A protein chip for early warning of liver cancer and application thereof

Abstract

This invention provides a protein chip for early warning of liver cancer and its application. The protein chip includes an antigen set; the antigen set includes antigens; the antibodies corresponding to the antigens are at least one of 61 selected from GNA11, GNAS, IDH1, MSH2, NPM1, PAX5, PTCH1, PTEN, SRSF2, Survivin, CAST, PAIP1, PRKCZ, RAD23A, RUNX1T1, SARS, TP53, APEX2, RCSD1, and AIF1; the protein chip is used for early warning assessment of liver cancer, and the early warning assessment steps include: collecting serum from the target population, using the protein chip to detect the serum to obtain detection data, and then determining whether they belong to a low-risk, medium-risk, or high-risk population based on a risk warning model; the protein chip systematically detects tumor-related autoantibody risk factor signals using high-throughput technology for early warning detection of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of liver cancer early warning, specifically to a protein chip for liver cancer early warning and its application. Background Technology

[0002] In today's society, the health big data situation is far from optimistic, showing a trend of an aging population and younger disease groups. Cardiovascular and cerebrovascular diseases and malignant tumors have become the leading killers of human lifespan. Therefore, it is the contemporary responsibility of health researchers to promote proactive health awareness, advocate healthy lifestyles, scientifically assess health risks, and prevent the occurrence of major diseases.

[0003] Cancer development is a long process, and establishing a three-tiered prevention and control system of early warning, early diagnosis, and early treatment is crucial for cancer prevention and control. Based on the characteristics of the cancer course, cancer prevention and control can be divided into stages such as cancer risk warning, early cancer diagnosis, and cancer treatment and prognosis monitoring. Cancer prevention and control emphasizes early warning, early diagnosis, and early treatment. Early cancer warning requires the detection and assessment of many risk factors for cancer development, providing alerts and warnings about the risk of cancer occurrence. Early cancer diagnosis involves the large-scale clinical testing and verification of some cancer risk factors, which are then applied to the medical diagnosis of individual cancer cases. Early cancer treatment involves timely intervention and treatment once a cancer has been diagnosed, stopping the cancer at its source, controlling its progression, and restoring health. Therefore, early cancer warning is an assessment and warning of cancer development risk, falling under the category of preventive medicine; early cancer diagnosis is a medical diagnosis of cancer development, falling under the category of diagnostic medicine; and early cancer treatment falls under the category of therapeutic medicine. Early warning and early diagnosis complement each other: early warning provides more comprehensive risk alerts and warnings for cancer development, providing a basis for early diagnosis; early diagnosis provides more accurate cancer diagnostic information at a specific cross-section, further supporting early warning. A three-tiered prevention and control system of early warning, early diagnosis, and early treatment for tumors forms a large funnel-shaped system for tumor prevention and control, which helps to better prevent and control tumors.

[0004] Liver cancer, also known as hepatocellular carcinoma, is a malignant tumor that occurs in the liver. It is mainly divided into two categories: primary liver cancer and secondary liver cancer. Primary liver cancer originates within the liver itself, such as from the transformation of hepatocellular or bile duct cells; secondary liver cancer develops from the metastasis of cancer cells from other organs to the liver. Early-stage liver cancer often presents with no obvious symptoms, while specific manifestations may appear in the middle and late stages, such as pain in the liver area, jaundice, and signs of cirrhosis.

[0005] However, there are currently no liver cancer risk warning detection products or intervention programs on the market that address early warning of liver cancer and control of risk factors.

[0006] Currently, from the perspective of "early detection and early treatment," the antigen chip that can be used for tumor risk early warning is a proteome antigen chip product developed and manufactured by CDI Corporation in the United States. This chip product contains proteins from the entire human proteome, thus it is suitable for detecting an individual's autoantibody profile. This product can be used to construct an individual's baseline and also for detecting autoantibodies and assessing tumor risk in an individual's serum. However, this proteome chip has several important limitations for risk assessment in preventative healthcare: (1) Low safety factor: This protein chip is a product manufactured and operated by CDI lab in the United States. Due to the global monopoly of this product, and considering the current great uncertainty in the international situation, there is a risk of supply disruption for this chip.

[0007] (2) High testing costs: This product involves the research and development and production of all histone proteins, which are extremely expensive. This product is a monopolistic product with a high import price, and domestic companies cannot effectively participate in pricing. Therefore, it is not suitable for large-scale screening of major health risks in China.

[0008] (3) Lack of standardization: The protein production of this proteome chip is a large-scale micro-production, and the main control factor is the soluble expression of the protein. Other key parameters of the protein raw materials (such as quantity and purity) cannot be controlled, so standardized production is not possible. The large uncertainty between batches of this chip product leads to chaotic health risk screening data, which is not conducive to health management and product iteration.

[0009] (4) Lack of Specificity: This proteome chip belongs to the field of omics research and development tools and plays an important role in the discovery of autoantibody risk factors in tumors. However, it is not specific for the detection and early warning of risk factors for specific cancer types. Therefore, using this product for general health risk detection will generate a lot of background information, resulting in a waste of human, material and financial resources. This product can be used for risk factor screening, but it cannot be used for general health screening of the population. Summary of the Invention

[0010] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a protein chip for liver cancer early warning, which screens antigens for liver cancer early warning and prepares a protein chip; the protein chip systematically detects tumor-related autoantibody risk factor signals using high-throughput technology for liver cancer risk early warning detection.

[0011] The second objective of this invention is to provide an application of the aforementioned protein chip for early warning of liver cancer.

[0012] The first objective of this invention can be achieved by adopting the following technical solution: a protein chip for liver cancer early warning, comprising an antigen set; the antigen set comprising antigens; the antibodies corresponding to the antigens being: GNA11, GNAS, IDH1, MSH2, NPM1, PAX5, PTCH1, PTEN, SRSF2, Survivin, CAST, PAIP1, PRKCZ, RAD23A, RUNX1T1, SARS, TP53, APEX2, RCSD1, AIF1, CDKN1B, EpCAM, MMP9, 14-3-3zeta, Bmi-1, c-myc, At least one of CyclinB1, ET-1, FGF21, HCC1, HDGF2, IL-17A, IL-26, LECT2, p62, p90, RhoA, SOD1, STMN4, SUMO-4, TXN, UBE2C, VCC1, ADCK5, ANKRD13D, DCAF4L2, RPLP0, TSPAN13, WTAP, ASAH1, CIAPIN1, EGFR, MAS1, SLC44A3, UBL7, ZNF428, CDC37L1, MAP3K14, PCNA, ZIC2, and AFP.

[0013] Furthermore, the antibodies are: GNA11, GNAS, IDH1, MSH2, NPM1, PAX5, PTCH1, PTEN, SRSF2, Survivin, CAST, PAIP1, PRKCZ, RAD23A, RUNX1T1, SARS, TP53, APEX2, RCSD1, AIF1, CDKN1B, EpCAM, MMP9, 14-3-3zeta, Bmi-1, c-myc, CyclinB1, ET-1, FGF21, and HCC. 1. HDGF2, IL-17A, IL-26, LECT2, p62, p90, RhoA, SOD1, STMN4, SUMO-4, TXN, UBE2C, VCC1, ADCK5, ANKRD13D, DCAF4L 2. RPLP0, TSPAN13, WTAP, ASAH1, CIAPIN1, EGFR, MAS1, SLC44A3, UBL7, ZNF428, CDC37L1, MAP3K14, PCNA, ZIC2 and AFP.

[0014] Furthermore, the antibodies are derived from humans.

[0015] Furthermore, protein chips also include control systems.

[0016] Furthermore, the control system includes IgG, IgM, BSA, GST tag, His Tag, biotin, and buffer.

[0017] Furthermore, the protein chip further includes at least one of the markers Cy3 and Cy5.

[0018] To achieve the second object of the present invention, the following technical solution can be adopted: An application of a protein chip for early warning of liver cancer, using the protein chip described above for early warning assessment of liver cancer.

[0019] Furthermore, the steps of early warning assessment include: collecting the serum of the population to be tested, using the protein chip to detect the serum to obtain detection data, and then judging whether it belongs to a low-risk, medium-risk or high-risk population according to the risk early warning model.

[0020] Furthermore, when the total number of antibody risks ≤ 5, the total antibody risk value Σ ≤ 15, and there is no antibody with risk values of 2 and 3, it is defined as low risk; when the total number of antibody risks ≤ 9, and there are a small number of antibodies with risk values of 2 and 3, it is defined as medium risk; when the total number of antibody risks ≥ 9, the total antibody risk value Σ ≥ 20, and there are a relatively large number of antibodies with risk values of 2 and 3, it is defined as high risk.

[0021] Furthermore, the median value of the signal foreground value in the 532nm channel is used to represent the signal value F532 of the antibody; the median value of the background value in the 532nm channel is used to represent the background value B532 of the antibody; the ratio of the foreground value to the background value of each protein, that is, F532 / B532, is defined as the signal-to-noise ratio SNR of the antibody; Risk value: when SNR ≤ 2, the risk value is assigned 0.1; when 2 < SNR ≤ 5, the risk value is assigned 1; when 5 < SNR ≤ 10, the risk value is assigned 2; when SNR > 10, the risk value is assigned 3; Total number of antibody risks: Antibodies with SNR ≥ 2 are counted; antibodies with SNR < 2 are not counted; the reference normal dynamic range of the antibody risk value is designed to be between 0 and 1.

[0022] Furthermore, the steps of early warning assessment also include continuous risk tracking and early warning: individual annual examinations form an individual baseline database, and each detection data is compared and analyzed with the previous detection data to establish a fingerprint map of autoantibodies, and accordingly track the antibodies with continuously increasing differences for early warning of the risk of liver cancer occurrence.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention starts from the immune abnormality carcinogenesis of liver nodules, screens the antigen group for early warning of liver cancer and prepares a protein chip; the protein chip systematically detects the signals of tumor-related autoantibody risk factors by high-throughput technology for early warning detection of liver cancer risk; 2. The protein chip of the present invention helps to construct a large model for ultra-early risk warning of liver cancer and serves the development of the big health industry; 3. The protein chip of the present invention is applied to the early warning assessment of liver cancer, and has the characteristics of convenient sampling, high detection throughput, short experimental time and standardized detection system. Attached Figure Description

[0024] Figure 1 This refers to the fabrication process of protein chips; Figure 2 A schematic diagram illustrating the principle of protein chip detection of autoantibodies; Figure 3 For the detection of protein chip health control chips; Figure 4 Illustration of protein chip-based detection of healthy liver cancer; Figure 5 A schematic diagram of risk assessment parameters for signal conversion detection using protein chips; Figure 6 A schematic diagram illustrating the ability of protein chip detection and evaluation parameters to distinguish between healthy individuals and liver cancer patients; Figure 7 This is a schematic diagram of an initial assessment and early warning system for the risk of liver cancer based on protein chip detection. Figure 8 This is a schematic diagram of a continuous tracking, assessment, and early warning system for the risk of liver cancer based on protein chip detection. Detailed Implementation The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0026] Unless otherwise specified, the instruments and strains used in this specific implementation method are from the following sources: Table 1. Instruments and strains used

[0027] Example 1: 1. Protein chip for liver cancer early warning, including an antigen set; the antigen set includes antigens.

[0028] Data source: Autoantibody data from normal individuals, individuals with liver nodules, and individuals with liver cancer were initially collected from the internet and self-developed databases. After screening, analysis, and summarization, 61 autoantibodies were finally obtained. The antigen numbers, corresponding gene names, and functional annotations in liver cancer are shown in Table 2.

[0029] Table 2. Antigen Risk Factors Associated with Hepatic Nodule Immune Dysregulation Leading to Carcinogenesis

[0030] 2. The fabrication steps of protein chips are as follows: Figure 1 As shown: 1) Construction of yeast expression strains: A risk factor (antibody) protein expression vector was constructed, and the expression vector was transfected into yeast to create a protein expression strain. The expression strain was then identified as effectively expressing the protein. The specific procedures are as follows: 1- The antigen gene expression cassette is directly constructed into the secretory protein expression vector through gene synthesis; 2- The expression plasmid was transfected into Saccharomyces cerevisiae INVSC1 competent cells via PEG / LiAc heat shock, plated on defective culture plates, screened and cultured for 3 days, and single clones were selected for storage and later use. At the same time, single clones were selected for activation culture after overnight culture. 3-Expression strains were induced to express proteins using galactose medium, and the supernatant was analyzed by Western blotting.

[0031] 2) Protein expression and purification: The expression strain was cultured in 50 mL of medium. After galactose-induced expression, the culture supernatant was collected for protein extraction and purification. 1-Scale-up expression: The INVSC1 strain expressing the target protein was inoculated into 50 mL of SD / URA3 galactose-induced medium and cultured at 220 rpm and 37 ℃ for 24 h. The bacterial cells and supernatant were then collected by centrifugation at 4000 rpm. 2. Protein extraction and purification: The protein supernatant was filtered through a 0.22 μm filter membrane and then purified by Ni column affinity enrichment. The purified protein was obtained by washing with 5 mM imidazole PBS and eluting with 300 mM imidazole PBS.

[0032] 3) Protein standardization: The purified protein was subjected to SDS-PAGE gel chromatography to ensure a purity greater than 90%, thus reducing background noise. After quantification using Qubit assay, the purified protein was aliquoted, lyophilized, and stored. The lyophilized protein underwent reconstitution stability and solubility testing to assess its suitability for subsequent protein chip fabrication.

[0033] 4) Protein chip fabrication: 1. Substrate Preparation: Glass slides are used as substrates for protein chip fabrication. The chip substrate is placed in a cleaning solution to remove surface grease and impurities. It is then rinsed thoroughly with deionized water and dried with nitrogen or allowed to air dry in a clean environment. 2. Protein sample preparation: Take standardized protein and reconstitute it using PBS; 3. Spotting Layout Design: Utilize specialized software to design the spotting layout, including the protein arrangement order, number of spotting replicates, and concentration gradient detection. A well-designed layout can improve experimental efficiency and accuracy. Label each protein's name, concentration, and other information on the designed layout for easy data analysis and result tracking.

[0034] 4. Spotting Procedure: Select an inkjet printer according to experimental requirements and spot the prepared protein sample according to the designed layout. Pay attention to the consistency of the spotting volume and the shape and size of the spots. Avoid air bubbles or missed spots. Maintain a stable environment during the spotting process and avoid the influence of temperature changes and wind speed on the spotting results.

[0035] 5. Chip Drying and Sealing: Place the fabricated chips in a temperature- and humidity-controlled environment, avoiding direct sunlight to prevent protein damage. Drying time depends on environmental conditions and the chip matrix, typically ranging from several hours to several days. Use BSA blocking solution to seal the chips, eliminating non-specific binding and improving signal specificity. Evenly coat the blocking solution onto the chip surface, allow it to stand for a period of time, and then rinse thoroughly with deionized water.

[0036] 6-Chip storage: Store chip products in a -80℃ freezer, clearly label the batch, and avoid repeated freeze-thaw cycles.

[0037] This protein chip contains antigen proteins corresponding to 61 autoantibody risk factors (antibodies) for liver cancer, system controls: IgG, IgM, BSA, GST tag, His Tag, biotin, Buffer, markers Cy3 and Cy5, and corresponding labels: LCProteinArray and Reneocare.Co.LTD.

[0038] Example 2: The steps involved in using protein chips for early warning assessment of liver cancer, including experimental testing of collected serum to obtain corresponding detection data (e.g.) Figure 2-4 (as shown) 1) Detection principle: Each protein chip can detect one serum sample. Specific antibodies (mainly IgG antibodies) bind to proteins immobilized on the chip. Unbound antibodies and other proteins are washed away, and then detected with anti-human IgG fluorescent secondary antibody (Cy3 labeled, appearing green). The signal is read by a fluorescence scanner, and the strength of the signal is positively correlated with the affinity and quantity of the antibody.

[0039] 2) Sample collection: Blood samples were collected from 54 healthy controls (including 30 individuals with liver nodules) and 90 individuals with liver cancer. 0.5 mL of serum was collected using brown procoagulant tubes. After standing for 1 hour, the samples were centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected as serum. Individual serum samples were aliquoted into 100 μL portions and stored at -80°C for later use.

[0040] 3) Detection method: 1-Rewarming: Remove the chip from the -80℃ freezer, place it in a 4℃ freezer for 30 minutes, and then at room temperature for 15 minutes; 2-Sealing: Secure the chip enclosure, add sealing solution to 14 blocks, place on a side-swinging shaker, and seal at room temperature for 3 hours; 3- Serum sample incubation: Discard the blocking solution, quickly add serum incubation solution (the serum is diluted 50 times with the incubation solution) to the 14 blocks, place on a side-shaking incubator, and incubate overnight at 4°C; 4- Cleaning: Discard the incubation solution, quickly add the cleaning solution, then discard the cleaning solution again. Repeat this cycle several times. Remove the enclosure, place the container on a horizontal shaker, and clean the container three times at room temperature, 10 minutes each time. 5. Secondary antibody incubation: Place on a side-swinging shaker and incubate at room temperature for 1 hour with the secondary antibody incubation solution (the secondary antibody is diluted 1000 times with the incubation solution). (From this step onwards, be careful to avoid light.) 6- Cleaning: Place on a horizontal shaker and clean with cleaning solution at room temperature 3 times, 10 min each time. After completion, clean with ddH2O 2 times, 10 min each time. 7-Drying; 8. Scanning: Operate according to the scanner's operating procedures and user manual; 9. Data Extraction: Obtain raw data using GenePix Pro v6.0 software.

[0041] 4) Quantitative analysis of test data: 1-Microarray detection: Protein microarray detection was performed on serum samples from 54 healthy controls and 90 liver cancer patients. The microarray scanning results were then archived for subsequent quantitative analysis of autoantibody data. 2-Data Quantification: The original data was obtained through GenePix Pro v6.0 software. The median value of the foreground signal value in the 532nm channel was used to represent the signal value F532 of the risk factor; the median value of the background value in the 532nm channel was used to represent the background value B532 of the risk factor. The ratio of the foreground value to the background value of each protein, i.e., F / B, was defined as the signal-to-noise ratio (SNR) of the risk factor; 5) Detection Signal Evaluation Parameters: 1-Risk Assessment Early Warning Variables, including the signal risk value of the risk factor and the number of risk factors: According to the signal value range of SNR, risk assignment was performed, and the risk value was used as the evaluation variable of the protein chip risk assessment system (as Figure 5 shown). Risk Factor Signal Risk Value Conversion Scale: Signal-to-noise ratio (SNR) ≤ 2, risk value (RF) was assigned 0.1; 2 < SNR ≤ 5, RF was assigned 1; 5 < SNR ≤ 10, RF was assigned 2; SNR > 10, RF was assigned 3. In addition, according to the positive SNR signal value, the number of risk factors was defined as a risk assessment variable. Risk Factor Quantity Counting Rule: Risk factors with SNR ≥ 2 were counted; risk factors with SNR < 2 were not counted. The reference normal dynamic range of the risk factor risk value was designed to be between 0 and 1.

[0042] 2-Sensitivity and Specificity of the Evaluation Parameters for Distinguishing Healthy Populations and Hepatocellular Carcinoma Populations: The number of values of risk factors with SNR ≥ 2 was statistically analyzed, and at the same time, statistical analysis was performed based on the total risk value of the risk factors. The correlations between the hepatocellular carcinoma group and the healthy group were compared. The results showed that the number of risk factors with signals in the hepatocellular carcinoma group was significantly greater than that in the healthy population, and the total risk value of the risk factors in the hepatocellular carcinoma group was significantly greater than that in the healthy population. Further, the specificity and sensitivity of the number of risk factors and the risk factor value for the risk of hepatocellular carcinoma occurrence were evaluated through the ROC curve. The results showed that the AUC value of the number of risk factors ROC was as high as 0.988, and the AUC of the total risk value of the risk factors was as high as 0.994 (as Figure 6 shown). These indicate that the number of risk factors and the total risk value of the risk factors can be used to warn of the risk of hepatocellular carcinoma occurrence.

[0043] 3-Hepatocellular Carcinoma Occurrence Risk Early Warning Assessment System: Early Warning Assessment of the First Detection of Hepatocellular Carcinoma Occurrence Risk: Based on the detection data of healthy populations and hepatocellular carcinoma populations, the total risk number and total risk value of risk factors for healthy populations and hepatocellular carcinoma patients were statistically analyzed, and an early warning assessment system for the first detection of hepatocellular carcinoma occurrence risk was established (as Figure 7 shown), specifically as follows: Low risk: Risk factors with a total number of risks ≤ 5, a total risk value Σ ≤ 15, and no risk factors with risk values ​​of 2 and 3 are defined as low risk. Medium risk: Risk factors with a total number of risks ≤ 9, and a small number of risk factors with risk values ​​of 2 and 3, are defined as medium risk; High risk: Risk factors with a total number of risks ≥ 9, a total risk value Σ ≥ 20, and a large number of risk factors with risk values ​​of 2 and 3 are defined as high risk.

[0044] The initial warning system states: The results of this protein chip test are prospective and preliminary, serving as an alert for the emergence and potential development of diseases. They do not imply a specific diagnosis for the individual. This protein chip can simultaneously detect multiple liver cancer-related indicators, enabling efficient and accurate disease risk assessment and subsequent intervention for sub-health conditions. However, the risk detection results cannot replace a doctor's diagnosis. Individuals can submit their results to health management experts for supplementary reference in subsequent examinations.

[0045] 4. Continuous tracking, assessment, and early warning of liver cancer risk: Due to significant individual differences, a single test cannot comprehensively assess disease risk and overall health. Annual testing is recommended to help establish a true individual autoantibody risk warning database (e.g., ...). Figure 8 (As shown). A heatmap is plotted using SNR values ​​to show the baseline differences in individual antibody levels, facilitating intuitive comparison and tracking.

[0046] The tracking and early warning system is explained as follows: Each test is based on the previous baseline data. By comparing and analyzing the fingerprint profiles of autoantibody risk factors from previous physical examinations, and comparing them with disease protein databases, it is possible to detect subtle changes in the early stages of many diseases in a timely manner. This allows for the detection of small lesions that are already developing or cannot be detected by routine hospital examinations 3-5 years in advance, ultimately achieving the goal of early detection, early diagnosis, and early treatment.

[0047] It is evident that protein chips for liver cancer early warning have the following characteristics: 1. Convenient sampling: Peripheral blood samples are easy to collect and require only a small amount, 0.1-0.5 mL each time; 2. High throughput: It can detect 61 antibody risk factors in a single run. Compared to existing protein chips used for disease diagnosis, which contain over 20,000 proteins, have numerous indicators, and are prone to missed diagnoses, making standardization and precision difficult, this protein chip is not suitable for direct use in early warning systems. In contrast, this chip has fewer indicators, acquires only 61 antibody risk factors, offers high accuracy, and has a moderate detection volume, avoiding excessively high testing costs and meeting the requirements for early warning systems. 3. Short testing time: The entire testing project can be completed within 1-2 days; 4. Standardization of the testing system: standardization of protein raw materials, standardization of protein chip preparation, standardization of testing procedures, standardization of data analysis, and standardization of test reports; 5. Personalized Risk Factor Tracking: Continuously track individual baselines, monitor and warn of baseline dynamics, and provide timely warnings of the risk of cancerous transformation of liver nodules in the population due to immune abnormalities.

[0048] The above description is merely a preferred embodiment of this application and an explanation of the technical principles and solutions employed. Furthermore, the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above-described features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A protein chip for early warning of liver cancer, characterized in that, The antigen set includes antigens; the corresponding antibodies for the antigens are: GNA11, GNAS, IDH1, MSH2, NPM1, PAX5, PTCH1, PTEN, SRSF2, Survivin, CAST, PAIP1, PRKCZ, RAD23A, RUNX1T1, SARS, TP53, APEX2, RCSD1, AIF1, CDKN1B, EpCAM, MMP9, 14-3-3zeta, Bmi-1, c-myc, CyclinB1, ET-1, FGF.

21. At least one of HCC1, HDGF2, IL-17A, IL-26, LECT2, p62, p90, RhoA, SOD1, STMN4, SUMO-4, TXN, UBE2C, VCC1, ADCK5, ANKRD13D, DCAF4L2, RPLP0, TSPAN13, WTAP, ASAH1, CIAPIN1, EGFR, MAS1, SLC44A3, UBL7, ZNF428, CDC37L1, MAP3K14, PCNA, ZIC2, and AFP.

2. The protein chip for liver cancer early warning as described in claim 1, characterized in that, The antibodies are: GNA11, GNAS, IDH1, MSH2, NPM1, PAX5, PTCH1, PTEN, SRSF2, Survivin, CAST, PAIP1, PRKCZ, RAD23A, RUNX1T1, SARS, TP53, APEX2, RCSD1, AIF1, CDKN1B, EpCAM, MMP9, 14-3-3zeta, Bmi-1, c-myc, CyclinB1, ET-1, FGF21, HCC1. , HDGF2, IL-17A, IL-26, LECT2, p62, p90, RhoA, SOD1, STMN4, SUMO-4, TXN, UBE2C, VCC1, ADCK5, ANKRD13D, DCAF4L2 , RPLP0, TSPAN13, WTAP, ASAH1, CIAPIN1, EGFR, MAS1, SLC44A3, UBL7, ZNF428, CDC37L1, MAP3K14, PCNA, ZIC2 and AFP.

3. The protein chip for liver cancer early warning as described in claim 1, characterized in that, The antibody is derived from human.

4. The protein chip for liver cancer early warning as described in claim 4, characterized in that, The protein chip also includes a control system.

5. The protein chip for liver cancer early warning as described in claim 1, characterized in that, The control system includes IgG, IgM, BSA, GST tag, His Tag, biotin, and buffer.

6. The protein chip for liver cancer early warning as described in claim 1, characterized in that, The protein chip also includes at least one of the markers Cy3 and Cy5.

7. An application of a protein chip for early warning of liver cancer, characterized in that, The protein chip as described in claim 1 is used for early warning assessment of liver cancer.

8. The application of the protein chip for liver cancer early warning as described in claim 7, characterized in that, The steps of the early warning assessment include: collecting serum from the population to be tested, using a protein chip to detect the serum to obtain test data, and then determining whether they belong to a low-risk, medium-risk, or high-risk population based on a risk warning model.

9. The application of the protein chip for liver cancer early warning as described in claim 8, characterized in that, When the total number of antibody risk numbers ≤ 5, the total antibody risk value Σ ≤ 15, and there are no antibodies with risk values of 2 and 3, it is defined as low risk; when the total number of antibody risk numbers ≤ 9, and there are a small number of antibodies with risk values of 2 and 3, it is defined as medium risk; When the total number of antibody risk numbers ≥ 9, the total antibody risk value Σ ≥ 20, and there are a relatively large number of antibodies with risk values of 2 and 3, it is defined as high risk.

10. The application of the protein chip for liver cancer early warning as described in claim 9, characterized in that, The median value of the signal foreground value under the 532nm channel is used to represent the signal value F532 of the antibody; the median value of the background value under the 532nm channel is used to represent the background value B532 of the antibody; the ratio of the foreground value to the background value of each protein, that is, F532 / B532, is defined as the signal-to-noise ratio SNR of the antibody; Risk value: When SNR ≤ 2, the risk value is assigned 0.1; when 2 < SNR ≤ 5, the risk value is assigned 1; when 5 < SNR ≤ 10, the risk value is assigned 2; when SNR > 10, the risk value is assigned 3; Total number of antibody risk numbers: Antibodies with SNR ≥ 2 are counted; antibodies with SNR < 2 are not counted; the reference normal dynamic range of the antibody risk value is designed to be between 0 and 1.

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