CN112213487B - Application of ASTE1 in detection of esophageal squamous carcinoma - Google Patents
Application of ASTE1 in detection of esophageal squamous carcinoma Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine and molecular biology, and relates to application of ASTE1 in preparation of an esophageal squamous cell carcinoma detection kit. The invention utilizes a gene chip kit to establish a differential gene expression profile between asymptomatic people and esophageal squamous carcinoma tissues, uses a real-time quantitative PCR technology to measure the ASTE1mRNA level in the asymptomatic people and the esophageal squamous carcinoma tissues, and uses a double-sandwich ELISA method to detect the ASTE1 content in serum samples of the asymptomatic people and the esophageal squamous carcinoma patients. The result proves that ASTE1 can be used as a new serum marker of esophageal squamous cell carcinoma, can be further used for preparing an ELISA diagnostic kit for detecting esophageal squamous cell carcinoma, and provides important reference information for preoperative or early-stage diagnosis of esophageal squamous cell carcinoma.
Description
Technical Field
The invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to application of ASTE1 in detection of esophageal squamous cell carcinoma.
Background
About 50 million patients with esophageal cancer newly occur in China every year, more than half of the patients occur in China, the state city of forest in Henan province is China, the incidence rate and death rate of esophageal cancer in the world are the highest, the incidence rate is 100 times higher than that of western countries, and the esophageal cancer is still the main cause of death related to tumors in the region. Because early patients lack specific symptoms, about 90 percent of the patients are in the middle and late stages at the first visit, and the 5-year survival rate of the patients in the middle and late stages is only about 10 percent, the prognosis of the patients with esophageal cancer is poor. However, the 5-year survival rate of early esophageal cancer patients can reach about 90%. Therefore, the screening and establishment of the index and the method for early detection and diagnosis of esophageal cancer in a large-scale population are the key points for improving the early detection rate of esophageal cancer and reducing the death rate.
The ASTE1 gene is located on chromosome 3q22.1, has 704 amino acids, encodes asteroid synprotein 1, and may play a role in Epidermal Growth Factor Receptor (EGFR) signaling, i.e., a complex pathway with important roles in intercellular communication, cell fate, and proliferation, through sequence similarity with the drosophila asteroid gene. There is no report of primary disease-related mutation in ASTE1, but in recent years, ASTE1 has been reported to be closely related to the occurrence of colorectal cancer, but the biological function of ASTE in esophageal squamous cell carcinoma has not been reported.
Disclosure of Invention
The invention aims to provide application of an esophageal squamous carcinoma related serum marker ASTE1 protein in preparation of an esophageal squamous carcinoma detection kit, and correspondingly, application of a molecule capable of specifically detecting the expression level of ASTE1 in preparation of the esophageal squamous carcinoma detection kit.
Based on the purpose, the invention adopts the following technical scheme:
application of molecules capable of specifically detecting ASTE1 expression level in preparation of esophageal squamous cell carcinoma detection kits.
In the above applications, the molecule capable of specifically detecting the expression level of ASTE1 is a nucleic acid or a protein.
The nucleotide sequence of the ASTE1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the ASTE1 protein is shown as SEQ ID NO. 2.
In the above application, a nucleic acid capable of specifically detecting the expression level of ASTE1 is used as a primer.
In the above application, the primer for detecting the expression level of ASTE1 comprises a forward primer and a reverse primer, wherein the forward primer is: 5'-GAGTTCTTCACTGATTTGAAGTTGC-3', respectively; the reverse primer is as follows: 5'-TTGTAAGCTTTTTATCTGAAATGTC-3' are provided.
In the above applications, the protein capable of specifically detecting the expression level of ASTE1 is an antibody.
As another claimed subject matter, the application of ASTE1 protein in preparing an esophageal squamous carcinoma detection kit.
The ASTE1 protein is used for preparing an esophageal squamous carcinoma detection kit, and the detection refers to detection by an enzyme-linked immunosorbent assay (ELISA).
As another claimed subject matter, a kit for detecting esophageal squamous carcinoma is an ELISA kit, and the kit comprises a solid carrier and a tumor associated antigen ASTE1 protein coated on the solid carrier.
Further, the kit further comprises a sample diluent, a second antibody diluent, negative control serum, positive control serum, a washing solution, a color development solution and a stop solution.
Further, the positive control serum was ASTE1 positive control serum; the negative control serum was ASTE1 negative control serum.
Compared with the prior art, the invention has the following beneficial effects:
the inventor of the application establishes a differential gene expression profile between asymptomatic people and esophageal squamous carcinoma tissues by arranging a large amount of cardia cancer clinical diagnosis and treatment, pathology and follow-up information bases and using an Affymetrix GeneChip Human U133Plus2.0 gene chip, and finds that ASTE1 is closely related to esophageal squamous carcinoma. Based on the findings, Primer ExpressTM1.0 software is used for designing oligonucleotide primers of the ASTE1 gene, real-time quantitative PCR is used for detecting the mRNA levels of the ASTE1 in cancer tissues and normal tissues, and the detection result shows that the ASTE1mRNA levels in tissues of esophageal squamous cell carcinoma patients are remarkably increased compared with tissues of asymptomatic people, so that the ASTE1 can be used as a detection marker for preparing a real-time quantitative PCR kit, and the real-time quantitative PCR kit can detect the expression level of the ASTE1mRNA in tissues to be detected, thereby providing important indication for preoperative or early diagnosis of the esophageal squamous cell carcinoma.
The detection takes ASTE1 expression product mRNA as a detection target, however, ASTE1 expression product ASTE1 protein is also expected to be a detection index for early detection, diagnosis, treatment and prognosis judgment of esophageal squamous cell carcinoma. Based on this, the ASTE1 protein is used as an antigen to be coated in an ELISA kit, the content of the autoantibody in human serum is detected by using the ELISA kit to detect esophageal squamous cell carcinoma, the detection sensitivity is up to 82.8%, the specificity is up to 90.0%, and the application prospect is good.
Drawings
FIG. 1 shows the relative expression amounts of mRNA in cancer tissue and normal tissue in example 1 and example 2, respectively, using a gene chip and real-time quantitative PCR.
FIG. 2 is the relative expression amounts of mRNA in serum of different esophageal cancer types and mRNA in serum of normal human in example 2.
FIG. 3 is the concentration of ASTE1 protein in the serum of patients with esophageal squamous carcinoma at different stages in example 3.
Detailed Description
Example 1 Gene profiling between asymptomatic individuals and esophageal squamous carcinoma tissue Using Gene chips
First, experiment sample
Through the standard arrangement of 50 ten thousand cases of esophageal cancer and cardiac cancer clinical diagnosis, pathology and follow-up information bases, 10 cases of esophageal squamous carcinoma samples are finally selected and included into the standard: firstly, receiving a radical operation treatment of esophageal squamous carcinoma; secondly, pathological diagnosis after operation is esophageal squamous carcinoma; thirdly, the follow-up visit data is complete; fourthly, preoperative serum and postoperative tissue specimens exist; all specimens come from the same municipal hospital. In 10 samples of esophageal squamous carcinoma, the diagnosis time of all patients is 1 month to 12 months in 2019, 6 cases in men and 4 cases in women; mean diagnostic age 60.5 ± 4.3 years; age range 55-75 years; wherein the composition is in the form of medulla 3, mushroom 2, ulcer 3, and narrowing 2. Normal esophageal tissue samples from 5 control groups, inclusion criteria: diagnosing a healthy outpatient through pathological biopsy; ② there is serum before biopsy and tissue specimen after biopsy. In 5 normal esophageal tissue samples, the diagnosis time is 1 month 2019 to 12 months 2019, 3 men and 2 women have the average diagnosis age of 56.5 +/-3.8 years and the age range is 51-70 years.
Second, Experimental methods
1. Total RNA in esophageal squamous carcinoma tissue was extracted by the one-step method using Trizol (Invitrogen, Gaithersburg, MD, USA), and further column-purified using RNeasy Micro kit (Qiagen), and quality and amount of RNA were evaluated using Agilent Bioanalyzer 2100 (Agilent Technologies); and simultaneously extracting total RNA in normal esophageal tissues as a control in chip analysis.
2. The RNA extracted and purified as described above was subjected to reverse transcription of total RNA using One-Cycle cDNA Synthesis kit (Affymetrix) to obtain cDNA, and the cDNA was purified using GeneChip Sample clear Module column (Affymetrix).
3. Second round amplification of double stranded cDNA the GeneChip IVT Label-ing kit (Affymetrix) was used to generate labeled cRNA. The labeled product was fragmented and hybridized with Affymetrix GeneChip Human U133Plus2.0array, which required 500ml of 42 ℃ hybridization solution (4 XSSC NaCl-Na citrate solution, 0.2% SDS sodium dodecyl sulfate, 25% formamide) overnight, finally, the labeled cRNA was stained on GeneChip Fluidics Station 450(Affymetrix), the dried slide was scanned with GeneChip Scanner 30007G (Affymetrix), scanning was performed with 635nm and 532nm channels, PMT value was adjusted to 600, and the scanned raw data was processed with GeneChip Operating Software (GC0S) 1.4.
4. Finally, a quality control report was made using Bioconductor and a data model was generated using rma (robust multiciphilaverage) method, genes with different expression were determined by setting the false discovery rate to <0.l, and the threshold levels were finally set to >2 and <0.5 fold difference by comparing the average expression levels of each group.
Third, experimental results
According to the invention, different gene expression profiles are established among healthy asymptomatic people and tissues of esophageal squamous cell carcinoma patients, and a molecular mechanism of esophageal squamous cell carcinoma pathogenesis can be disclosed on a transcription level. The results of the gene chip are shown in FIG. 1, and the expression of ASTE1 gene in esophageal squamous cell carcinoma is 67 times that of healthy asymptomatic people.
Fourth, conclusion
The expression of the ASTE1 gene in esophageal cancer tissues is higher than that of healthy asymptomatic human esophageal tissues, so that ASTE1 can be used as an esophageal cancer detection marker, and the indicating effect of ASTE1 as the esophageal cancer detection marker is further verified by the ASTE1mRNA expression level in a tissue sample or the ASTE1 protein expression level in a serum sample.
Example 2 application of primer sequences of ASTE1 in real-time quantitative PCR kit
First, experiment sample
Through the standard arrangement of 50 ten thousand cases of esophageal cancer and cardiac cancer clinical diagnosis, pathology and follow-up information bases, 10 cases of esophageal squamous carcinoma samples are finally selected and included into the standard: firstly, receiving a radical operation treatment of esophageal squamous carcinoma; secondly, pathological diagnosis after operation is esophageal squamous carcinoma; thirdly, the follow-up visit data is complete; fourthly, preoperative serum and postoperative tissue specimens exist; all specimens come from the same municipal hospital. In 10 samples of esophageal squamous carcinoma, the diagnosis time of all patients is 1 month to 12 months in 2019, 6 cases in men and 4 cases in women; mean diagnostic age 60.5 ± 4.3 years; age range 55-75 years; wherein the composition is in the form of medulla 3, mushroom 2, ulcer 3, and narrowing 2. Normal esophageal tissue samples from 5 control groups, inclusion criteria: diagnosing a healthy outpatient through pathological biopsy; ② there is serum before biopsy and tissue specimen after biopsy. In 5 normal esophageal tissue samples, the diagnosis time is 1 month 2019 to 12 months 2019, 3 men and 2 women have the average diagnosis age of 56.5 +/-3.8 years and the age range is 51-70 years.
Second, Experimental methods
Total RNA was isolated from the tissue sample using Trizol reagent from Invi trogen, and then synthesized into complementary DNA using QuantiTect reverse transcription system (Qiagen).
Oligonucleotide primers for the human ASTE1 gene were designed by Primer expression TM1.0 software (Applied Biosystems), and the sequences of the oligonucleotide primers included forward and reverse primers, and the specific sequences were as follows:
the sequence of the forward primer is: 5'-GAGTTCTTCACTGATTTGAAGTTGC-3', respectively;
the reverse primer has the sequence: 5'-TTGTAAGCTTTTTATCTGAAATGTC-3' are provided.
PCR amplification was performed using QuantiTect SYBR green PCR kit (Qiagen). All reactions were performed on a MicroAmp Optical96-well platform using a 7500Sequence Detector (applied Biosystems) and SYBR fluorescence was measured in each amplification phase, Cycle Threshold (CT) was used to determine the relative amount of RNA, distilled water was used as a negative control for the entire procedure, the relative amount of target gene at each sample transcript level was expressed using 2-ACT, CT reflected the difference between target gene and reference gene (human GAPDH), and all real-time quantitative PCR reactions were repeated twice.
Third, experimental results
The real-time quantitative PCR results are shown in FIG. 1, and the results show that the ASTE1mRNA level of esophageal squamous carcinoma is 89 times higher than that of healthy asymptomatic people, which is completely consistent with the results of the gene chip.
Further, the level of ASTE1mRNA in the tissues of different general types of esophageal squamous carcinomas including medullary, mushroom, ulcer and narrowing types was examined, and the results are shown in FIG. 2, which confirmed that all of the 4 general types of esophageal squamous carcinomas have high level of ASTE1mRNA expression. Wherein ASTE1mRNA levels of medullary, mushroom, ulcer and narrowed esophageal squamous carcinoma are 89(P <0.001), 92(P <0.001), 97(P <0.001) and 90(P <0.001) times of tissues of healthy asymptomatic population, respectively.
Fourth, conclusion
The ASTE1 gene has higher expression in the esophageal cancer tissues of medullary esophageal squamous carcinoma, mushroom-type esophageal squamous carcinoma, ulcer-type esophageal squamous carcinoma and narrowed esophageal squamous carcinoma than the esophageal cancer tissues of a healthy asymptomatic human body, so that the ASTE1 specific primer sequence can be applied to a real-time quantitative PCR kit, and the real-time quantitative PCR kit is utilized to screen and diagnose the esophageal cancer.
Example 3 application of ASTE1 protein in ELISA detection kit
Through the standard arrangement of 50 ten thousand cases of esophageal cancer and cardiac cancer clinical diagnosis, pathology and follow-up information bases, 1000 cases of esophageal squamous carcinoma samples are finally selected and included into the standard: firstly, receiving a radical operation treatment of esophageal squamous carcinoma; secondly, pathological diagnosis after operation is esophageal squamous carcinoma; thirdly, the follow-up visit data is complete; fourthly, preoperative serum and postoperative tissue specimens exist; all specimens come from the same municipal hospital. In 1000 samples of esophageal squamous carcinoma, the diagnosis time of all patients is 1 month to 12 months in 2018, 546 patients in men and 454 patients in women; mean diagnostic age 60.5 ± 7.3 years; the age range is 52-86 years; among them, there are 196 cases in stage I, 514 cases in stage II, 222 cases in stage III and 68 cases in stage IV of esophageal cancer. Esophageal squamous carcinoma 100 control normal esophageal tissue samples, inclusion criteria: diagnosing a healthy outpatient through pathological biopsy; ② there is serum before biopsy and tissue specimen after biopsy. In 100 esophageal tissue samples, the diagnosis time is 1 month to 12 months in 2018, 69 men and 31 women have the average diagnosis age of 58.2 +/-5.4 years and the age range is 51-78 years.
The indirect enzyme-linked immunity method is characterized by that the antigen (ASTE 1 protein) is linked to solid-phase carrier, the antibody to be tested in the sample is combined with it to form solid-phase antigen tested compound, then the enzyme-labeled secondary antibody is combined with the antibody in the solid-phase antigen tested antibody compound to form solid-phase antigen tested antibody enzyme-labeled secondary antibody compound, then the colour development degree after adding colour-developing agent is measured, and the content of antibody to be tested is determined. The invention prepares an ELISA kit for detecting esophageal squamous cell carcinoma related serum markers according to the principle of indirect enzyme-linked immunosorbent assay.
Reagents and materials
1. Preparation of ASTE1 protein
(1) Constructing a recombinant prokaryotic expression plasmid of esophageal squamous carcinoma related protein ASTE1 by using a gene cloning technology;
(2) respectively transforming the constructed recombinant prokaryotic expression plasmids into escherichia coli B121 (DE 3), and inducing the expression of the target protein by IPTG (isopropyl thiogalactoside);
(3) according to the label carried by the target protein, the target protein is purified by adopting a traditional corresponding purification scheme;
(4) protein concentration was measured by Bradford method, and immunological activity of the purified protein was identified by Western Blot
2. Antigen coating solution: carbonate buffer, pH = 9.6;
3. sealing liquid: PBST buffer containing 2% (W/V) BSA;
4. enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein;
5.96-well ELISA plate: purchased from Abcam corporation;
6. color developing solution A: 0.02% (W/V) TMB, formulation: dissolving 0.005g of methylbenzidine (TMB) in 25ml of deionized water;
7. color developing solution B: 0.006% (W/V) carbamide peroxide, formulation: fully dissolving 4.665g of citric acid and 4.40g of Na2HPO418 in 400ml of deionized water, adding 3.2ml of 0.75% urea hydrogen peroxide, adjusting the pH value to 5.0-5.5, adding deionized water to a constant volume of 500ml, uniformly mixing, and storing at 4 ℃;
8. positive control serum: inactivated ASTE1 antibody positive human serum;
9. negative control serum: inactivated ASTE1 antibody negative human serum;
ASTE1 antibody standard.
Secondly, preparing an antigen-coated ELISA plate
(1) Preparing an esophageal squamous carcinoma related antigen solution: dissolving ASTE1 protein in an antigen coating solution, fully and uniformly mixing, and preparing into an antigen solution, wherein the concentration of the ASTE1 protein is 0.75 mu g/ml;
(2) coating an enzyme label plate: adding the prepared ASTE1 antigen solution into spotting wells of a 96-well enzyme label plate, wherein the loading amount is 100 mul/well, 3 positive control wells and 4 negative control wells are arranged, and 1 blank control well is reserved. ASTE1 antigen solution is coated in 3 positive control wells and 4 negative control wells, and only coating solution is added in 1 blank control well; removing the coating solution after overnight standing at 4 ℃;
(3) and (3) sealing: adding a sealing solution into the coated sample application holes of the 96-hole enzyme label plate, incubating for 2 hours at room temperature, and removing the redundant sealing solution;
(4) and (3) placing the treated 96-hole enzyme label plate in a drying box at 37 ℃ for drying, packaging, and storing at 4 ℃ for later use.
Composition of ELISA kit
(1) 96-hole enzyme label plate coated by antigen;
(2) ASTE1 antibody standard;
(3) positive control serum: inactivated ASTE1 antibody positive human serum;
(4) negative control serum: inactivated ASTE1 antibody negative human serum;
(5) sample diluent: PBST buffer containing 1% (W/V) BSA;
(6) enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein, purchased from Abcam;
(7) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;
(8) color development liquid: the developing solution consists of a developing solution A and a developing solution B, wherein the developing solution A is 0.02% (W/V) TMB, and the developing solution B is 0.006% (W/V) carbamide peroxide;
(9) stopping liquid: 10% concentrated sulfuric acid;
(10) washing liquid: PBST (phosphate) buffer 0.01M, pH 7.2.
Fourth, detection
(1) Preparing reagents in an ELISA kit, and diluting 1000 cases of esophageal squamous cell carcinoma patients, 100 cases of healthy human serum samples and control serum by using sample diluent in a 10-fold manner;
(2) and adding 100 microliters of diluted positive control serum into the positive control hole, adding 100 microliters of diluted negative control serum into the negative control hole, taking the rest reaction holes coated with ASTE1 antigen as sample holes to be detected, and adding 100 microliters of sample to be detected into each reaction hole. The blank control hole is not added with the sample, and the operation of the rest steps is the same as that of the sample hole to be detected. 5 standard solutions with different concentrations are prepared by using the sample diluent and an ASTE1 antibody standard, and a group of 5 standard holes are simultaneously arranged, and 100 microliters of a series of standard solutions with different concentrations are respectively added into the standard holes for drawing a standard curve. Incubating for 2 hours at 37 ℃ after the sample loading is finished;
(3) removing redundant liquid in the reaction hole, and washing for 5 times by using a washing solution;
(4) adding 0.1ml of freshly diluted enzyme-labeled secondary antibody into each reaction hole, and incubating at 37 ℃ for 1 hour;
(5) removing redundant liquid in the reaction hole, and washing for 5 times by using a washing solution;
(6) mixing the color development liquid A and the color development liquid B in an equal volume of 1:1, adding 100 microliters of the mixed color development liquid into each reaction hole, and reacting for 30min at 37 ℃;
(7) add 90. mu.l of stop solution to each reaction well, stop the color development, and read the absorbance A at 450 nm.
(8) And (4) judging a result: and connecting the values of the concentrations point by taking the concentration of the standard substance as an abscissa and the absorbance as an ordinate to obtain an S-shaped standard curve for quantitative determination.
The threshold for serum ASTE1 antibody levels was set at 0pg/ml, and any serum with ASTE1 antibody concentrations greater than this was considered positive.
Fifth, experimental results
The serum concentrations of ASTE1 antibody in 100 healthy asymptomatic persons and 1000 patients with esophageal squamous cell carcinoma are shown in table 1 and fig. 3, and the detection positive rate of ASTE1 antibody in 100 healthy asymptomatic persons is only 9%. In 196 serum samples of the patients with esophageal squamous carcinoma of stage I, 154 serum samples detected ASTE1 antibody, and the positive rate was 78.6%; 431 serum samples of 514 esophageal squamous carcinoma patients in the II stage detect ASTE1 antibody, and the positive rate is 83.9%; ASTE1 antibody was detected in 181 sera of 222 patients with esophageal squamous carcinoma at stage III, and the positive rate was 81.5%; ASTE1 antibody was detected in 62 sera of 68 stage IV esophageal squamous carcinoma patients. As can be seen from FIG. 3, the concentration of ASTE1 protein in the serum of patients with esophageal squamous carcinoma is much higher than that of healthy asymptomatic people, and the concentration of ASTE1 protein in the serum of patients shows an increasing trend as the malignancy of esophageal squamous carcinoma is increased.
From the results of Table 1: of 1000 patients with esophageal squamous carcinoma, 828 patients were positive and 172 patients were negative, and 100 patients with esophageal squamous carcinoma were negative and 9 patients with 91 patients and 91 patients with normal control. From the data, the expression level of ASTE1 antibody in serum is detected by ELISA method to diagnose esophageal squamous cell carcinoma, the sensitivity is as high as 82.8%, and the specificity is as high as 90.0%.
Sixth, conclusion
The results show that the ASTE1 protein can also be used as a detection marker of esophageal squamous cell carcinoma, and the content of the ASTE1 antibody in the serum to be detected is detected by using an ELISA kit prepared from the ASTE1 protein, so that the esophageal squamous cell carcinoma can be quickly, simply and quickly diagnosed, the detection sensitivity of the esophageal squamous cell carcinoma reaches 82.8 percent, and the detection specificity reaches 90.0 percent.
Sequence listing
<110> first subsidiary Hospital of Zhengzhou university
The First Affiliated Hospital of Xinjiang Medical University
ZHEJIANG CANCER Hospital
Linzhou People's Hospital
Application of <120> ASTE1 in detection of esophageal squamous carcinoma
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2115
<212> DNA
<213> human (Homo sapiens)
<400> 1
atgggtatcc gaggactaat gagttttgtg gaagatcata gtaatgagtt cttcactgat 60
ttgaagttgc gggacacaaa aattgtcatt gatggttatg ctcttttcca ccgtctttgc 120
ttcagttcaa acttggatct ccggtatgga ggggactatg attcttttgc agatgttgta 180
caaaaattct ttgaatcact gtttgcttgt aatatatgcc catatgttgt attagatgga 240
ggatgtgaca tttcagataa aaagcttaca actttaaagg atagagctag agagaagatc 300
cagatggccc attccctttc tgttggtggg agtgggtacg tatgtccctt actcatccgg 360
gaagtattca tacaggtttt gatcaagctg cgggtgtgtt ttgtccagtg cttttcagaa 420
gcagatcggg acattatgac acttgctaac cattggaatt gccctgtgtt atcatcagat 480
agtgactttt gcatttttga cctgaaaact gggttttgcc cattgaatag ctttcagtgg 540
agaaatatga acactattaa gggcacacaa aactatatcc ctgccaaatg cttttccctt 600
gatgcattct gccatcactt cagcaatatg aataaagctc tactacctct ctttgcggtg 660
ctatgtggaa atgaccatgt taatctaccc atcatggaga cattcttaag taaagcgcgt 720
cttcctcttg gagctaccag ttctaaaggg aggagacacc accgaatcct gggacttctg 780
aattggttgt ctcattttgc caaccctacc gaagcactag ataatgttct gaaatacctc 840
ccaaaaaagg atcgagaaaa tgttaaggaa cttctctgct gttccatgga agaataccaa 900
cagtcccagg tgaagctaca ggacttcttc cagtgtggta cttatgtctg tccagatgcc 960
ttgaatcttg gtttaccaga atgggtatta gtggctttag ctaaaggcca gctatctcct 1020
ttcatcagtg atgctttggt cctaagacgg accattcttc ccacacaggt ggaaaacatg 1080
cagcaaccaa atgcccacag aatatctcag cccatcaggc aaatcatcta tgggcttctt 1140
ttaaatgcct caccacatct ggacaagaca tcctggaatg cattgcctcc tcagcctcta 1200
gctttcagtg aagtggaaag gattaataaa aatatcagaa cctcaatcat tgatgcagta 1260
gaactggcca aggatcattc tgacttaagc agattgactg agctctcctt gaggaggcgg 1320
cagatgcttc tgttagaaac cctgaaggtg aaacagacca ttctggagcc aatccctact 1380
tcactgaagt tgcccattgc tgtcagttgc tactggttgc agcacaccga gaccaaagca 1440
aagctacatc atctacaatc cttactgctc acaatgctag tggggccctt gattgccata 1500
atcaacagcc ctggtaagga agagctgcag gaagatggtg ctaagatgtt gtatgcagag 1560
ttccaaagag tgaaggcgca gacacggctg ggcacaagac tggacttaga cacagctcac 1620
atcttctgtc agtggcagtc ctgtctccag atggggatgt atctcaacca gctgctgtcc 1680
actcctctcc cagagccaga cctaactcgc cccagtgcct tcaatacttt gaatgaggat 1740
atatggacgc atacaaaata tggtgggact ttacatgttt caagactgta cagtggaagc 1800
ctggtgcacg gactatgcca gcaactgcta gcatcgacct ctgtagaaag tctcctgagc 1860
atatgtcctg aggctaagca actttatgaa tatctattca atgccacaag gtcatatgcc 1920
cccgctgaaa tattcctacc aaaaggtaga tcaaattcaa aaaaaaaaag gcagaagaaa 1980
cagaatacca gctgttctaa gaacagaggg agaaccactg cacacaccaa gtgttggtat 2040
gagggaaaca accggtttgg gttgttaatg gttgaaaact tagaggaaca tagtgaggcc 2100
tccaacattg aataa 2115
<210> 2
<211> 704
<212> PRT
<213> human (Homo sapiens)
<400> 2
Met Gly Ile Arg Gly Leu Met Ser Phe Val Glu Asp His Ser Asn Glu
1 5 10 15
Phe Phe Thr Asp Leu Lys Leu Arg Asp Thr Lys Ile Val Ile Asp Gly
20 25 30
Tyr Ala Leu Phe His Arg Leu Cys Phe Ser Ser Asn Leu Asp Leu Arg
35 40 45
Tyr Gly Gly Asp Tyr Asp Ser Phe Ala Asp Val Val Gln Lys Phe Phe
50 55 60
Glu Ser Leu Phe Ala Cys Asn Ile Cys Pro Tyr Val Val Leu Asp Gly
65 70 75 80
Gly Cys Asp Ile Ser Asp Lys Lys Leu Thr Thr Leu Lys Asp Arg Ala
85 90 95
Arg Glu Lys Ile Gln Met Ala His Ser Leu Ser Val Gly Gly Ser Gly
100 105 110
Tyr Val Cys Pro Leu Leu Ile Arg Glu Val Phe Ile Gln Val Leu Ile
115 120 125
Lys Leu Arg Val Cys Phe Val Gln Cys Phe Ser Glu Ala Asp Arg Asp
130 135 140
Ile Met Thr Leu Ala Asn His Trp Asn Cys Pro Val Leu Ser Ser Asp
145 150 155 160
Ser Asp Phe Cys Ile Phe Asp Leu Lys Thr Gly Phe Cys Pro Leu Asn
165 170 175
Ser Phe Gln Trp Arg Asn Met Asn Thr Ile Lys Gly Thr Gln Asn Tyr
180 185 190
Ile Pro Ala Lys Cys Phe Ser Leu Asp Ala Phe Cys His His Phe Ser
195 200 205
Asn Met Asn Lys Ala Leu Leu Pro Leu Phe Ala Val Leu Cys Gly Asn
210 215 220
Asp His Val Asn Leu Pro Ile Met Glu Thr Phe Leu Ser Lys Ala Arg
225 230 235 240
Leu Pro Leu Gly Ala Thr Ser Ser Lys Gly Arg Arg His His Arg Ile
245 250 255
Leu Gly Leu Leu Asn Trp Leu Ser His Phe Ala Asn Pro Thr Glu Ala
260 265 270
Leu Asp Asn Val Leu Lys Tyr Leu Pro Lys Lys Asp Arg Glu Asn Val
275 280 285
Lys Glu Leu Leu Cys Cys Ser Met Glu Glu Tyr Gln Gln Ser Gln Val
290 295 300
Lys Leu Gln Asp Phe Phe Gln Cys Gly Thr Tyr Val Cys Pro Asp Ala
305 310 315 320
Leu Asn Leu Gly Leu Pro Glu Trp Val Leu Val Ala Leu Ala Lys Gly
325 330 335
Gln Leu Ser Pro Phe Ile Ser Asp Ala Leu Val Leu Arg Arg Thr Ile
340 345 350
Leu Pro Thr Gln Val Glu Asn Met Gln Gln Pro Asn Ala His Arg Ile
355 360 365
Ser Gln Pro Ile Arg Gln Ile Ile Tyr Gly Leu Leu Leu Asn Ala Ser
370 375 380
Pro His Leu Asp Lys Thr Ser Trp Asn Ala Leu Pro Pro Gln Pro Leu
385 390 395 400
Ala Phe Ser Glu Val Glu Arg Ile Asn Lys Asn Ile Arg Thr Ser Ile
405 410 415
Ile Asp Ala Val Glu Leu Ala Lys Asp His Ser Asp Leu Ser Arg Leu
420 425 430
Thr Glu Leu Ser Leu Arg Arg Arg Gln Met Leu Leu Leu Glu Thr Leu
435 440 445
Lys Val Lys Gln Thr Ile Leu Glu Pro Ile Pro Thr Ser Leu Lys Leu
450 455 460
Pro Ile Ala Val Ser Cys Tyr Trp Leu Gln His Thr Glu Thr Lys Ala
465 470 475 480
Lys Leu His His Leu Gln Ser Leu Leu Leu Thr Met Leu Val Gly Pro
485 490 495
Leu Ile Ala Ile Ile Asn Ser Pro Gly Lys Glu Glu Leu Gln Glu Asp
500 505 510
Gly Ala Lys Met Leu Tyr Ala Glu Phe Gln Arg Val Lys Ala Gln Thr
515 520 525
Arg Leu Gly Thr Arg Leu Asp Leu Asp Thr Ala His Ile Phe Cys Gln
530 535 540
Trp Gln Ser Cys Leu Gln Met Gly Met Tyr Leu Asn Gln Leu Leu Ser
545 550 555 560
Thr Pro Leu Pro Glu Pro Asp Leu Thr Arg Pro Ser Ala Phe Asn Thr
565 570 575
Leu Asn Glu Asp Ile Trp Thr His Thr Lys Tyr Gly Gly Thr Leu His
580 585 590
Val Ser Arg Leu Tyr Ser Gly Ser Leu Val His Gly Leu Cys Gln Gln
595 600 605
Leu Leu Ala Ser Thr Ser Val Glu Ser Leu Leu Ser Ile Cys Pro Glu
610 615 620
Ala Lys Gln Leu Tyr Glu Tyr Leu Phe Asn Ala Thr Arg Ser Tyr Ala
625 630 635 640
Pro Ala Glu Ile Phe Leu Pro Lys Gly Arg Ser Asn Ser Lys Lys Lys
645 650 655
Arg Gln Lys Lys Gln Asn Thr Ser Cys Ser Lys Asn Arg Gly Arg Thr
660 665 670
Thr Ala His Thr Lys Cys Trp Tyr Glu Gly Asn Asn Arg Phe Gly Leu
675 680 685
Leu Met Val Glu Asn Leu Glu Glu His Ser Glu Ala Ser Asn Ile Glu
690 695 700
Claims (6)
1. Application of molecules capable of specifically detecting ASTE1 expression level in preparation of esophageal squamous cell carcinoma detection kits.
2. The use of claim 1, wherein the molecule is a nucleic acid or a protein.
3. The use of claim 2, wherein the nucleic acid is a primer.
4. The use of claim 2, wherein the protein is an antibody.
Application of ASTE1 protein in preparation of an esophageal squamous cell carcinoma detection kit.
6. The use of claim 5, wherein said detection is by enzyme-linked immunosorbent assay.
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| CN114324897A (en) * | 2022-01-10 | 2022-04-12 | 亚科因(武汉)生物技术有限公司 | Method for producing human reference protein for western-blot experiment and application thereof |
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| US20110318738A1 (en) * | 2008-12-23 | 2011-12-29 | University Of Utah Research Foundation | Identification and regulation of a novel dna demethylase system |
| CN103149369B (en) * | 2013-03-20 | 2015-08-19 | 江苏元化生命科技有限公司 | A kind of protein-chip and kit thereof detecting esophageal squamous cell carcinoma mark |
| PL3752501T3 (en) * | 2018-02-13 | 2023-08-21 | Gilead Sciences, Inc. | Pd-1/pd-l1 inhibitors |
| CN110785499A (en) * | 2018-05-25 | 2020-02-11 | 伊鲁米那股份有限公司 | Circulating RNA identification specific for preeclampsia |
| CN111323586B (en) * | 2020-02-27 | 2020-10-09 | 郑州大学第一附属医院 | ELISA kit for early diagnosis of esophageal squamous cell carcinoma |
| CN111551545B (en) * | 2020-05-25 | 2020-11-06 | 郑州大学第一附属医院 | Liquid biopsy ELISA kit for early screening of high risk group of esophageal cancer |
| CN111665358B (en) * | 2020-06-17 | 2021-01-05 | 郑州大学第一附属医院 | Application of NALCN protein in prognosis prediction of esophageal squamous cell carcinoma |
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