阅读说明：本技术 一种用于食管鳞癌早期筛查和诊断的血清蛋白标志物、试剂盒及检测方法 (Serum protein marker, kit and detection method for early screening and diagnosis of esophageal squamous carcinoma ) 是由 张建营 王鹏 叶华 代丽萍 史健翔 王晓 孙桂英 姜国忠 赵志华 于 2019-10-23 设计创作，主要内容包括：本发明公开一种用于食管鳞癌早期筛查和诊断的血清蛋白标志物,属于生物医学技术领域,血清蛋白标志物为P53、GNA11、GNAS、PTEN、ACVR1B、FBXW7、EGFR、PDGFRA、SRSF2、MEN1、DAXX或CASP8基因编码的蛋白中的任意一种或两种以上的联合。本发明基于癌症驱动基因在肿瘤发生和发展中所起的作用,定制138个癌症驱动基因编码的人类蛋白质芯片,共包含180个人源重组蛋白质,用以筛选潜在的可用于诊断或其他表征癌症的标志物,先通过蛋白质芯片初步筛选出食管鳞癌的早期检测血清标志物,再经过ELISA间接法实验进行验证,最终筛选出可用于食管鳞癌早期筛查和诊断的一组食管鳞癌联合血清蛋白标志物,用于辅助食管鳞癌的临床诊断,具有较好的参考价值。(The invention discloses a serum protein marker for early screening and diagnosis of esophageal squamous carcinoma, which belongs to the technical field of biomedicine, and the serum protein marker is any one or combination of more than two of proteins encoded by P53, GNA11, GNAS, PTEN, ACVR1B, FBXW7, EGFR, PDGFRA, SRSF2, MEN1, DAXX or CASP8 genes. Based on the role of the cancer driver genes in the occurrence and development of tumors, the invention customizes 138 human protein chips coded by the cancer driver genes, which contain 180 human source recombinant proteins in total and are used for screening potential markers capable of being used for diagnosis or other characterization of cancers, firstly, early detection serum markers of esophageal squamous cell carcinoma are preliminarily screened out through the protein chips, then, the early detection serum markers are verified through ELISA indirect method experiments, and finally, a group of esophageal squamous cell carcinoma combined serum protein markers capable of being used for early screening and diagnosis of esophageal squamous cell carcinoma are screened out for assisting clinical diagnosis of esophageal squamous cell carcinoma, so that the invention has better reference value.)

1. A serum protein marker for early screening and diagnosis of esophageal squamous carcinoma, which is characterized in that: the serum protein marker is any one or combination of more than two of proteins encoded by P53, GNA11, GNAS, PTEN, ACVR1B, FBXW7, EGFR, PDGFRA, SRSF2, MEN1, DAXX or CASP8 genes;

the protein coded by the P53 gene has an amino acid sequence shown in SEQ ID NO. 1;

the protein coded by the GNA11 gene has an amino acid sequence shown in SEQ ID NO. 2;

the protein coded by the GNAS gene has an amino acid sequence shown as SEQ ID NO. 3;

the protein coded by the PTEN gene has an amino acid sequence shown as SEQ ID NO. 4;

the protein coded by the ACVR1B gene has an amino acid sequence shown in SEQ ID NO. 5;

the protein coded by the FBXW7 gene has an amino acid sequence shown as SEQ ID NO. 6;

the protein coded by the EGFR gene has an amino acid sequence shown as SEQ ID NO. 7;

the PDGFRA gene coded protein has an amino acid sequence shown as SEQ ID NO. 8;

the protein coded by the SRSF2 gene has an amino acid sequence shown in SEQ ID NO. 9;

the protein coded by the MEN1 gene has an amino acid sequence shown in SEQ ID NO. 10;

the protein coded by the DAXX gene has an amino acid sequence shown in SEQ ID NO. 11;

the protein coded by the CASP8 gene has an amino acid sequence shown as SEQ ID NO. 12.

2. The serum protein marker for early screening and diagnosis of esophageal squamous carcinoma according to claim 1, wherein: the serum protein marker is any one or combination of more than two of proteins encoded by P53, GNA11, GNAS, EGFR, SRSF2, MEN1 or DAXX genes.

3. The serum protein marker for early screening and diagnosis of esophageal squamous carcinoma according to claim 2, wherein: the serum protein markers are combinations of proteins encoded by the P53, GNA11, GNAs, EGFR, SRSF2, MEN1, and DAXX genes.

4. A kit, characterized in that: comprising the serum protein marker for early screening and diagnosis of esophageal squamous carcinoma according to any one of claims 1-3.

5. The kit of claim 4, wherein: the serum protein marker is coated on a solid phase carrier.

6. The kit of claim 5, wherein: the solid phase carrier is made of polyvinyl chloride, polystyrene, polyacrylamide or cellulose.

7. The kit of any one of claims 4 to 6, wherein: the kit also comprises any one or the combination of more than two of positive control serum, negative control serum, confining liquid, sample diluent, a second antibody, second antibody diluent, washing liquid, developing liquid or stopping liquid.

8. The detection method using the serum protein marker for early screening and diagnosis of esophageal squamous carcinoma according to claim 3, characterized in that: the method comprises the following steps:

1) coating and sealing each serum protein marker, and then cleaning;

2) performing primary antibody incubation and cleaning with the diluted serum to be detected, and performing secondary antibody incubation and cleaning;

3) stopping the reaction after the color development of the color development system, and measuring the absorbance value;

4) by OD₄₅₀-OD₆₂₀The relative OD value is obtained, then the blank contrast is deducted, the absorbance value is substituted into the following formula to calculate the predicted probability P value,

P＝1/(1+Exp(7.412-6.101×OD_P53-12.763×OD_GNA11-10.307×OD_GNAS-11.469×OD_EGFR-10.270×OD_SRSF2+5.737×OD_MEN1+14.533×OD_DAXX))；

OD in the formula_P53、OD_GNA11、OD_GNAS、OD_EGFR、OD_PTEN、OD_SRSF2、OD_MEN1、OD_DAXXThe relative OD value of each serum protein marker is subtracted by the absorbance value of a blank control;

when the P value is more than or equal to 0.5, the esophageal squamous carcinoma sample is preliminarily judged;

and when the P value is less than 0.5, the sample is preliminarily judged to be a normal sample.

9. The detection method using serum protein markers for early screening and diagnosis of esophageal squamous carcinoma according to claim 8, characterized in that: further comprising the step 5) of calculating the positive rate, the sensitivity specificity, the john index, the positive predictive value, the negative predictive value, the positive likelihood ratio and the negative likelihood ratio of each serum protein marker;

step 6), parallel joint detection: and (3) constructing a parallel joint detection model of the serum protein marker by using a logistic regression model, and calculating a joint diagnosis result.

Technical Field

The invention belongs to the technical field of biomedicine.

Background

Esophageal Cancer (EC) is one of the common digestive tract malignant tumors of human beings, and is mainly divided into esophageal squamous cell carcinoma (esophageal squamous carcinoma) and esophageal adenocarcinoma from the tissue type, and the latest global tumor prevalence data (Globocan 2018) published by the international cancer institute under the world health organization show that 57.2 ten thousand cases of global esophageal cancer in 2018 are located at the 7 th position of malignant tumor; 50.9 ten thousand cases of esophageal cancer death in the same period are positioned at the 6 th position of malignant tumor death, and the incidence rates of esophageal cancer in different regions of the world can differ by 2-3 times. China is one of five regions with the highest incidence of esophageal cancer worldwide, about 53% of new esophageal cancer cases in the world are Chinese, and according to the latest tumor statistical data, the incidence of esophageal cancer in China is 6 th of malignant tumor incidence, the mortality is 4 th of malignant tumor death cause, 24.6 ten thousand of new esophageal cancer cases and 18.8 ten thousand of esophageal cancer death causes exist in 2015, wherein more than 80% of esophageal cancer patients are esophageal squamous carcinoma patients. The early stage of the esophageal cancer is mostly hidden, the symptoms are more and less typical or asymptomatic, and the esophageal cancer is easy to ignore, and when the typical symptoms such as progressive dysphagia appear in a patient, the cancer is usually in the middle and late stages, and the optimal treatment time is lost.

At present, operations, radiotherapy and chemotherapy are common treatment means for tumors including esophageal cancer, for early esophageal cancer patients, a radical treatment mode mainly comprising operations and preoperative or postoperative chemoradiotherapy is mainly adopted, and for middle and late esophageal cancer patients losing the time of operation treatment, a radiotherapy or chemotherapy treatment mode is mainly adopted. The esophageal cancer detection method widely applied in clinic at present is upper gastrointestinal endoscopy, but endoscopy has the characteristic of invasiveness due to operation, has certain pain, and lacks compliance and possibility of popularization and application; and the esophagus X-ray barium meal examination and the CT image scanning are not suitable for screening in large-scale population due to respective limitations. Based on the defects of the current clinical esophageal cancer screening method, if esophageal cancer markers with ideal sensitivity and specificity can be found, and the detection means is low in price and convenient for screening high risk groups, huge social values can be created, medical resources and economic values can be saved, and the survival rate and the survival time of patients can be improved.

In the field of esophageal cancer biomarkers, numerous scholars at home and abroad have already conducted a lot of research and exploration, and some traditional tumor markers are also commonly used clinically at present for diagnosing esophageal cancer, such as carcinoembryonic antigen, cancer antigen 125, cancer antigen 199 and the like, but the tumor markers have low sensitivity and specificity in esophageal cancer diagnosis, and particularly have a limited diagnostic value for early esophageal cancer patients. In recent years, in the field of human oncology, it has been found that the serum of cancer patients contains a unique set of cellular proteins that induce autoantibody responses, called tumor-associated antigens (TAAs), and the antibodies that they induce are called anti-TAA antibodies (autoantibodies). The proposal of the concept guides a new direction for the early diagnosis research of the esophageal cancer. Studies have shown that there are different classes of anti-TAAs autoantibodies in the serum of patients with esophageal cancer, probably as a result of an immune response against certain intracellular antigens during the process of esophageal epithelial cell carcinogenesis. Many studies also provide basis and feasibility for the anti-TAAs autoantibodies for early diagnosis of esophageal cancer, but based on the complexity of the tumorigenesis process, the single diagnosis index has relatively poor capability for tumor diagnosis, and generally cannot meet the clinical tumor diagnosis requirement, for example, studies show that the frequency of the single anti-TAAs autoantibody appearing in the serum of esophageal cancer patients is very low, generally not more than 20%, and the application of the single anti-TAA autoantibody in tumor diagnosis is limited. Researches show that the diagnosis of tumors is carried out by jointly using a group of carefully screened anti-TAAs autoantibody combinations, the tumor diagnosis specificity is ensured, and meanwhile, the sensitivity of tumor diagnosis is greatly improved.

Subsequent studies over a decade have attempted to find more sensitive and specific anti-TAA autoantibodies for the diagnosis of esophageal cancer, optimizing the combination for the diagnosis of esophageal cancer. There are two common methods for finding valuable TAA autoantibodies: one is serological screening of recombinant cDNA expression library (serological analysis of recombinant cDNA expressionlibraries, SEREX); the other is proteomics technology. In contrast to SEREX, proteomics technology enables screening of multiple tumor sera and enables screening of TAAs with post-translational modifications. During the development of tumors, hundreds of thousands of mutations of genes are involved, but only some key genes, called cancer driver genes, are mutated to cause the development of tumors. Studies suggest that different types of tumorigenesis generally contain 2-8 driver genes, and that mutations in these genes lead to preferential tumor growth, and that these genes can be divided into 12 signaling pathways by regulating the cell cycle, cell survival and genome to maintain 3 cell core processes. 138 cancer driver genes (see Vogelstein B. science. (2013)339(6127):1546-1558), including 74 cancer suppressor genes and 64 cancer genes, are currently found in a variety of tumor whole genome sequencing studies. The protein coded based on the cancer driving gene can also induce the body to generate corresponding autoantibodies in circulating blood of the body, and the research on the protein coded by the cancer driving gene and the autoantibodies in serum induced by the protein can reveal the occurrence, development or prognosis of tumors to a certain extent.

Disclosure of Invention

The invention aims to provide a serum protein marker for early screening and diagnosis of esophageal squamous carcinoma, and simultaneously provides a kit containing the serum protein marker for early screening and diagnosis of esophageal squamous carcinoma and a corresponding detection method.

Based on the purpose, the invention adopts the following technical scheme:

a serum protein marker for early screening and diagnosis of esophageal squamous carcinoma, the serum protein marker is any one or combination of more than two of proteins encoded by P53, GNA11, GNAS, PTEN, ACVR1B, FBXW7, EGFR, PDGFRA, SRSF2, MEN1, DAXX or CASP8 genes;

the protein coded by the P53 gene has an amino acid sequence shown in SEQ ID NO. 1;

the protein coded by the GNA11 gene has an amino acid sequence shown in SEQ ID NO. 2;

the protein coded by the GNAS gene has an amino acid sequence shown as SEQ ID NO. 3;

the protein coded by the PTEN gene has an amino acid sequence shown as SEQ ID NO. 4;

the protein coded by the ACVR1B gene has an amino acid sequence shown in SEQ ID NO. 5;

the protein coded by the FBXW7 gene has an amino acid sequence shown as SEQ ID NO. 6;

the protein coded by the EGFR gene has an amino acid sequence shown as SEQ ID NO. 7;

the PDGFRA gene coded protein has an amino acid sequence shown as SEQ ID NO. 8;

the protein coded by the SRSF2 gene has an amino acid sequence shown in SEQ ID NO. 9;

the protein coded by the MEN1 gene has an amino acid sequence shown in SEQ ID NO. 10;

the protein coded by the DAXX gene has an amino acid sequence shown in SEQ ID NO. 11;

the protein coded by the CASP8 gene has an amino acid sequence shown as SEQ ID NO. 12.

The serum protein marker is any one or combination of more than two of proteins encoded by P53, GNA11, GNAS, EGFR, SRSF2, MEN1 or DAXX genes.

The serum protein markers are combinations of proteins encoded by the P53, GNA11, GNAs, EGFR, SRSF2, MEN1, and DAXX genes.

A kit comprising serum protein markers for early screening and diagnosis of esophageal squamous carcinoma.

The serum protein marker is coated on a solid phase carrier.

The solid phase carrier is made of polyvinyl chloride, polystyrene, polyacrylamide or cellulose.

The kit also comprises any one or the combination of more than two of positive control serum, negative control serum, confining liquid, sample diluent, a second antibody, second antibody diluent, washing liquid, developing liquid or stopping liquid.

A detection method using a serum protein marker for early screening and diagnosis of esophageal squamous carcinoma, characterized in that: the method comprises the following steps:

1) coating and sealing each serum protein marker, and then cleaning;

2) performing primary antibody incubation and cleaning with the diluted serum to be detected, and performing secondary antibody incubation and cleaning;

3) stopping the reaction after the color development of the color development system, and measuring the absorbance value;

4) by OD₄₅₀-OD₆₂₀The relative OD value is obtained, then the blank contrast is deducted, the absorbance value is substituted into the following formula to calculate the predicted probability P value,

P＝1/(1+Exp(7.412-6.101×OD_P53-12.763×OD_GNA11-10.307×OD_GNAS-11.469×OD_EGFR-10.270×OD_SRSF2+5.737×OD_MEN1+14.533×OD_DAXX))；

OD in the formula_P53、OD_GNA11、OD_GNAS、OD_EGFR、OD_PTEN、OD_SRSF2、OD_MEN1、OD_DAXXThe relative OD value of each serum protein marker is subtracted by the absorbance value of a blank control;

when the P value is more than or equal to 0.5, the esophageal squamous carcinoma sample is preliminarily judged;

and when the P value is less than 0.5, the sample is preliminarily judged to be a normal sample.

Further comprising the step 5) of calculating the positive rate, the sensitivity specificity, the john index, the positive predictive value, the negative predictive value, the positive likelihood ratio and the negative likelihood ratio of each serum protein marker;

step 6), parallel joint detection: and (3) constructing a parallel joint detection model of the serum protein marker by using a logistic regression model, and calculating a joint diagnosis result.

Compared with the prior art, the invention has the following beneficial effects:

1) the invention is based on the role of cancer driving gene in the generation and development of tumor, customizes 138 human protein chips coded by cancer driving gene, totally comprises 180 human source recombinant proteins, the screening method is used for screening potential markers which can be used for diagnosing or other characterizing cancers, firstly, early detection serum markers of the esophageal squamous carcinoma are preliminarily screened out through a protein chip, then, ELISA indirect method experiments are carried out for verification, and finally, a group of esophageal squamous carcinoma serum protein markers which can be used for early screening and diagnosing the esophageal squamous carcinoma, in particular the combination of proteins coded by P53, GNA11, GNAS, EGFR, SRSF2, MEN1 and DAXX genes, is screened out, when the area under the ROC curve of the combined diagnosis of the esophageal squamous cell carcinoma reaches 0.85 and 95% CI is 0.77-0.92, and the specificity is ensured to be 90.0%, the sensitivity is 56.3%, the consistency rate reaches 79.1%, and the kit is used for assisting the clinical diagnosis of esophageal squamous cell carcinoma and has a better reference value;

2) the detection method has the characteristics of high sensitivity, strong specificity, low cost and the like, is simple and quick to operate, and provides a basis for early diagnosis of the esophageal squamous cell carcinoma.

Drawings

FIG. 1 is a schematic diagram of the detection of a focused array-based human protein chip in an experimental example;

FIGS. 2-1 and 2-2 are ROC curve analysis charts of 12 TAAs screened by the protein chip in the experimental example for individual diagnosis of esophageal squamous cell carcinoma;

FIG. 3 is a scattergram of SNR values of 12 TAAs selected by the protein chip in the experimental example;

FIG. 4 is a schematic diagram of indirect ELISA detection in an experimental example;

FIGS. 5-1 and 5-2 are ROC curve analysis graphs of ELISA verified 12 TAAs for diagnosing esophageal squamous cell carcinoma alone in experimental examples;

FIG. 6 is a graph showing the distribution of OD value scatter of 12 TAAs verified by ELISA in the experimental examples;

FIG. 7 is a ROC graph of the training set data for ELISA verification of 7 TAAs combined diagnosis of esophageal squamous carcinoma in experimental examples;

FIG. 8 is a ROC graph of the data in the validation set of the ELISA validated 7 TAAs for the combined diagnosis of esophageal squamous carcinoma in the experimental examples.

Detailed Description

Examples of the experiments

1 preparation of serum samples

1.1 serum samples for protein chip experiments

Primary esophageal squamous carcinoma patients (esophageal squamous carcinoma pathologically diagnosed) were collected at the beijing youan hospital and the first subsidiary hospital of zhengzhou university, with patient consent and approval by the institutional review board and the hospital ethics committee. All samples are collected by a red blood collection tube for 5-10 mL of whole blood of a research object, the whole blood is placed at room temperature for 2 hours, then the sample is centrifuged at 1000Xg for 15 minutes, supernatant is taken, each sample is subpackaged with a plurality of samples, labels are attached to the samples, and the samples are stored in a low-temperature refrigerator at minus 80 ℃ so as to avoid repeated freeze thawing.

According to epidemiological analysis, 86 primary esophageal squamous carcinoma sera and 50 normal control sera from YouAn hospital for the same period of physical examination were finally collected in the study for primary chip screening. Of the 86 patients with primary esophageal squamous carcinoma, 50 (58.1%) men and 36 (41.9%) women had a mean age of 64 + -8 years and an age range of 44-88 years; in the 50 cases of normal serum, there were 23 (46.0%) cases of males and 27 (54.0%) cases of females, with the average age of 40 ± 13 years and the age range of 20-71 years. All esophageal squamous carcinoma patient sera were collected when the patient was initially diagnosed with esophageal squamous carcinoma who had not received any chemoradiotherapy and surgery, and were diagnosed between 4 months 2015 and 2016-5 months. The normal human serum is from the physical examination population participating in the annual health physical examination and free of any malignant tumor symptoms.

1.2 serum samples for experimental validation of ELISA Indirect methods

(1) Serum samples were collected from the Beijing Youran Hospital and the first subsidiary Hospital of Zhengzhou university (see section 1.1 above for details).

(2) From the first subsidiary hospital of Zhengzhou university and tumor hospital of Henan province (190 new cases of esophageal squamous carcinoma) and cardiovascular survey program of Jinshui district of Zhengzhou city (190 normal persons), among 190 new cases of esophageal squamous carcinoma, 133 (70%) cases were shared among 190 cases of new cases of esophageal squamous carcinoma, 57 (30%) cases were shared among women, the average age was 64 + -8 years, and the age range was 41-87 years; in 190 normal sera, 133 (70%) male and 57 (30%) female were observed, with the mean age of 64 + -9 years and the age range of 40-88 years. All esophageal squamous carcinoma patient sera were diagnosed at 2015 4 months to 2016 5 months when the patient was initially diagnosed with esophageal squamous carcinoma who had not received any chemoradiotherapy. The normal human serum is from the physical examination population participating in the annual health physical examination and free of any malignant tumor symptoms.

2 protein chip customization for screening esophageal squamous carcinoma diagnosis marker

Proteins (180 total human recombinant proteins) encoded by 138 cancer driver genes (see Vogelstein B. science. (2013)339(6127):1546-1558) were immobilized on protein chips for screening of tumor markers. The protein chip for screening tumor markers was a HuProtTM human protein chip custom-made by Biotech, Inc. of Bo Chong, Guangzhou.

3 protein chip experiment

See figure 1 for experimental principles.

3.1 reagents required for the experiment:

1) sealing liquid: 3mL of 10% BSA, 7mL of 1 XPBS solution, mixed well and placed on ice.

2) Serum incubation liquid: 1mL of 10% BSA was added to 9mL of 1 XPBST solution, mixed well and placed on ice.

3) Cleaning solution: 1 XPBST solution, stored in a refrigerator at 4 ℃.

4) Secondary antibody incubation solution: including a fluorescently-labeled anti-human IgM secondary antibody (cy 5-labeled, appearing red) and a fluorescently-labeled anti-human IgG secondary antibody (cy 3-labeled, appearing green).

3.2 specific Experimental procedures for protein chips

(a) Rewarming: taking out the chip from a refrigerator at-80 deg.C, re-warming in a refrigerator at 4 deg.C for half an hour, and re-warming at room temperature for 15 min.

(b) And (3) sealing: and fixing the rewarming chip in 14blocks in a fence, adding sealing liquid into each block, placing the blocks on a side swing shaking bed, and sealing for 3 hours at room temperature.

(c) Incubation of serum samples: after the blocking was completed, the blocking solution was poured out, then the prepared serum incubation solution was added quickly, 14 samples were incubated per chip (samples were frozen and thawed in a 4 ℃ chromatography cabinet and diluted 1:50 with 1% BSA in 1 XPBST solution), the loading volume of each serum sample was 200. mu.L, the shaking table was set aside at 20rpm, and the incubation was carried out overnight at 4 ℃.

(d) Cleaning: taking out the chip and the chip clamp together, sucking out the sample, then quickly adding an equal volume of 1 XPBST solution, and circulating for a plurality of times to ensure that no cross contamination exists among the serum samples when the chip clamp is detached. After the chip clamp was removed, the chip was placed in a chip washing cassette containing washing solution, and washed on a horizontal shaker at room temperature at 80rpm for 3 times, each time for 10 min.

(e) And (3) secondary antibody incubation: the chip was transferred to an incubation box containing 3mL of secondary antibody incubation solution, and the shaking table was shaken laterally at 40rpm, protected from light, and left at room temperature for 60 min.

(f) Cleaning: the chip was removed (note that the upper surface of the chip was not touched or scratched), placed in a chip washing cassette to which a washing solution was added, and washed 3 times at 80rpm for 10min each time on a horizontal shaker. After completion with ddH₂O washing for 10min 2 times.

(g) And (3) drying: the chip is placed in a chip drier for centrifugal drying.

(h) Scanning: operating according to the operating specifications and instructions of the scanner.

(i) Data extraction: and aligning the chip image and each array of the result as a whole, pressing an automatic alignment button, and extracting and storing data.

(j) And carrying out data preprocessing.

(k) And (3) carrying out data analysis to obtain a final esophageal squamous carcinoma serum marker, and screening the following serum protein markers by the protein chip experiment: the proteins encoded by the cancer drivers P53, GNA11, GNAS, PTEN, ACVR1B, FBXW7, EGFR, PDGFRA, SRSF2, MEN1, DAXX and CASP8 (FIGS. 2-1, 2-2 are ROC curve analysis charts of 12 TAAs screened by the above protein chip for diagnosing esophageal squamous carcinoma alone, and ROC curves of proteins encoded by P53, GNA11, GNAS, PTEN, ACVR1B, FBXW7, EGFR, PDGFRA, SRSF2, MEN1, DAXX and CASP8 in order of 1-1, 2-2; FIG. 3 is a dot plot value dispersion of the above 12 TAAs, and N in FIG. 3 is Normal, E is a healthy, Esophagagealsquamomous cell carcinos, instant phosphorus tube cancer case). Wherein, proteins coded by the genes P53, GNA11, GNAS, PTEN, ACVR1B, FBXW7, EGFR, PDGFRA, SRSF2, MEN1, DAXX and CASP8 sequentially have amino acid sequences shown in SEQ ID NO. 1-12. 4ELISA Indirect method experimental verification

See figure 4 for experimental principles.

The specific experimental steps are as follows:

a) coating: coating was performed at the concentrations in Table 1 at 100. mu.L/well overnight at 4 ℃.

b) And (3) sealing: a1 XPBST solution (PBS, Tween20 Solebao, Beijing) of 2% BSA (Solebao, Beijing, analytical grade) was applied at 200. mu.L/well overnight at 4 ℃.

c) Cleaning: wash 3 times with 350 μ L/well 1 × PBST solution.

d) Primary antibody incubation: after the serum was diluted 1:100 volume ratio with 1% BSA in 1 XPBST solution, 100. mu.L/well was placed in a 37 ℃ half water bath for 1 h.

e) Cleaning: wash 5 times with 350 μ L/well 1 × PBST solution.

f) And (3) secondary antibody incubation: HRP-labeled mouse anti-human IgG (Olympic, Wuhan) was diluted 1:10000 in 1 XPBST solution containing 1% BSA and then treated with 100. mu.L/well in a half-water bath at 37 ℃ for 1 h.

g) Cleaning: wash 5 times with 350 μ L/well 1 × PBST solution.

h) Color development: TMB color development System, solution A (200 mgTMB.2HCl in 1L deionized water, Solebao, Beijing, analytical grade) and solution B (9.2g citric acid, 37g Na)₂HPO₄·12H₂O and 8ml of 0.75% H₂O₂Dissolved in 1L of deionized water) and mixed according to the volume ratio of 1:1, 100 mu L/hole are carried out at room temperatureAnd (4) irradiating to reach the expected color (about 5-15 min).

i) And (4) terminating: absorbance was measured within 10min after 50. mu.L/well of 10% concentrated sulfuric acid.

j) Measuring the absorbance: by OD₄₅₀-OD₆₂₀For relative OD values, the blank control was then subtracted, and IgG was normalized and then subjected to subsequent data processing (details of data processing are shown in "5 data processing" sections b) -d) described below).

The coating concentrations of the 12 TAAs screened by the protein chip experiment when the 12 TAAs are subjected to ELISA experiment verification are shown in table 1 below, and the arrangement table of the 96-well plate of the ELISA experiment is shown in table 2 below. In table 2, the positive quality control refers to serum with a higher OD value of the ELISA experiment and positive corresponding antibody through Western Blot experiment verification, the negative quality control refers to serum with an OD value near the mean value of the ELISA experiment in normal control population and negative through Western Blot verification, the blank is serum diluent, IgG 1-IgG 8 are human IgG antibodies diluted in a gradient manner, and the concentrations are 10, 20, 50, 100, 150, 200, 250 and 300ng/ml in sequence.

Coating concentrations of each of the 112 TAAs in Table

Table 2 96-well plate arrangement for ELISA experiments

The experimental results are as follows: 12 TAAs were detected by ELISA-mediated assay, and the results are shown in FIGS. 5-1, 5-2 and 6. FIGS. 5-1 and 5-2 are ROC curve analysis graphs of 12 TAAs for independent diagnosis of esophageal squamous carcinoma in ELISA validation experiments, and in the graphs, 1-12 are ROC curves of proteins encoded by P53, GNA11, GNAS, PTEN, ACVR1B, FBXW7, EGFR, PDGFRA, SRSF2, MEN1, DAXX and CASP8 for independent diagnosis of esophageal squamous carcinoma; FIG. 6 is a graph showing the distribution of OD value scatter over 12 TAAs in ELISA validation experiments, where N is Normal, i.e., healthy Normal serum, and E is Ehalophagalsquamous cell carcinoma, i.e., ductal phosphocarcinoma case.

As can be seen from FIGS. 5-1 and 5-2, the area under the ROC curve of the single index diagnosis of esophageal squamous cell carcinoma is 0.51-0.70, and the sensitivity range is 13.1% -35.3% when the lowest specificity is ensured to be 90%. Wherein, the area under the curve of GNA11 is maximum, which is 0.70, the sensitivity reaches 33.7%, and the specificity is 90.5%; the area under the ROC curve of PTEN is 0.67, the sensitivity reaches 26.8 percent, and the specificity is 91.6 percent; MEN1 has the smallest area under the ROC curve of 0.51, sensitivity of 16.8% and specificity of 90.5%. As can be seen from FIG. 6, the OD values of the 12 indexes are distributed between 0 and 1, and the OD values of the median are substantially distributed between 0.2 and 0.4. 5 data processing

The differential expression protein is screened out by using the focused array human protein chip in an esophageal squamous carcinoma group and an NC normal control group through statistical data analysis, and the specific method is as follows:

(1) the initial screening result of the chip is obtained through Focused Array protein chip experiment.

(2) And (3) stability analysis: in the experimental process, the test samples test are repeated according to different time, different chips and different positions so as to evaluate the stability of different chips at different time.

(3) Data analysis and results: samples after high background and extreme sample interference were rejected and 180 proteins of each of the IgG and IgM response types were subjected to consistent statistical analysis with the following analysis logic:

a) in order to eliminate the situation of signal nonuniformity caused by inconsistent background values among different protein points in the same chip, the background normalization method is used for processing, the ratio of the foreground value to the background value of each protein, namely F/B, is realized, SNR (signal to noise ratio), namely the mean value of the F/B of two repeated proteins, is defined on the basis, and subsequent statistical analysis is carried out.

b) Assuming that samples needing to be aligned are respectively from two identical populations, and whether the two groups of variances needing to be aligned are homogeneous is determined through an F test, then the F test result is selected to correspond to a t test, and the t test result is characterized by P-value. By definition, when p-value <0.05, the original hypothesis is rejected, i.e. there is a significant difference between the two.

c) For any protein, fold change, which is the difference between the cancer group and the normal group, was calculated to indicate the difference between the two groups.

d) For any protein, according to the diagnostic significance of the two groups compared, firstly, defining cutoff-1.5 as a positive judgment threshold, namely, when the SNR of a sample on the protein is more than or equal to 1.5, the protein is a positive protein; then, based on the control group, setting a proper cutoff threshold, calculating the difference of the positive rate of the cancer group and the control group at the cutoff threshold, and taking the maximum difference as the positive rate of the protein in the compared cancer group to search the high response protein specific to the control group in the cancer group, and finally, defining that the positive rate is not lower than 15%.

e) Based on the logic, the esophageal squamous carcinoma group (86 primary esophageal squamous carcinoma patient sera collected from the first subsidiary hospital of Beijing Youran Hospital and Zheng State university) and the Youran control group (50 normal sera of Youran Hospital) are compared, differential proteins which are obviously higher than the control group in the esophageal squamous carcinoma group are screened out to serve as esophageal squamous carcinoma candidate markers, and finally 12 serum protein markers (P53, GNA11, GNAS, PTEN, ACVR1B, FBXW7, EGFR, PDGFRA, SRSF2, MEN1, DAXX and CASP8) are selected through a chip to evaluate the diagnostic value of the esophageal squamous carcinoma. Wherein, the protein coded by the P53 gene has an amino acid sequence shown as SEQ ID NO.1, the protein coded by the GNA11 gene has an amino acid sequence shown as SEQ ID NO.2, the protein coded by the GNAS gene has an amino acid sequence shown as SEQ ID NO.3, the protein coded by the PTEN gene has an amino acid sequence shown as SEQ ID NO.4, the protein coded by the ACVR1B gene has an amino acid sequence shown as SEQ ID NO.5, the protein coded by the FBXW7 gene has an amino acid sequence shown as SEQ ID NO.6, the protein coded by the EGFR gene has an amino acid sequence shown as SEQ ID NO.7, the protein coded by the PDGFRA gene has an amino acid sequence shown as SEQ ID NO.8, the protein coded by the SRSF2 gene has an amino acid sequence shown as SEQ ID NO.9, the protein coded by the MEN1 gene has an amino acid sequence shown as SEQ ID NO.10, and the protein coded by the DAXX gene has an amino acid sequence shown as SEQ ID NO.11, the protein coded by the CASP8 gene has an amino acid sequence shown in SEQ ID NO. 12. The information sources of the above 12 genes are shown in Table 3 below.

Information sources of the 312 genes in Table

(4) The ELISA experiment verification is carried out on 12 serum protein markers screened by the protein chip: the method comprises the steps of verifying the samples of the submission chip and verifying the samples collected outside the submission chip again, thereby realizing the verification of the protein chip and ensuring the popularization.

(5) The experimental results are as follows: ELISA experimental verification is carried out on 12 serum protein markers screened by a protein chip, 70% of total population is extracted as a training set by using a random sampling method for all verified population, a disease prediction model is constructed by using binary logistic regression, indexes are screened by using three methods of Forward (Forward: conditional), Backward (Backward: conditional) and direct input (Enter), 7 and 12 proteins Enter the model respectively, and the corresponding area under the ROC curve (AUC), sensitivity (Se) and specificity (Sp) are shown in the following table 4.

TABLE 4 model indices screened by different screening methods

The diagnostic value and economic benefit analysis of the model constructed above shows that the model containing 7 indexes (P53, GNA11, GNAS, EGFR, SRSF2, MEN1 and DAXX) has the best effect, and the model is verified in the rest 30% of people (verification set), as shown in FIGS. 7 and 8, the area under the ROC curve of the combined diagnosis of esophageal squamous cell carcinoma reaches 0.85, 95% CI is 0.77-0.92, the sensitivity is 56.3% and the consistency reaches 79.1% when the specificity is ensured to be 90.0%.