CN110716043A

CN110716043A - Serum protein marker, kit and detection method for early screening and diagnosis of breast cancer

Info

Publication number: CN110716043A
Application number: CN201911014029.4A
Authority: CN
Inventors: 张建营; 叶华; 王鹏; 王晓; 史健翔; 代丽萍; 邱翠鹏; 黎天东
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2020-01-21
Anticipated expiration: 2039-10-23
Also published as: CN110716043B

Abstract

The invention discloses a serum protein marker for early screening and diagnosis of breast cancer, 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 CEBPA, RalA, p62, PTCH1, BRCA2, HRAS, FUBP1, GATA3, FGFR3, ALK, HISTIH3B or p53 genes. Also discloses a kit comprising the serum protein marker and a detection method. Based on the functions of cancer driver genes in tumorigenesis and development, the invention customizes 138 human protein chips coded by the cancer driver genes, which contain 180 human recombinant proteins in total, firstly preliminarily screens out serum markers for early detection of breast cancer through the protein chips, then verifies through ELISA indirect method experiments, and finally screens out a group of serum protein markers of breast cancer for early screening and diagnosis of the breast cancer, wherein the area under a ROC curve for combined diagnosis of the breast cancer reaches 0.886, 95% CI is 0.847-0.925, and when the specificity is 92.2%, the sensitivity is 67.9%, and the consistency rate reaches 80.1%.

Description

Serum protein marker, kit and detection method for early screening and diagnosis of breast cancer

Technical Field

The invention belongs to the technical field of biomedicine.

Background

The breast cancer is one of the most common malignant tumors in the world, the incidence rate of the breast cancer in most Asian countries including China is about 11.6% of that of other malignant tumors in the whole body, and the incidence rate of the breast cancer in China is obviously higher than that in Western countries, thereby seriously threatening the life health of people. Breast cancer is insidious, and most patients have advanced into the breast cancer at the time of initial diagnosis, thus losing the opportunity for treatment. The main reason is that most of breast cancer is painless mass in the early stage, which is not easy to attach importance and does not have typical symptoms in the early stage, or the small mass is difficult to detect by imaging examination. At present, the latest early detection methods for malignant tumors include a multiple tumor marker protein chip detection system (C12 chip), a tumor gene mutation detection kit, six tumor marker assay kits, and a human malignant Tumor Specific Growth Factor (TSGF) enzyme-linked immunoassay (ELISA) kit. However, the detection false positive of the multi-tumor marker protein chip detection system (C12 chip) is higher, and no targeted breast cancer detection marker exists, so that the detection efficiency is low; the tumor gene mutation detection reagent depends on the complicated operation of Polymerase Chain Reaction (PCR) and is easy to generate false positive; the six tumor marker determination kits and the human malignant Tumor Specific Growth Factor (TSGF) enzyme-linked immunosorbent assay (ELISA) kit can only detect that a patient has a tumor, but cannot judge the specific type of the tumor.

In recent years, in the field of human oncology, many studies have found that the sera of cancer patients contain 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 research of early diagnosis of the breast cancer. The tumor-associated antigen p62 can stimulate human body to generate immune response and generate autoantibody, and has important significance in the occurrence time of the anti-p 62 autoantibody in the circulation system of a liver cancer patient: the use of the sequential serum from the development of chronic hepatitis to cirrhosis to liver cancer shows that the anti-p 62 autoantibody does not appear in the chronic hepatitis stage of a patient, does not appear in the early stage and the middle stage of cirrhosis, the content of the autoantibody sharply rises for a period of time before the liver cancer occurs in the late stage of the cirrhosis stage, the clinical symptoms and signs of the liver cancer do not appear at the moment, the liver cancer cannot be found clinically, and the phenomenon is repeated in the sequential serum of a plurality of liver cancer patients. This suggests that the anti-p 62 autoantibody is a potential serological marker for early diagnosis of liver cancer. The detection of p62 autoantibody by serum detection of liver cancer, liver cirrhosis, chronic hepatitis patients and normal persons in small samples shows that the specificity of the autoantibody is high, but the sensitivity cannot reach 20%. Obviously, if the anti-p 62 autoantibody is used as a marker for diagnosing liver cancer alone, the diagnostic value is low. In a study of 2003 in which 7 anti-TAA autoantibodies were used to detect liver cancer, the sensitivity of a single marker did not exceed 20%, but by paralleling these 7 TAA autoantibodies, the sensitivity of detection could reach 56.9%, while the specificity of diagnosis was still high. The combination of multiple TAA autoantibodies has higher diagnostic value and is a strategy with application potential. The research provides a thought for early diagnosis of the breast cancer, so that the search of a group of TAA autoantibody combinations with higher diagnostic value for early screening and diagnosis of the breast cancer also has application potential.

There are two common methods for finding valuable TAA autoantibodies: the first is serological screening of recombinant cDNA expression library (serological analysis of recombinant cDNA expression libraries, 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. In order to find the anti-TAAs autoantibody index having diagnostic value for tumor, a protein chip technology, also called protein microarray, can be used to rapidly detect a plurality of tumor-associated antigens or autoantibodies on one chip at the same time, and has the advantages of automation, rapidness, high sensitivity and the like. The protein chip technology mainly utilizes the combination of high specificity of antigen and antibody to detect, is a novel scientific research technology for researching proteomics and protein interaction, can quickly analyze protein in blood by using the high-flux analysis technology, greatly accelerates the research process of tumor biomarkers, and gradually becomes a mainstream technology for screening and evaluating the tumor markers. 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.

Therefore, the invention preliminarily detects 138 related antigen antibodies by a protein chip technology, screens out differential expression proteins, namely potential TAAs, and designs and optimizes the tumor related antigen combination for breast cancer diagnosis by measuring the expression levels of the indexes in breast cancer and normal control serum.

Disclosure of Invention

The invention aims to provide a serum protein marker for early screening and diagnosis of breast cancer, and also provides a kit and a detection method comprising the serum protein marker.

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

a serum protein marker for early screening and diagnosis of breast cancer is any one or combination of more than two of CEBPA, RalA, p62, PTCH1, BRCA2, HRAS, FUBP1, GATA3, FGFR3, ALK, HISTIH3B or p53 gene encoded proteins.

The serum protein marker is any one or combination of more than two of proteins coded by p62, PTCH1, BRCA2, HRAS, FUBP1 or GATA3 genes.

The serum protein markers are combinations of proteins encoded by p62, PTCH1, BRCA2, HRAS, FUBP1 and GATA3 genes.

The protein coded by the p62 gene has an amino acid sequence shown in SEQ ID NO. 3; the protein coded by the PTCH1 gene has an amino acid sequence shown as SEQ ID NO. 4; the protein coded by the BRCA2 gene has an amino acid sequence shown as SEQ ID NO. 5; the protein coded by the HRAS gene has an amino acid sequence shown as SEQ ID NO. 6; the protein coded by the FUBP1 gene has an amino acid sequence shown in SEQ ID NO. 7; the protein coded by the GATA3 gene has an amino acid sequence shown in SEQ ID NO. 8.

A kit comprising serum protein markers for early screening and diagnosis of breast cancer.

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 serum protein markers for early screening and diagnosis of breast cancer, comprising the steps of:

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(-(-9.295+16.882×OD_p62+6.528×OD_PTCH1+8.975×OD_BRCA2-29.232×OD_HRAS+25.514×OD_FUBP1+13.090×OD_GATA3)))；

OD in the formula_p62、OD_PTCH1、OD_BRCA2、OD_HRAS、OD_FUBP1、OD_GATA3The 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 breast cancer 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 breast cancer are preliminarily screened out through a protein chip, then, ELISA indirect method experiments are carried out for verification, and finally, a group of serum protein markers of breast cancer which can be used for early screening and diagnosing of breast cancer are screened out, especially the combination of proteins coded by p62, PTCH1, BRCA2, HRAS, FUBP1 and GATA3 genes, the area under the ROC curve of the combined diagnosis breast cancer reaches 0.886, 95% CI is 0.847-0.925, when the specificity is ensured to be 92.2%, the sensitivity is 67.9%, the consistency rate reaches 80.1%, and the method can assist clinical diagnosis of breast cancer 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 can provide a basis for early diagnosis of the breast cancer.

Drawings

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

FIG. 2 is ROC curve analysis chart of 12 TAAs individually diagnosed with breast cancer screened by protein chip in experimental example;

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;

FIG. 5 is ROC curve analysis chart of ELISA for verifying 12 TAAs for breast cancer 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 data in the training set of ELISA-verified 6 TAAs for combined diagnosis of breast cancer in experimental examples;

FIG. 8 is a ROC plot of the data in the validation set of ELISA validated 6 TAAs for the combined diagnosis of breast cancer in the experimental examples.

Detailed Description

Examples of the experiments

1 preparation of serum samples

1.1 serum samples for protein chip experiments

Primary breast cancer patients (breast cancer pathologically diagnosed) were collected at the first subsidiary hospital of the university of zhengzhou and the beijing youan hospital, 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 for 2 hours at room temperature, 1000g of the whole blood is centrifuged for 15 minutes, supernatant is taken, each sample is subpackaged with a plurality of parts, labels are attached to the parts, and the parts are stored in a low-temperature refrigerator at minus 80 ℃ so as to avoid repeated freeze thawing.

Based on epidemiological analysis, 50 primary breast cancer sera were finally collected and 27 of the Yogan hospital normal control sera of the same physical examination were subjected to preliminary chip screening. 50 patients with primary breast cancer were all women, with a mean age of 48.16 ± 10.99 years, ranging from 29 to 71 years; the 27 normal sera were all women, with a mean age of 40.20 + -10.48 years, ranging from 21-58 years. All breast cancer patient sera were collected at a time when the patient was initially diagnosed with breast cancer and had not received any radiotherapy, chemotherapy, or surgical treatment. 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 ELISA Experimental validation

(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, breast surgery (184 cases of primary breast cancer) and cardiovascular research projects (184 cases of normal persons) in Jinshui district of Zhengzhou City, 184 cases of primary breast cancer patients were all female, with the average age of 50.10 ± 10.33 years and the age range of 29-81 years; the 184 normal sera were all from women with a mean age of 49.47 ± 10.98 years and an age range of 29-81 years. All breast cancer patient sera were collected at a time when the patient was initially diagnosed with breast cancer and had not received any radiotherapy, chemotherapy, or surgical treatment.

2 protein chip customization for screening breast cancer 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 is HuProt customized by Guangzhou Bo Chong Biotechnology Co., Ltd^TMHuman protein chips.

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 (the serum samples were frozen and thawed in a 4 ℃ chromatography cabinet, 1 XPBST solution containing 1% BSA was diluted at a ratio of 1: 50), 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: take out the chip (notice notCan touch or scratch the upper surface of the chip), placed in a chip washing box with a washing solution 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 breast cancer serum marker, and screening the following serum protein markers by the protein chip experiment: the proteins encoded by cancer driver genes CEBPA, RalA, p62, PTCH1, BRCA2, HRAS, FUBP1, GATA3, FGFR3, ALK, HISTIH3B and p53 (FIG. 2 is a ROC curve analysis chart of the individual diagnosis of breast cancer by 12 TAAs screened out by the above protein chip, FIG. 2 is a ROC curve of the individual diagnosis of breast cancer by the proteins encoded by CEBPA, RalA, p62, PTCH1, BRCA2, HRAS, FUBP1, GATA3, FGFR3, ALK, HISTIH3B and p53 in the order from 1 to 12; FIG. 3 is a SNR scatter diagram of the above 12 TAAs, N in FIG. 3 is Normal, namely, healthy Normal serum, B is a case of breast cancer (namely brestcer), wherein the sequences of the proteins encoded by CEBPA, RalA, p62, PTCH1, BRCA2, BRCA 1, GAAS 869, GATA 863, HRTA 8653 and HRP 8427 have the amino acid sequences of the individual diagnosis of breast cancer protein encoded proteins encoded by the aforementioned HAP B and HRPD.

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) containing 2% BSA (Solebao, Beijing, analytical grade) was dispensed 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: the serum was diluted 1:100 with 1% BSA in 1 XPBST and placed in a half water bath at 37 ℃ for 1h at 100. mu.L/well.

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 at the ratio of 1:1, and then 100 mu L/hole is put away from light at room temperature to reach the expected color (about 5-15min is needed).

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 the following table 1, and the arrangement table of the 96-well plate of the ELISA experiment is shown in the following table 2. 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 and the results are shown in FIGS. 5 and 6. FIG. 5 is a ROC curve analysis diagram of 12 TAAs individually diagnosing breast cancer in ELISA validation experiment, in which 1-12 are ROC curves of proteins encoded by CEBPA, RalA, p62, PTCH1, BRCA2, HRAS, FUBP1, GATA3, FGFR3, ALK, HISTIH3B and p53 individually diagnosing breast cancer; FIG. 6 is a graph showing the distribution of OD value scatter of 12 TAAs in ELISA-verified experiments, in which N indicates Normal, i.e., healthy Normal serum, and B indicates Breast cancer, i.e., breast cancer case.

As can be seen from FIG. 5, the area under the ROC curve of breast cancer diagnosis by a single index is 0.634-0.737, and the sensitivity range is 25.9% -46.2% when the lowest specificity is ensured. Wherein the area under the curve of BRCA2 is 0.737, the sensitivity reaches 46.2%, and the specificity is 90.2%; the area under the ROC curve of RalA is 0.736, the sensitivity reaches 25.9 percent, and the specificity is 91.7 percent; the area under the ROC curve of ALK is minimum, and is 0.643, the sensitivity reaches 28.2 percent, and the specificity is 90.2 percent. As can be seen from FIG. 6, the OD values of the 12 indexes are distributed between 0 and 0.8, the medium OD values are basically distributed between 0.2 and 0.4, and the differences between the healthy control and the breast cancer cases have statistical significance.

5 data processing

The differential expression protein is screened out by using the focused array human protein chip in a breast cancer 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 above logic, the breast cancer group (50 primary breast cancer patient sera collected from the first subsidiary hospital of the university of zheng and beijing youan) and the youan control group (27 normal sera of youan hospital) are compared, differential proteins which are obviously higher than the control group in the breast cancer group are screened out to be used as breast cancer candidate markers, and finally 12 serum protein markers (CEBPA, RalA, p62, PTCH1, BRCA2, HRAS, FUBP1, GATA3, FGFR3, ALK, HISTIH3B and p53) are selected by a chip to evaluate the diagnostic value of the breast cancer. Wherein, the protein coded by CEBPA gene has an amino acid sequence shown as SEQ ID NO.1, the protein coded by RalA gene has an amino acid sequence shown as SEQ ID NO.2, the protein coded by p62 gene has an amino acid sequence shown as SEQ ID NO.3, the protein coded by PTCH1 gene has an amino acid sequence shown as SEQ ID NO.4, the protein coded by BRCA2 gene has an amino acid sequence shown as SEQ ID NO.5, the protein coded by HRAS gene has an amino acid sequence shown as SEQ ID NO.6, the protein coded by FUBP1 gene has an amino acid sequence shown as SEQ ID NO.7, the protein coded by GATA3 gene has an amino acid sequence shown as SEQ ID NO.8, the protein coded by FGFR3 gene has an amino acid sequence shown as SEQ ID NO.9, the protein coded by ALK gene has an amino acid sequence shown as SEQ ID NO.10, the protein coded by HISTIH3B gene has an amino acid sequence shown as SEQ ID NO.11, the protein coded by the p53 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

Name of Gene	Uniprot database accession number	NCBI reference sequence accession number
			CEBPA	P49715	NM_004364
RalA	P11233	NM_005402
			P62	Q9Y6M1-6	NM_006548
PTCH1	Q13635	NM_000264
			BRCA2	P51587	NM_000059
HRAS	P01112	NM_005343
			FUBP1	Q96AE4-2	NM_003902
GATA3	P23771	NM_002051
			FGFR3	P22607	NM_000142
ALK	Q9UM73	NM_004304
			HISTIH3B	P68431	NM_003529
TP53	P04637	NM_000546

(4) The 12 serum protein markers screened by the protein chip are subjected to ELISA indirect method experimental verification: 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 method (Enter), 6, 8 and 8 types of entry models are respectively selected, 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 diagnosis value and economic benefit analysis of the model constructed above shows that the model containing 6 indexes (p62, PTCH1, BRCA2, HRAS, FUBP1 and GATA3) has the best effect, and the model is verified in the rest 30% of people (verification set), as shown in fig. 7 and 8, the area under the ROC curve of the combined diagnosis breast cancer reaches 0.886, 95% CI is 0.847-0.925, and when the specificity is ensured to be 92.2%, the sensitivity is 67.9%, and the consistency rate reaches 80.1%.

Examples

A serum protein marker for early screening and diagnosis of breast cancer is the combination of proteins encoded by p62, PTCH1, BRCA2, HRAS, FUBP1 and GATA3 genes. The protein coded by the p62 gene has an amino acid sequence shown in SEQ ID NO. 3; the protein coded by the PTCH1 gene has an amino acid sequence shown as SEQ ID NO. 4; the protein coded by the BRCA2 gene has an amino acid sequence shown in SEQ ID NO. 5; the protein coded by the HRAS gene has an amino acid sequence shown as SEQ ID NO. 6; the protein coded by the FUBP1 gene has an amino acid sequence shown in SEQ ID NO. 7; the protein coded by the GATA3 gene has an amino acid sequence shown in SEQ ID NO. 8.

A kit comprises a serum protein marker for early screening and diagnosis of breast cancer, wherein the serum protein marker is coated on a solid phase carrier, and the solid phase carrier is a concave hole flat plate made of polyvinyl chloride. The kit also comprises positive control serum, negative control serum, confining liquid, sample diluent, a second antibody, second antibody diluent, washing liquid, developing liquid and stopping liquid.

The positive control serum is serum which has a higher ELISA experiment OD value and is verified to be positive by a Western Blot experiment, the negative control serum is serum which has an ELISA experiment OD value near the mean value and is verified to be negative by a Westernblot experiment in a normal control population, the blocking solution is a 1 XPBST solution containing 2% BSA, the serum diluent and the second antibody diluent are both 1 XPBST solutions containing 1% BSA, the second antibody is HRP-labeled mouse anti-human IgG, the washing solution is a 1 XPBST solution, the developing solution is an A solution (200 mgTMB.2 HCl is dissolved in 1L deionized water, Solaibao, Beijing, analytically pure) and a B solution (9.2g citric acid, 37gNa Na₂HPO₄·12H₂O and 8ml of 0.75% H₂O₂Dissolved in 1L of deionized water) and mixed solution with the volume ratio of 1:1, and the stop solution is concentrated sulfuric acid with the concentration of 10 percent.

1) coating each serum protein marker (the coating concentration is shown in the table 1 above), 100 mu L/hole, overnight at 4 ℃, and cleaning for 3 times by using a 350 mu L/hole cleaning solution after sealing (200 mu L/hole by using a sealing solution and overnight at 4 ℃);

2) performing primary antibody incubation (100 mu L/hole, 37 ℃ half water bath for 1h) with diluted serum to be detected (the diluted serum to be detected and serum diluent are diluted according to the volume ratio of 1: 100), washing with 350 mu L/hole washing solution for 5 times, performing secondary antibody incubation (100 mu L/hole, 37 ℃ half water bath for 1h and washing with 350 mu L/hole washing solution for 5 times after the second antibody and the second antibody diluent are diluted according to the volume ratio of 1: 10000);

3) color development of a color development system: a TMB color development system with 100 mu L/hole of color development liquid is protected from light at room temperature to reach the expected color; and (3) terminating the reaction: stopping with 50 μ L/well, and measuring absorbance within 10 min;

4) by OD₄₅₀-OD₆₂₀The relative OD value is obtained, then the blank control is deducted, the absorbance value of each index (namely the serum protein marker) is substituted into the following formula, the predicted probability P value is calculated,

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

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

The result shows that the P value of the serum to be detected is more than 0.5, and the sample is judged to be suspected breast cancer sample preliminarily.

Since the measurement result of the method of this embodiment can only be information of intermediate results, and it cannot be directly determined whether the patient has breast cancer, it is necessary to finally determine the diseased state of the patient by combining information of clinical symptoms, imaging, histopathology, and the like.

<110> Zhengzhou university

<120> serum protein marker, kit and detection method for early screening and diagnosis of breast cancer

<160>12

<170>SIPOSequenceListing 1 .0

<211>358

<212>PRT

<213> human

<221> protein encoded by CEBPA gene

<400>1

MESADFYEAE PRPPMSSHLQ SPPHAPSSAA FGFPRGAGPA QPPAPPAAPE PLGGICEHET 60

SIDISAYIDP AAFNDEFLAD LFQHSRQQEK AKAAVGPTGG GGGGDFDYPG APAGPGGAVM 120

PGGAHGPPPG YGCAAAGYLD GRLEPLYERV GAPALRPLVI KQEPREEDEA KQLALAGLFP 180

YQPPPPPPPS HPHPHPPPAH LAAPHLQFQI AHCGQTTMHL QPGHPTPPPT PVPSPHPAPA 240

LGAAGLPGPG SALKGLGAAH PDLRASGGSG AGKAKKSVDK NSNEYRVRRE RNNIAVRKSR 300

DKAKQRNVET QQKVLELTSD NDRLRKRVEQ LSRELDTLRG IFRQLPESSL VKAMGNCA 358

<211>206

<212>PRT

<213> human

<221> protein encoded by RalA Gene

<400>2

MAANKPKGQN SLALHKVIMV GSGGVGKSAL TLQFMYDEFV EDYEPTKADS YRKKVVLDGE 60

EVQIDILDTA GQEDYAAIRD NYFRSGEGFL CVFSITEMES FAATADFREQ ILRVKEDENV 120

PFLLVGNKSD LEDKRQVSVE EAKNRAEQWN VNYVETSAKT RANVDKVFFD LMREIRARKM 180

EDSKEKNGKK KRKSLAKRIR ERCCIL 206

<211>598

<212>PRT

<213> human

<221> protein encoded by p62 gene

<400>3

MNKLYIGNLS PAVTADDLRQ LFGDRKLPLA GQVLLKSGYA FVDYPDQNWA IRAIETLSGK 60

VELHGKIMEV DYSVSKKLRS RKIQIRNIPP HLQWEVLDGL LAQYGTVENV EQVNTDTETA 120

VVNVTYATRE EAKIAMEKLS GHQFENYSFK ISYIPDEEVS SPSPPQRAQR GDHSSREQGH 180

APGGTSQARQ IDFPLRILVP TQFVGAIIGK EGLTIKNITK QTQSRVDIHR KENSGAAEKP 240

VTIHATPEGT SEACRMILEI MQKEADETKL AEEIPLKILA HNGLVGRLIG KEGRNLKKIE 300

HETGTKITIS SLQDLSIYNP ERTITVKGTV EACASAEIEI MKKLREAFEN DMLAVNQQAN 360

LIPGLNLSAL GIFSTGLSVL SPPAGPRGAP PAAPYHPFTT HSGYFSSLYP HHQFGPFPHH 420

HSYPEQEIVN LFIPTQAVGA IIGKKGAHIK QLARFAGASI KIAPAEGPDV SERMVIITGP 480

PEAQFKAQGR IFGKLKEENF FNPKEEVKLE AHIRVPSSTA GRVIGKGGKT VNELQNLTSA 540

EVIVPRDQTP DENEEVIVRI IGHFFASQTA QRKIREIVQQ VKQQEQKYPQ GVASQRSK 598

<211>1447

<212>PRT

<213> human

<221> protein encoded by PTCH1 Gene

<400>4

MASAGNAAEP QDRGGGGSGC IGAPGRPAGG GRRRRTGGLR RAAAPDRDYL HRPSYCDAAF 60

ALEQISKGKA TGRKAPLWLR AKFQRLLFKL GCYIQKNCGK FLVVGLLIFG AFAVGLKAAN 120

LETNVEELWV EVGGRVSREL NYTRQKIGEE AMFNPQLMIQ TPKEEGANVL TTEALLQHLD 180

SALQASRVHV YMYNRQWKLE HLCYKSGELI TETGYMDQII EYLYPCLIIT PLDCFWEGAK 240

LQSGTAYLLG KPPLRWTNFD PLEFLEELKK INYQVDSWEE MLNKAEVGHG YMDRPCLNPA 300

DPDCPATAPN KNSTKPLDMA LVLNGGCHGL SRKYMHWQEE LIVGGTVKNS TGKLVSAHAL 360

QTMFQLMTPK QMYEHFKGYE YVSHINWNED KAAAILEAWQ RTYVEVVHQS VAQNSTQKVL 420

SFTTTTLDDI LKSFSDVSVI RVASGYLLML AYACLTMLRW DCSKSQGAVG LAGVLLVALS 480

VAAGLGLCSL IGISFNAATT QVLPFLALGV GVDDVFLLAH AFSETGQNKR IPFEDRTGEC 540

LKRTGASVAL TSISNVTAFF MAALIPIPAL RAFSLQAAVV VVFNFAMVLL IFPAILSMDL 600

YRREDRRLDI FCCFTSPCVS RVIQVEPQAY TDTHDNTRYS PPPPYSSHSF AHETQITMQS 660

TVQLRTEYDP HTHVYYTTAE PRSEISVQPV TVTQDTLSCQ SPESTSSTRD LLSQFSDSSL 720

HCLEPPCTKW TLSSFAEKHY APFLLKPKAK VVVIFLFLGL LGVSLYGTTR VRDGLDLTDI 780

VPRETREYDF IAAQFKYFSF YNMYIVTQKA DYPNIQHLLY DLHRSFSNVK YVMLEENKQL 840

PKMWLHYFRD WLQGLQDAFD SDWETGKIMP NNYKNGSDDG VLAYKLLVQT GSRDKPIDIS 900

QLTKQRLVDA DGIINPSAFY IYLTAWVSND PVAYAASQAN IRPHRPEWVH DKADYMPETR 960

LRIPAAEPIE YAQFPFYLNG LRDTSDFVEA IEKVRTICSN YTSLGLSSYP NGYPFLFWEQ 1020

YIGLRHWLLL FISVVLACTF LVCAVFLLNP WTAGIIVMVL ALMTVELFGM MGLIGIKLSA 1080

VPVVILIASV GIGVEFTVHV ALAFLTAIGD KNRRAVLALE HMFAPVLDGA VSTLLGVLML 1140

AGSEFDFIVR YFFAVLAILT ILGVLNGLVL LPVLLSFFGP YPEVSPANGL NRLPTPSPEP 1200

PPSVVRFAMP PGHTHSGSDS SDSEYSSQTT VSGLSEELRH YEAQQGAGGP AHQVIVEATE 1260

NPVFAHSTVV HPESRHHPPS NPRQQPHLDS GSLPPGRQGQ QPRRDPPREG LWPPPYRPRR 1320

DAFEISTEGH SGPSNRARWG PRGARSHNPR NPASTAMGSS VPGYCQPITT VTASASVTVA 1380

VHPPPVPGPG RNPRGGLCPG YPETDHGLFE DPHVPFHVRC ERRDSKVEVI ELQDVECEER 1440

PRGSSSN 1447

<211>3418

<212>PRT

<213> human

<221> protein encoded by BRCA2 gene

<400>5

MPIGSKERPT FFEIFKTRCN KADLGPISLN WFEELSSEAP PYNSEPAEES EHKNNNYEPN 60

LFKTPQRKPS YNQLASTPII FKEQGLTLPL YQSPVKELDK FKLDLGRNVP NSRHKSLRTV 120

KTKMDQADDV SCPLLNSCLS ESPVVLQCTH VTPQRDKSVV CGSLFHTPKF VKGRQTPKHI 180

SESLGAEVDP DMSWSSSLAT PPTLSSTVLI VRNEEASETV FPHDTTANVK SYFSNHDESL 240

KKNDRFIASV TDSENTNQRE AASHGFGKTS GNSFKVNSCK DHIGKSMPNV LEDEVYETVV 300

DTSEEDSFSL CFSKCRTKNL QKVRTSKTRK KIFHEANADE CEKSKNQVKE KYSFVSEVEP 360

NDTDPLDSNV ANQKPFESGS DKISKEVVPS LACEWSQLTL SGLNGAQMEK IPLLHISSCD 420

QNISEKDLLD TENKRKKDFL TSENSLPRIS SLPKSEKPLN EETVVNKRDE EQHLESHTDC480

ILAVKQAISG TSPVASSFQG IKKSIFRIRE SPKETFNASF SGHMTDPNFK KETEASESGL 540

EIHTVCSQKE DSLCPNLIDN GSWPATTTQN SVALKNAGLI STLKKKTNKF IYAIHDETSY 600

KGKKIPKDQK SELINCSAQF EANAFEAPLT FANADSGLLH SSVKRSCSQN DSEEPTLSLT 660

SSFGTILRKC SRNETCSNNT VISQDLDYKE AKCNKEKLQL FITPEADSLS CLQEGQCEND 720

PKSKKVSDIK EEVLAAACHP VQHSKVEYSD TDFQSQKSLL YDHENASTLI LTPTSKDVLS 780

NLVMISRGKE SYKMSDKLKG NNYESDVELT KNIPMEKNQD VCALNENYKN VELLPPEKYM 840

RVASPSRKVQ FNQNTNLRVI QKNQEETTSI SKITVNPDSE ELFSDNENNF VFQVANERNN 900

LALGNTKELH ETDLTCVNEP IFKNSTMVLY GDTGDKQATQ VSIKKDLVYV LAEENKNSVK 960

QHIKMTLGQD LKSDISLNID KIPEKNNDYM NKWAGLLGPI SNHSFGGSFR TASNKEIKLS 1020

EHNIKKSKMF FKDIEEQYPT SLACVEIVNT LALDNQKKLS KPQSINTVSA HLQSSVVVSD 1080

CKNSHITPQM LFSKQDFNSN HNLTPSQKAE ITELSTILEE SGSQFEFTQF RKPSYILQKS 1140

TFEVPENQMT ILKTTSEECR DADLHVIMNA PSIGQVDSSK QFEGTVEIKR KFAGLLKNDC 1200

NKSASGYLTD ENEVGFRGFY SAHGTKLNVS TEALQKAVKL FSDIENISEE TSAEVHPISL 1260

SSSKCHDSVV SMFKIENHND KTVSEKNNKC QLILQNNIEM TTGTFVEEIT ENYKRNTENE 1320

DNKYTAASRN SHNLEFDGSD SSKNDTVCIH KDETDLLFTD QHNICLKLSG QFMKEGNTQI 1380

KEDLSDLTFL EVAKAQEACH GNTSNKEQLT ATKTEQNIKD FETSDTFFQT ASGKNISVAK 1440

ESFNKIVNFF DQKPEELHNF SLNSELHSDI RKNKMDILSY EETDIVKHKI LKESVPVGTG 1500

NQLVTFQGQP ERDEKIKEPT LLGFHTASGK KVKIAKESLD KVKNLFDEKE QGTSEITSFS 1560

HQWAKTLKYR EACKDLELAC ETIEITAAPK CKEMQNSLNN DKNLVSIETV VPPKLLSDNL 1620

CRQTENLKTS KSIFLKVKVH ENVEKETAKS PATCYTNQSP YSVIENSALA FYTSCSRKTS 1680

VSQTSLLEAK KWLREGIFDG QPERINTADY VGNYLYENNS NSTIAENDKN HLSEKQDTYL 1740

SNSSMSNSYS YHSDEVYNDS GYLSKNKLDS GIEPVLKNVE DQKNTSFSKV ISNVKDANAY 1800

PQTVNEDICV EELVTSSSPC KNKNAAIKLS ISNSNNFEVG PPAFRIASGK IVCVSHETIK 1860

KVKDIFTDSF SKVIKENNEN KSKICQTKIM AGCYEALDDS EDILHNSLDN DECSTHSHKV 1920

FADIQSEEIL QHNQNMSGLE KVSKISPCDV SLETSDICKC SIGKLHKSVS SANTCGIFST 1980

ASGKSVQVSD ASLQNARQVF SEIEDSTKQV FSKVLFKSNE HSDQLTREEN TAIRTPEHLI 2040

SQKGFSYNVV NSSAFSGFST ASGKQVSILE SSLHKVKGVL EEFDLIRTEH SLHYSPTSRQ 2100

NVSKILPRVD KRNPEHCVNS EMEKTCSKEF KLSNNLNVEG GSSENNHSIK VSPYLSQFQQ 2160

DKQQLVLGTK VSLVENIHVL GKEQASPKNV KMEIGKTETF SDVPVKTNIE VCSTYSKDSE 2220

NYFETEAVEI AKAFMEDDEL TDSKLPSHAT HSLFTCPENE EMVLSNSRIG KRRGEPLILV 2280

GEPSIKRNLL NEFDRIIENQ EKSLKASKST PDGTIKDRRL FMHHVSLEPI TCVPFRTTKE 2340

RQEIQNPNFT APGQEFLSKS HLYEHLTLEK SSSNLAVSGH PFYQVSATRN EKMRHLITTG 2400

RPTKVFVPPF KTKSHFHRVE QCVRNINLEE NRQKQNIDGH GSDDSKNKIN DNEIHQFNKN 2460

NSNQAAAVTF TKCEEEPLDL ITSLQNARDI QDMRIKKKQR QRVFPQPGSL YLAKTSTLPR 2520

ISLKAAVGGQ VPSACSHKQL YTYGVSKHCI KINSKNAESF QFHTEDYFGK ESLWTGKGIQ 2580

LADGGWLIPS NDGKAGKEEF YRALCDTPGV DPKLISRIWV YNHYRWIIWK LAAMECAFPK 2640

EFANRCLSPE RVLLQLKYRY DTEIDRSRRS AIKKIMERDD TAAKTLVLCV SDIISLSANI 2700

SETSSNKTSS ADTQKVAIIE LTDGWYAVKA QLDPPLLAVL KNGRLTVGQK IILHGAELVG 2760

SPDACTPLEA PESLMLKISA NSTRPARWYT KLGFFPDPRP FPLPLSSLFS DGGNVGCVDV 2820

IIQRAYPIQW MEKTSSGLYI FRNEREEEKE AAKYVEAQQK RLEALFTKIQ EEFEEHEENT 2880

TKPYLPSRAL TRQQVRALQD GAELYEAVKN AADPAYLEGY FSEEQLRALN NHRQMLNDKK 2940

QAQIQLEIRK AMESAEQKEQ GLSRDVTTVW KLRIVSYSKK EKDSVILSIW RPSSDLYSLL 3000

TEGKRYRIYH LATSKSKSKS ERANIQLAAT KKTQYQQLPV SDEILFQIYQ PREPLHFSKF 3060

LDPDFQPSCS EVDLIGFVVS VVKKTGLAPF VYLSDECYNL LAIKFWIDLN EDIIKPHMLI 3120

AASNLQWRPE SKSGLLTLFA GDFSVFSASP KEGHFQETFN KMKNTVENID ILCNEAENKL 3180

MHILHANDPK WSTPTKDCTS GPYTAQIIPG TGNKLLMSSP NCEIYYQSPL SLCMAKRKSV 3240

STPVSAQMTS KSCKGEKEID DQKNCKKRRA LDFLSRLPLP PPVSPICTFV SPAAQKAFQP 3300

PRSCGTKYET PIKKKELNSP QMTPFKKFNE ISLLESNSIA DEELALINTQ ALLSGSTGEK 3360

QFISVSESTR TAPTSSEDYL RLKRRCTTSL IKEQESSQAS TEECEKNKQD TITTKKYI 3418

<211>189

<212>PRT

<213> human

<221> protein encoded by HRAS gene

<400>6

MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG 60

QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHQYREQI KRVKDSDDVP MVLVGNKCDL 120

AARTVESRQA QDLARSYGIP YIETSAKTRQ GVEDAFYTLV REIRQHKLRK LNPPDESGPG 180

CMSCKCVLS 189

<211>653

<212>PRT

<213> human

<221> protein encoded by FUBP1 gene

<400>7

MADYSTVPPP SSGSAGGGGG GGGGGGVNDA FKDALQRARQ IAAKIGGDAG TSLNSNDYGY 60

GGQKRPLEDG DQPDAKKVAP QNDSFGTQLP PMHQQQRSVM TEEYKVPDGM VGFIIGRGGE 120

QISRIQQESG CKIQIAPDSG GLPERSCMLT GTPESVQSAK RLLDQIVEKG RPAPGFHHGD 180

GPGNAVQEIM IPASKAGLVI GKGGETIKQL QERAGVKMVM IQDGPQNTGA DKPLRITGDP 240

YKVQQAKEMV LELIRDQGGF REVRNEYGSR IGGNEGIDVP IPRFAVGIVI GRNGEMIKKI 300

QNDAGVRIQF KPDDGTTPER IAQITGPPDR CQHAAEIITD LLRSVQAGNP GGPGPGGRGR 360

GRGQGNWNMG PPGGLQEFNF IVPTGKTGLI IGKGGETIKS ISQQSGARIE LQRNPPPNAD 420

PNMKLFTIRG TPQQIDYARQ LIEEKIGGPV NPLGPPVPHG PHGVPGPHGP PGPPGPGTPM 480

GPYNPAPYNP GPPGPAPHGP PAPYAPQGWG NAYPHWQQQA PPDPAKAGTD PNSAAWAAYY 540

AHYYQQQAQP PPAAPAGAPT TTQTNGQGDQ QNPAPAGQVD YTKAWEEYYK KMGQAVPAPT 600

GAPPGGQPDY SAAWAEYYRQ QAAYYAQTSP QGMPQHPPAP QCRFDPASIE LAL 653

<211>443

<212>PRT

<213> human

<221> GATA3 Gene-encoded protein

<400>8

MEVTADQPRW VSHHHPAVLN GQHPDTHHPG LSHSYMDAAQ YPLPEEVDVL FNIDGQGNHV 60

PPYYGNSVRA TVQRYPPTHH GSQVCRPPLL HGSLPWLDGG KALGSHHTAS PWNLSPFSKT 120

SIHHGSPGPL SVYPPASSSS LSGGHASPHL FTFPPTPPKD VSPDPSLSTP GSAGSARQDE 180

KECLKYQVPL PDSMKLESSH SRGSMTALGG ASSSTHHPIT TYPPYVPEYS SGLFPPSSLL 240

GGSPTGFGCK SRPKARSSTG RECVNCGATS TPLWRRDGTG HYLCNACGLY HKMNGQNRPL 300

IKPKRRLSAA RRAGTSCANC QTTTTTLWRR NANGDPVCNA CGLYYKLHNI NRPLTMKKEG 360

IQTRNRKMSS KSKKCKKVHD SLEDFPKNSS FNPAALSRHM SSLSHISPFS HSSHMLTTPT 420

PMHPPSSLSF GPHHPSSMVT AMG 443

<211>391

<212>PRT

<213> human

<221> FGFR3 gene coded protein

<400>9

MGAPACALAL CVAVAIVAGA SSESLGTEQR VVGRAAEVPG PEPGQQEQLV FGSGDAVELS 60

CPPPGGGPMG PTVWVKDGTG LVPSERVLVG PQRLQVLNAS HEDSGAYSCR QRLTQRVLCH 120

FSVRVTDAPS SGDDEDGEDE AEDTGVDTGA PYWTRPERMD KKLLAVPAAN TVRFRCPAAG 180

NPTPSISWLK NGREFRGEHR IGGIKLRHQQ WSLVMESVVP SDRGNYTCVV ENKFGSIRQT 240

YTLDVLERSP HRPILQAGLP ANQTAVLGSD VEFHCKVYSD AQPHIQWLKH VEVNGSKVGP 300

DGTPYVTVLK TAGANTTDKE LEVLSLHNVT FEDAGEYTCL AGNSIGFSHH SAWLVVLPAE 360

EELVEADEAG SVYAGILSYG VGFFLFILVV AAVTLCRLRS PPKKGLGSPT VHKISRFPLK 420

RQVSLESNAS MSSNTPLVRI ARLSSGEGPT LANVSELELP ADPKWELSRA RLTLGKPLGE 480

GCFGQVVMAE AIGIDKDRAA KPVTVAVKML KDDATDKDLS DLVSEMEMMK MIGKHKNIIN 540

LLGACTQGGP LYVLVEYAAK GNLREFLRAR RPPGLDYSFD TCKPPEEQLT FKDLVSCAYQ 600

VARGMEYLAS QKCIHRDLAA RNVLVTEDNV MKIADFGLAR DVHNLDYYKK TTNGRLPVKW 660

MAPEALFDRV YTHQSDVWSF GVLLWEIFTL GGSPYPGIPV EELFKLLKEG HRMDKPANCT 720

HDLYMIMREC WHAAPSQRPT FKQLVEDLDR VLTVTSTDEY LDLSAPFEQY SPGGQDTPSS 780

SSSGDDSVFA HDLLPPAPPS SGGSRT 806

<211>1620

<212>PRT

<213> human

<221> protein encoded by ALK Gene

<400>10

MGAIGLLWLL PLLLSTAAVG SGMGTGQRAG SPAAGPPLQP REPLSYSRLQ RKSLAVDFVV 60

PSLFRVYARD LLLPPSSSEL KAGRPEARGS LALDCAPLLR LLGPAPGVSW TAGSPAPAEA 120

RTLSRVLKGG SVRKLRRAKQ LVLELGEEAI LEGCVGPPGE AAVGLLQFNL SELFSWWIRQ 180

GEGRLRIRLM PEKKASEVGR EGRLSAAIRA SQPRLLFQIF GTGHSSLESP TNMPSPSPDY 240

FTWNLTWIMK DSFPFLSHRS RYGLECSFDF PCELEYSPPL HDLRNQSWSW RRIPSEEASQ 300

MDLLDGPGAE RSKEMPRGSF LLLNTSADSK HTILSPWMRS SSEHCTLAVS VHRHLQPSGR 360

YIAQLLPHNE AAREILLMPT PGKHGWTVLQ GRIGRPDNPF RVALEYISSG NRSLSAVDFF 420

ALKNCSEGTS PGSKMALQSS FTCWNGTVLQ LGQACDFHQD CAQGEDESQM CRKLPVGFYC 480

NFEDGFCGWT QGTLSPHTPQ WQVRTLKDAR FQDHQDHALL LSTTDVPASE SATVTSATFP 540

APIKSSPCEL RMSWLIRGVL RGNVSLVLVE NKTGKEQGRM VWHVAAYEGL SLWQWMVLPL 600

LDVSDRFWLQ MVAWWGQGSR AIVAFDNISI SLDCYLTISG EDKILQNTAP KSRNLFERNP 660

NKELKPGENS PRQTPIFDPT VHWLFTTCGA SGPHGPTQAQ CNNAYQNSNL SVEVGSEGPL 720

KGIQIWKVPA TDTYSISGYG AAGGKGGKNT MMRSHGVSVL GIFNLEKDDM LYILVGQQGE 780

DACPSTNQLI QKVCIGENNV IEEEIRVNRS VHEWAGGGGG GGGATYVFKM KDGVPVPLII 840

AAGGGGRAYG AKTDTFHPER LENNSSVLGL NGNSGAAGGG GGWNDNTSLL WAGKSLQEGA 900

TGGHSCPQAM KKWGWETRGG FGGGGGGCSS GGGGGGYIGG NAASNNDPEM DGEDGVSFIS 960

PLGILYTPAL KVMEGHGEVN IKHYLNCSHC EVDECHMDPE SHKVICFCDH GTVLAEDGVS 1020

CIVSPTPEPH LPLSLILSVV TSALVAALVL AFSGIMIVYR RKHQELQAMQ MELQSPEYKL 1080

SKLRTSTIMT DYNPNYCFAG KTSSISDLKE VPRKNITLIR GLGHGAFGEV YEGQVSGMPN 1140

DPSPLQVAVK TLPEVCSEQD ELDFLMEALI ISKFNHQNIV RCIGVSLQSL PRFILLELMA 1200

GGDLKSFLRE TRPRPSQPSS LAMLDLLHVA RDIACGCQYL EENHFIHRDI AARNCLLTCP 1260

GPGRVAKIGD FGMARDIYRA SYYRKGGCAM LPVKWMPPEA FMEGIFTSKT DTWSFGVLLW 1320

EIFSLGYMPY PSKSNQEVLE FVTSGGRMDP PKNCPGPVYR IMTQCWQHQP EDRPNFAIIL 1380

ERIEYCTQDP DVINTALPIE YGPLVEEEEK VPVRPKDPEG VPPLLVSQQA KREEERSPAA 1440

PPPLPTTSSG KAAKKPTAAE ISVRVPRGPA VEGGHVNMAF SQSNPPSELH KVHGSRNKPT 1500

SLWNPTYGSW FTEKPTKKNN PIAKKEPHDR GNLGLEGSCT VPPNVATGRL PGASLLLEPS 1560

SLTANMKEVP LFRLRHFPCG NVNYGYQQQG LPLEAATAPG AGHYEDTILK SKNSMNQPGP 1620

<211>136

<212>PRT

<213> human

<221> protein encoded by HISTIH3B gene

<400>11

MARTKQTARK STGGKAPRKQ LATKAARKSA PATGGVKKPH RYRPGTVALR EIRRYQKSTE 60

LLIRKLPFQR LVREIAQDFK TDLRFQSSAV MALQEACEAY LVGLFEDTNL CAIHAKRVTI 120

MPKDIQLARR IRGERA 136

<211>393

<212>PRT

<213> human

<221> protein encoded by p53 gene

<400>12

MEEPQSDPSV EPPLSQETFS DLWKLLPENN VLSPLPSQAM DDLMLSPDDI EQWFTEDPGP 60

DEAPRMPEAA PPVAPAPAAP TPAAPAPAPS WPLSSSVPSQ KTYQGSYGFR LGFLHSGTAK 120

SVTCTYSPAL NKMFCQLAKT CPVQLWVDST PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE 180

RCSDSDGLAP PQHLIRVEGN LRVEYLDDRN TFRHSVVVPY EPPEVGSDCT TIHYNYMCNS 240

SCMGGMNRRP ILTIITLEDS SGNLLGRNSF EVRVCACPGR DRRTEEENLR KKGEPHHELP 300

PGSTKRALPN NTSSSPQPKK KPLDGEYFTL QIRGRERFEM FRELNEALEL KDAQAGKEPG 360

GSRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD 393

Claims

1. A serum protein marker for early screening and diagnosis of breast cancer, characterized by: the serum protein marker is any one or combination of more than two of proteins encoded by CEBPA, RalA, p62, PTCH1, BRCA2, HRAS, FUBP1, GATA3, FGFR3, ALK, HISTIH3B or p53 genes.

2. The serum protein marker for early screening and diagnosis of breast cancer according to claim 1, wherein: the serum protein marker is any one or combination of more than two of proteins coded by p62, PTCH1, BRCA2, HRAS, FUBP1 or GATA3 genes.

3. The serum protein marker for early screening and diagnosis of breast cancer according to claim 2, wherein: the serum protein markers are combinations of proteins encoded by p62, PTCH1, BRCA2, HRAS, FUBP1 and GATA3 genes.

4. The serum protein marker for early screening and diagnosis of breast cancer according to claim 3, wherein: the protein coded by the p62 gene has an amino acid sequence shown in SEQ ID NO. 3; the protein coded by the PTCH1 gene has an amino acid sequence shown as SEQ ID NO. 4; the protein coded by the BRCA2 gene has an amino acid sequence shown in SEQ ID NO. 5; the protein coded by the HRAS gene has an amino acid sequence shown as SEQ ID NO. 6; the protein coded by the FUBP1 gene has an amino acid sequence shown in SEQ ID NO. 7; the protein coded by the GATA3 gene has an amino acid sequence shown in SEQ ID NO. 8.

5. A kit, characterized in that: comprising a serum protein marker as defined in any one of claims 1-4 for early screening and diagnosis of breast cancer.

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

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

8. The kit of any one of claims 5 to 7, 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.

9. The detection method using the serum protein marker for early screening and diagnosis of breast cancer according to claim 3, characterized in that: the method comprises the following steps:

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

10. The assay method using serum protein markers for early screening and diagnosis of breast cancer according to claim 9, wherein: 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;