. If the control tissue section does not stain optimally, the results of the test tissue section are unreliable. 3) The EBV in situ hybridization technique is complex and expensive, increasing patient medical costs and hindering widespread screening. Other EBV infection detection methods include PCR and immunohistochemistry. PCR can quantitatively detect EBV DNA but cannot identify infected cells or their localization within tissues. Immunohistochemistry can only detect viral protein expression and has poor sensitivity for detecting low intracellular EBV copies. Therefore, establishing a more specific EBVaGC detection method and achieving accurate diagnosis of EBVaGC based on this method would provide new insights into improved treatment options.

[0035] Protein chip technology and phage display technology provide powerful tools for this purpose. The EBV whole protein chip technology utilized in this invention can be used to screen for EBV proteins that interact with serum antibodies. It offers global, high-throughput screening capabilities, enabling the establishment of a network of interactions between serum antibodies and EBV proteins at the proteomic level, providing an experimental basis for high-throughput screening of target proteins that interact with serum antibodies. An EBV phage display library was constructed in the early stages of this patent application, featuring a large screening capacity and high sensitivity. Phage display technology can be used to screen for phages that specifically bind to serum antibodies, thereby obtaining the antigenic epitopes recognized by these antibodies and enabling further analysis.

[0036] Specifically, the present invention screened EBVaGC diagnostic markers, LF2 and / or BFRF3, using an EBV proteome chip. These markers are used as protein indicators for the diagnosis of EBVaGC. The EBV proteome chip encompasses 85 proteins in the EBV proteome and consists of 85 EBV proteins and a chip substrate.

[0037] The 85 EBV proteins are unique resources of the present invention and are obtained by gene synthesis and high-throughput expression and purification.

[0038] The substrate was an optical grade epoxy substrate purchased from Beijing Bio Biochip Co., Ltd.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0040] Example 1. Screening of serum markers

[0041] In this example, in order to study the correlation between EBV-related proteins and EBV-associated gastric cancer (EBVaGC), an EBV proteome chip was used to screen for EBVaGC diagnostic markers. The specific steps are as follows:

[0042] (1) Sample collection from gastric cancer patients

[0043] This project established a gastric cancer-specific cohort at the Union Hospital Affiliated to Fujian Medical University, the First Affiliated Hospital of Fujian Medical University, and the Fujian Provincial Cancer Hospital, and collected nearly 1,000 gastric cancer serum and tissue samples. At the same time, a gastric disease-specific cohort was established in Changle, a high-incidence area of ​​gastric cancer in Fujian, and 1,500 patients covering different stages of gastric disease were enrolled. Tissues were used for EBER in situ hybridization to determine EBV infection status, and serum was used for marker screening.

[0044] From the gastric cancer-specific cohort collected above, 31 cases of EBV-positive gastric cancer and 33 cases of EBV-negative gastric cancer (a total of 64 gastric cancer patients) were further selected for the next step of EBVaGC diagnostic marker screening.

[0045] (II) Screening of EBVaGC diagnostic markers using proteomic chips

[0046] 1. The preparation method of the EBV proteome chip refers to the method in Example 1 of the specification of the applicant's previous patent document CN115125623A. The EBV proteome chip contains 85 protein sequences shown in Table 1.

[0047] Table 1

[0048]

[0049]

[0050] 2. Interaction between EBV protein chip and serum samples from 64 screened gastric cancer patients

[0051] (1) Closed

[0052] Prepare 30 ml of blocking buffer (1% BSA in PBS buffer, see Table 2) in a 4-chip cassette. Remove the chip from -80°C and warm it to 4°C and room temperature. (Note: For PATH substrates, do not remove individual chips, as this will cause moisture to accumulate on the chip surface and affect sample locations; ensure the entire process is sealed.) Incubate on a rocker at 20-30 rpm for 1 hour at room temperature. Discard the blocking buffer and rinse once with 1x PBS and then 0.2x PBS for 5 minutes each. Then, centrifuge to dry (800 rpm, 1 minute). Install the enclosure and set aside.

[0053] (2) Sample incubation

[0054] Remove the serum sample from -80°C and thaw on ice or at 4°C until completely thawed. Centrifuge the serum sample at 4°C at 10,000 rpm for 10 minutes. Remove the supernatant for analysis. (After centrifugation, a layer of white material, likely lipids, is often found on the surface. Remove this material by aspiration before removing the clear, pale yellow serum. If the serum sample is precious, aspirate the lower layer directly, but be careful to avoid removing the white material.) Dilute the sample with incubation buffer (1% BSA in PBST, 0.25 mg / mL E. coli lysate) at a 1:200 dilution ratio (4 μl serum to 800 μl incubation buffer). Add 600 μl to the chip and place in a humidified chamber on a rocker with side-to-side movement at 20-30 rpm for 2 hours at room temperature.

[0055] (3) Cleaning

[0056] This step is critical because it can easily cause contamination between samples in different blocks. Discard the serum sample, add 1 mL of PBST wash buffer (see Table 3), and shake on a shaker at 120 rpm for 5 minutes. Repeat this wash three times. After removing the clamps, place the sample in a large box, add 30 mL of PBST wash buffer, and shake on a shaker at 70 rpm for 5 minutes. Repeat this wash three times.

[0057] (4) Incubation with fluorescently labeled IgG / IgM secondary antibody

[0058] Prepare incubation solution containing fluorescently labeled IgG / IgM secondary antibodies (Cy3-conjugated AffiniPure Goat Anti-Human IgG; Alexa Fluor 647-conjugated AffiniPure Donkey Anti-Human IgM) in advance (mix the fluorescently labeled IgG / IgM secondary antibody with the incubation solution at a ratio of 1:1000, using 1% BSA in PBST). The volume of incubation solution depends on the number of microarrays. For a single microarray, use a microarray-specific incubation box with a 3 mL volume. For 3-4 microarrays, use a wash box with a 15 mL volume. Incubate on a side-by-side shaker at 20-30 rpm, protected from light, at room temperature for 1 hour.

[0059] (5) Cleaning

[0060] Place the chip treated in step (4) in a chip cleaning box containing 30 ml of PBST. Shake the chip 10-15 times, replace the cleaning solution, and shake again 10-15 times. Replace the cleaning solution with 20-25 ml of PBST and wash three times on a horizontal shaker at 100-110 rpm for 10 minutes each time. Protect from light during washing. After completion, wash the chip with ddH2O for 5 minutes twice, rinsing for 10 seconds.

[0061] (6) Drying

[0062] The chip was placed in a chip dryer and centrifuged to dry (800 rpm, 1 min).

[0063] (7) Scan

[0064] Follow the scanner's operating specifications and instructions and set the parameters as follows: 635nm, Power 100%, PMT value 550; 532nm, Power 100%, PMT value 550.

[0065] (8) Data extraction

[0066] Open the corresponding GAL file, align the chip image and each array in the GAL file as a whole, press the automatic alignment button, extract the data and save the GPR file.

[0067] The compositions of the reagents used in the above processes are shown in Tables 2 to 4.

[0068] Table 2 Blocking solution (1% BSA in PBS buffer; pH 7.4)

[0069]

[0070] Table 3 Washing solution (PBST solution; pH 7.4)

[0071]

[0072] Table 4 Incubation solution

[0073]

[0074] (3) Using differential analysis, T-test analysis, etc., to select differentially expressed proteins as candidate markers.

[0075] After normalizing the protein chip signals within and between chips using R language, candidate markers were selected using T test, fold difference analysis, and multivariate linear regression analysis.

[0076] The results showed that 64 gastric cancer patient samples were screened using EBV proteome chips, and the chip and patient serum reaction results were as follows: Figure 1 As shown ( Figure 1 A is the result of EBV-positive gastric cancer chip. Figure 1 B is the result of EBV-negative gastric cancer chip), and serological markers for diagnosing EBV-related gastric cancer were identified, among which LF2 and BFRF3 ranked in the top two in terms of signal differences. The analysis results of the two proteins are shown in Table 5.

[0077] Table 5 Analysis of EBV protein chip screening results

[0078]

[0079] Example 2: Independent sample validation of marker diagnostic efficacy

[0080] To further validate the diagnostic efficacy of LF2 and BFRF3 proteins as EBVaGC markers, the identified serological markers were further validated in 345 gastric cancer samples, including 18 EBV-positive gastric cancers and 327 EBV-negative gastric cancers. EBV-positive gastric cancers accounted for approximately 5% of the total sample, a proportion consistent with the true incidence of EBV-associated gastric cancer.

[0081] The specific verification steps are as follows:

[0082] 1. Protein dilution: Dilute LF2 protein and BFRF3 protein separately with TBST. The dilution multiples are as follows:

[0083] Dilution 0: 100ul protein

[0084] Dilute 2 times: 50ul protein + 50ul dilution

[0085] Dilute 4 times: 25ul protein + 75ul dilution

[0086] Dilute 8 times: 12.5ul protein + 87.5ul dilution

[0087] 2. Dilution of standard Human IgG (50ug / uL):

[0088] ① Take 1 μL of Human IgG, add 499 μL of protein diluent, and mix thoroughly by pipetting to obtain a standard stock solution with a concentration of 0.1 μg / μl.

[0089] ② Dilute to 50pg / ul:

[0090] Take four 1.5ml EP tubes

[0091] Preparation of Standard A: Prepare 500ul of standard stock solution + 500ul of protein diluent to a concentration of 50ng / ul

[0092] Preparation of Standard B: Prepare 100ul of Standard A + 900ul of protein diluent to a concentration of 5ng / ul

[0093] Preparation of Standard C: Prepare 100ul of Standard B + 900ul of protein diluent to a concentration of 500pg / μL

[0094] Preparation of Standard D: Prepare 100ul of Standard C + 900ul of protein diluent, with a concentration of 50pg / μL

[0095] The above protein diluent is: PBS containing 5% glycerol (42.75ml PBS + 2.25ml glycerol)

[0096] Take standard D, and then dilute it into 8 standard solutions with gradient concentrations in the following order: 100ul of standard D, 100ul of standard D + 100ul of protein diluent, 50ul of standard D + 100ul of protein diluent, 25ul of standard D + 100ul of protein diluent, 12.5ul of standard D + 100ul of protein diluent, 6.25ul of standard D + 100ul of protein diluent, 3.125ul of standard D + 100ul of protein diluent, and 1.5625ul of standard D + 100ul of protein diluent.

[0097] 3. Antigen coating: Add 50 μL of the corresponding diluted protein from step 1 or the 8 gradient concentrations of each standard solution prepared in step 2 to each well and incubate at 4°C overnight.

[0098] 4. Washing: Pour out the liquid in the wells and wash each well once with PBST. Each wash should add at least half the height of the well (about 200ul). Pat clean.

[0099] 5. Blocking: Add 100 μL of blocking solution (3% BSA in TBST) to each well and incubate at room temperature for 1 h.

[0100] 6. Washing: Pour out the liquid in the wells, wash each well once with PBST, pour out the liquid in the wells, and pat clean.

[0101] 7. Sample addition: (standard curve + PBST)

[0102] 1) The preparation of serum diluent is shown in Table 6.

[0103] Table 6 Serum dilution (45 mL):

[0104]

[0105] 2) Serum treatment: 12000r, 4°C, 20min.

[0106] 3) Serum dilution: dilute the serum treated in step 2) to 25 times, 50 times,

[0107] 100 times, 200 times.

[0108] 4) Add 150 μl of diluted serum to the corresponding wells.

[0109] Dilution calculation: 650ul per 4 wells

[0110] Dilute 25 times: 26ul treated serum + 624ul serum diluent

[0111] Dilute 50 times: 13ul treated serum + 637ul serum diluent

[0112] Dilute 100 times: 6.5ul treated serum + 643.5ul serum diluent

[0113] Dilute 200 times: 3.25ul treated serum + 646.75ul serum diluent

[0114] 8. Incubation: Place in a 37°C incubator and incubate for 1.5 hours.

[0115] 9. Washing: Pour out the liquid in the wells, wash 5 times with PBST, and pat clean.

[0116] 10. Enzyme-labeled antibody incubation: Add 100 μL of enzyme-labeled antibody (Rabbit HRP-Anti Human IgG antibody) diluted 1:10,000 in PBST to each well and incubate in a 37°C incubator for 35 min.

[0117] 11. Washing: Pour out the liquid in the wells, add 200 μL PBST to each well, wash 5 times, and pat clean.

[0118] 12. Color development: Add 100 μL of the color development substrate TMB to each well and incubate at 37°C in the dark for 15 min (observe every 5 minutes).

[0119] 13. Termination: Add 100 μL 1M H2SO4 to each well to terminate the reaction. (In order)

[0120] 14. Microplate reader reading: Measure the optical density (OD450) of each well at a wavelength of 450 nm using a single-channel microplate reader.

[0121] 15. Data Processing and Analysis

[0122] The results showed that the diagnostic AUC using LF2 protein as a single indicator was 0.873 (e.g. Figure 2 As shown); BFRF3 protein single indicator, the diagnostic AUC is 0.758 (as shown Figure 3 The combined diagnosis of LF2 and BFRF3 proteins has an AUC of 0.911 (as shown in Figure 4 shown).

[0123] Application Example 1

[0124] Based on the results of the above examples, LF2 protein and BFRF3 protein were used as diagnostic markers for the detection and diagnosis of EB-related gastric cancer. The same steps as in Example 2 were used. The diagnostic criteria of the obtained results were as follows:

[0125] The unit value of anti-LF2 protein IgG antibody (LF2_IgG) is ≥1.5 and the unit value of anti-BFRF3 protein IgG antibody (BFRF3_IgG) is ≥1.3: The patient can be preliminarily diagnosed as EB-related gastric cancer patient.

[0126] The above application examples demonstrate the feasibility of the method of the present invention.

[0127] It should be noted that the LF2 protein described in the present invention has the amino acid sequence shown in SEQ ID NO. 1. The BFRF3 protein described in the present invention has the amino acid sequence shown in SEQ ID NO. 2. Both LF2 and BFRF3 proteins have been reported for use in the diagnosis of nasopharyngeal carcinoma. In previous research by the applicant, as shown in Example 2 of the specification of patent document CN115125623A, LF2 was found to be associated with healthy controls when using an EBV proteome chip to screen for proteins interacting with gastric cancer patient serum, and its expression was low in healthy controls. However, the role and expression level of LF2 in EBV-positive and EBV-negative gastric cancers have not been elucidated. Therefore, the mechanisms underlying the pathogenicity and screening significance of LF2 and BFRF3 proteins in EBV-related gastric cancer remain unclear, and no relevant reports have been published. Therefore, the present invention is the first to discover and validate the potential of these two proteins as diagnostic standards for EBV-related gastric cancer, providing a new direction for the early diagnosis of EBV-related gastric cancer.

[0128] Amino acid sequence of LF2 protein (SEQ ID NO.1):

[0129] MAEAYPGGAHAALASRRSSFRNSLRRLRPTEKPDTSFMRGVWKYEIFPSYVRVTNKQVLQLDAQCQELPPCPSVGQILSFKLPSFSFNTTTYGSRYFTVAFLFFGAE DNEVFLKPFFVMHSDQDIVLSVLNPRSLFIEKGKFTWYIVPIRLVKNPYLYLQILPGQSDIQLTRSCTQSGDKLNTSEPQIFLSGSPVTSQDECLPYLLAQHTPPFL KSYARIHTFPGKVCPVNAIRRGKGYVRVSVDTPDLKREGPLNVKVGMTLLDDVIIAFRYNPYPKSHWRWDGESTDIRYFGSPVIIPPNFITELEYNNTYEAPLSSKI TAVVVSHSSNPVFYVYPQEWKPGQTLKLTVRNISNNPITIVTGQSMAQAFFIYAGDPSISTIMRRYIQRQGCALTLPGNIVVESSSLPTFERINKTFNGNIVASEGTL

[0130] Amino acid sequence of BFRF3 protein (SEQ ID NO.2):

[0131] MARRLPKPTLQGRLEADFPDSPLLPKFQELNQNNLPNDVFREAQRSYLVFLTSQFCYEEYVQRTFGVPRRQRAIDKRQRASVAGAGAHAHLGGSSATPVQQAQAAASAGTGALASSAPSTAVAQSATPSVSSSISSLRAATSGATAAASAAAAVDTGSGGGGQPHDTAPRGARKKQ

[0132] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, several improvements can be made without departing from the principles of the present invention, and these improvements should also be considered as the scope of protection of the present invention.

Claims

1. Use of a diagnostic marker in the preparation of a product for diagnosing EBV-related gastric cancer, characterized in that: The diagnostic marker is a combination of LF2 protein and BFRF3 protein; The product is a detection kit, which includes reagents for detecting the level of IgG antibodies against LF2 protein and / or BFRF3 protein in a sample to be tested.

2. The use according to claim 1, characterized in that The amino acid sequence of the LF2 protein is shown in SEQ ID NO.1; The amino acid sequence of the BFRF3 protein is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The LF2 protein and BFRF3 protein-related antibodies are highly expressed in EBV-related gastric cancer patients.

4. The use according to claim 1, characterized in that The sample to be tested is any one of whole blood, plasma, and serum.

5. The use according to claim 1, characterized in that The reagents include standard substances, antigen coating solution, blocking solution, sample diluent, stop solution, enzyme labeling reagent, color development reagent and washing solution.

6. The use according to claim 5, characterized in that The standard substance is IgG antibody; The antigen coating solution is TBST; The blocking solution is TBST containing 3% BSA; The sample diluent includes a protein diluent and a serum diluent; the protein diluent is PBS containing 5% glycerol; the serum diluent is PBST containing 10% BSA; The stop solution is 1M H2SO4 solution; The enzyme-labeled reagent contains PBST containing enzyme-labeled antibodies; The color developing reagent is TMB; The washing solution is PBST.

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