CN110305963A

CN110305963A - A kind of cancer of the esophagus molecular marker and its application

Info

Publication number: CN110305963A
Application number: CN201910663015.9A
Authority: CN
Inventors: 李秀敏; 周孝峰; 陈志国; 王伟隆; 庞丹; 卢奎; 张爱佳; 王素杰; 侯静晗; 高灿; 吕本杰; 闫子怡
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2019-07-22
Filing date: 2019-07-22
Publication date: 2019-10-08
Anticipated expiration: 2039-07-22
Also published as: CN110305963B

Abstract

The invention belongs to the technical field of immunoassay of cancer more particularly to a kind of cancer of the esophagus molecular marker and its applications.The present invention has differences expression using organization chip discovery PARK2 gene in patient with esophageal carcinoma cancerous tissue and Carcinoma side normal tissue, PARK2 is significantly reduced compared with cancer beside organism in the expression of human esophageal carcinoma, and the expression of PARK2 and cancer of the esophagus neoplasm staging are negatively correlated, in addition, by comparing the above differential expression of TCGA database cross validation.PARK2 is a kind of E3 ubiquitin ligase of RING-BETWEEN-RING (RBR) family, present invention firstly discovers that PARK2 gene expression is related to the cancer of the esophagus, pass through the expression of PARK2 in detection subject esophageal tissue, it can be determined that whether subject suffers from the cancer of the esophagus or judge that subject whether there is the risk with the cancer of the esophagus.

Description

A kind of cancer of the esophagus molecular marker and its application

Technical field

The invention belongs to the technical field of immunoassay of cancer more particularly to a kind of cancer of the esophagus molecular marker and its answer With.

Background technique

China is one of highest country of Incidence of esophageal cancer, the death rate in the world.It is saved including Henan, Hebei, Shanxi etc. One band of Taihang mountain range of some areas is the traditional Esophageal Cancer region in China.However with oesophagus epidemiology severe situation Corresponding is the shortage of cancer of the esophagus early diagnosis, and most of patient has been in the cancer of the esophagus middle and advanced stage stage when medical, although There are many improvement in treatment means at present, however 5 years survival rates of patient are totally still hovered 15% or so, particularly with evening The effect of the phase cancer of the esophagus is not satisfactory.The early detection and treatment of the cancer of the esophagus are the effective means for improving the survival rate of patient.Cause This, a kind of method for finding early diagnosis cancer of the esophagus is of great significance to the control and intervention of the cancer of the esophagus.

Publication No. is to disclose a kind of molecular marked compound of esophageal squamous cell carcinoma in CN105734159A Chinese invention patent application, It is pointed out that the detection primer of CCKBR gene can be used as the diagnostic products of esophageal squamous cell carcinoma, but it is only using a small number of clinical The QPCR data of oesophagus sample illustrate in patients with esophageal squamous cell carcinoma body that CCKBR is in low expression, and and TCGA big-sample data is not used Library carries out cross validation, and lacks the verifying of protein level, still lacks accurately and effectively cancer of the esophagus molecular marker at present.

Summary of the invention

In view of the problems of the existing technology, the present invention provides a kind of cancer of the esophagus molecular marker and its applications.

The invention is realized in this way a kind of cancer of the esophagus molecular marker, the molecular marker includes PARK2 gene And/or the expression product of PARK2 gene.

Further, the coded sequence of the PARK2 gene is DNA sequence dna shown in SEQ ID NO.1；Or with SEQ ID The DNA sequence dna of DNA sequence dna hybridization and coding identical function protein that NO.1 is limited；Or have with two class DNA sequence dnas of restriction 70% or more homology, and encode the DNA molecular of identical function protein.

Further, the expression product of the PARK2 gene includes PARK2mRNA and/or PARK2 albumen.

Further, the PARK2 albumen includes the functional equivalent of PARK2 albumen and/or PARK2 albumen.

Further, the amino acid sequence of the PARK2 albumen is sequence shown in SEQ ID NO.2；Or by SEQ ID Amino acid sequence shown in NO.2 passes through the substitution and/or deletion and/or addition of several amino acid residues, or by SEQ ID Derived from amino acid sequence shown in NO.2, and with amino acid sequence amino with the same function shown in SEQ ID NO.2 Acid sequence.

A kind of cancer of the esophagus molecular marker a method as claimed in any one of claims 1 to 5 detects correlation RT-PCR in the preparation cancer of the esophagus Kit or real-time quantitative PCR kit or in situ hybridization kit or immunity detection reagent or genetic chip or protein chip Or the application in the drug of the treatment cancer of the esophagus.

Further, the RT-PCR kit or real-time quantitative PCR kit or in situ hybridization kit or high pass measure The testing product of sequence detection of platform is the product of PARK2 gene mRNA levels expression.

Further, the RT-PCR kit or real-time quantitative PCR kit include drawing for specific amplified PARK2 gene Object, the primer sequence of the specific amplified PARK2 gene is as shown in SEQ ID NO.3 and SEQ ID NO.4.

Further, the in situ hybridization kit includes the probe hybridized with PARK2 gene nucleic acid sequence；The gene core Piece includes the probe with the nucleic acid array hybridizing of PARK2 gene；The protein chip includes in conjunction with PARK2 protein-specific Antibody.

The drug of the treatment cancer of the esophagus includes PARK2 gene and/or the promotor of its expression product；The promotor is Promote the reagent of PARK2 gene expression or promotes the reagent of PARK2 gene expression product stability or improve PARK2 gene table Up to the reagent of lytic activity or the reagent of enhancing PARK2 gene expression product function；The examination for promoting PARK2 gene expression Agent is to promote the reagent of PARK2 genetic transcription or promote the reagent of PARK2 gene translation or promote the examination of PARK2 protein content The carrier of agent or reagent or carrying PARK2 gene containing PARK2 gene is formed by reagent or carries PARK2 gene Host cell is formed by reagent or the reagent containing PARK2 protein.

In conclusion advantages of the present invention and good effect are as follows:

PARK2 albumen belongs to a kind of E3 ubiquitin ligase of RING-BETWEEN-RING (RBR) family, and the present invention passes through TCGA database obtains the tumor tissues and Carcinoma side normal tissue transcript profile sequencing data of cancer of the esophagus tumor patient；According to acquisition Transcript profile sequencing data carries out analysis of gene differential expression, it is found that expression quantity of the PARK2 gene in cancerous tissue is substantially less than cancer Side tissue.PARK2 gene and PARK2 albumen can be used as the molecular marker of cancer of the esophagus early diagnosis as a result, use gene mark Will object, which carrys out diagnosis of esophageal cancer, has the characteristics that timeliness, specificity and sensitivity, to make patient that can know in disease early stage Disease risks take corresponding prevention and treatment measure for risk height.

The present invention is by a large amount of cancer of the esophagus cases analysis shows PARK2 gene is in patient with esophageal carcinoma and normal esophageal tissue Expression is had differences, present invention firstly discovers that PARK2 gene expression is related to the cancer of the esophagus, passes through detection human esophageal carcinoma chip The expression of middle PARK2, it can be determined that whether subject suffers from the cancer of the esophagus or judge that subject whether there is with the cancer of the esophagus Risk, so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject.The present invention is clinical esophageal squamous cell carcinoma (food The main Types of pipe cancer) accurate diagnosis provide potential molecular target, so that it is pre- to instruct clinician to provide to subject Anti- scheme or therapeutic scheme have important practical application value.Of the invention cancer of the esophagus detection, diagnose from molecular level into Row detection, compared to traditional detection means, gene diagnosis is more timely, more special, sensitiveer, and the early stage that can be realized the cancer of the esophagus examines It is disconnected.

The testing product of the PARK2 mrna expression is the RT-PCR testing product of PARK2 gene, determines in real time Measure PCR testing product, in situ hybridization testing product or high-flux sequence detection of platform product, the PARK2 expressing quantity Testing product be PARK2 albumen immune detection product or high-flux sequence detection of platform product.

The present invention chooses sample and carries out organization chip detection, TCGA database human esophageal carcinoma transcript profile sequencing data point Analysis, testing result are consistent.Therefore the mRNA that PARK2 gene in detection subject esophageal tissue can be tested by these expresses water Flat or PARK2 expressing quantity, and then may determine that whether subject suffers from the cancer of the esophagus or judge that subject whether there is Risk with the cancer of the esophagus.

The in situ hybridization testing product of PARK2 gene of the present invention includes the spy hybridized with PARK2 gene nucleic acid sequence Needle.In conjunction with the mRNA that in situ hybridization probe can be obtained with PARK2 genetic transcription, how much judgement samples of the probe of combination passed through The mRNA expression of PARK2 gene.

The high-flux sequence detection of platform product of PARK2 gene of the present invention includes genetic chip, wherein genetic chip Probe including the nucleic acid array hybridizing with PARK2 gene.Genetic chip generally comprises solid phase carrier and is fixed on solid phase load The oligonucleotide probe of body is particularly visited for detecting the oligonucleotides for PARK2 gene of PARK2 gene transcription level Needle, high-flux sequence platform is a kind of tool of special diagnosis of esophageal cancer, with the development of high throughput sequencing technologies, to one The detection of the PARK2 gene expression amount of people will become very easily work.

The high-flux sequence detection of platform testing product of PARK2 albumen of the present invention includes protein chip, wherein albumen Chip includes the antibody in conjunction with PARK2 protein-specific.High-flux sequence platform is a kind of work of special diagnosis of esophageal cancer Tool will become very easily the detection of the expression quantity of the PARK2 albumen of a people with the development of high throughput sequencing technologies Work.

Present invention finds the targets that PARK2 gene and its expression product can be used as esophageal carcinoma therapy, new for instructing The research and development of medicine.The drug of the treatment cancer of the esophagus of the invention includes PARK2 gene and/or the promotor of its expression product, promotor On the one hand the missing or deficiency of endogenic PARK2 albumen can be supplemented, to treat by the expression of raising PARK2 albumen Because of the cancer of the esophagus caused by PARK2 hypoproteinosis；On the other hand it can be used for promoting the activity or function of PARK2 albumen, thus Treat the cancer of the esophagus.

Promotor of the present invention is the reagent for promoting PARK2 gene expression, promotes PARK2 gene expression product stability Reagent, improve the active reagent of PARK2 gene expression product or enhance the reagent of PARK2 gene expression product function.It is logical It crosses the expression quantity for improving PARK2 albumen or improves the stability and validity of PARK2 albumen, can have and efficiently reach treatment Purpose.

The reagent of the present invention for promoting PARK2 gene expression is the reagent for promoting PARK2 genetic transcription, promotes PARK2 The reagent of gene translation or the reagent for promoting PARK2 protein content.By promoting a certain step in PARK2 Gene Expression Pathway The expression quantity of PARK2 gene can be effectively increased.

The reagent of the present invention for promoting PARK2 gene expression is the reagent containing PARK2 gene, carries PARK2 gene Carrier be formed by reagent, the host cell for carrying PARK2 gene is formed by reagent, or contains PARK2 protein Reagent.The level of patient's body PARK2 protein can directly or indirectly be increased by the means, and then improve or cure food Pipe cancer.

Detailed description of the invention

Fig. 1 is expression of the organization chip detection PARK2 gene in human esophageal carcinoma and normal esophageal tissue in embodiment 1 Situation comparison diagram；

Fig. 2 is that organization chip detects PARK2 albumen in the human esophageal carcinoma expression of different neoplasm stagings in embodiment 1 Comparison diagram；

Fig. 3 is to utilize TCGA database cross validation PARK2 in human esophageal carcinoma and normal esophageal tissue in embodiment 2 Differential expression figure.

Specific embodiment

In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention It is further elaborated, equipment used in each embodiment and test example and reagent unless otherwise specified, can be from business Approach obtains.Described herein specific examples are only used to explain the present invention, is not intended to limit the present invention.

The present invention discloses a kind of cancer of the esophagus molecular marker and its application, shown in each embodiment specific as follows.

Human esophageal carcinoma used in test example is that cancer of the esophagus emphasis open laboratory, Henan Province provides in the present invention, thoracic surgery Surgical resection therapy, preoperative without chemotherapy or radiotherapy, the primary cancerous tissue of 214 pieces of cancer of the esophagus and wherein 84 pieces of pairings are just Often feeding pipe epithelial tissue excision rear portion tissue be placed in formalin it is fixed after paraffin embedding and be sliced, remaining part in It is put into liquid nitrogen container and saves in operation half an hour.

Example 1 group knits the differential expression of chip detection PARK2 gene

1. collecting 214 patient with esophageal carcinoma human esophageal carcinomas and 84 corresponding cancer beside organism samples

All patients are preoperative without chemotherapy or radiotherapy, and diagnosis is confirmed through postoperative pathological result.By the cancer of the esophagus and cancer Tissue is unified to be fixed through 4% paraformaldehyde, and paraffin embedding is placed in 4 DEG C of preservations.The above wax stone entrusts Wuhan Seville biotech firm It makes organization chip (Tissue microarray, TMA).

1) dewaxing, aquation

Before dewaxing, organization chip is placed at room temperature 60 minutes or 60 DEG C of insulating boxs in toast 20 minutes.Detailed process Are as follows: organization chip, which is placed in dimethylbenzene, to be impregnated 10 minutes, is impregnated again 10 minutes after replacing dimethylbenzene；5 points are impregnated in dehydrated alcohol Clock；It is impregnated 5 minutes in 95% ethyl alcohol；It is impregnated 5 minutes in 70% ethyl alcohol.

2) antigen retrieval

Water-bath heat 0.01M sodium citrate buffer solution (pH6.0) to 95 DEG C or so, be put into organization chip heating 10~ 15 minutes.

3) immunohistochemical staining

Specific experiment scheme are as follows: PBS wash 2-3 times it is 5 minutes each；Normal Goat Serum confining liquid is added dropwise, room temperature 20 minutes, gets rid of Remove surplus liquid；Be added dropwise 50 μ l of primary antibody, be stored at room temperature 1 hour or 4 DEG C overnight or 37 DEG C 1 hour；4 DEG C overnight after need to be multiple at 37 DEG C Temperature 45 minutes；PBS washes 3 times 2 minutes every time；Drip secondary antibody 45-50 μ l, be stored at room temperature or 37 DEG C 1 hour；It can be added in secondary antibody 0.05% tween-20；PBS wash 3 times it is 5 minutes each；DAB develops the color 5-10 minutes, grasps dye levels under the microscope；PBS or Tap water rinses 10 minutes；Haematoxylin redyeing 2 minutes, hydrochloride alcohol differentiation；Tap water rinses 10-15 minutes；Be dehydrated, be transparent, Mounting, microscopy.

4) immunohistochemistry results are analyzed

For positive cell premised on the cell of quasi- label, positive expression must be in the specific antigen portion of cell in diagnosis Position, if the positive expression and untargeted cells at position where antigen not should not be used as judgment basis the positive yet.Institute as above PARK2 expression is stated in endochylema.According to cell cytosol positive coloring degree (antigenic content), weakly positive (+) can be divided into --- 1 point；In Etc. the positives (++) --- 2 points；Strong positive (+++) --- 3 points.According to cell cytosol positive cell quantity, can be divided into weakly positive (+, sun Property total number of cells are below 25%)；Moderate positive (++, positive cell sum is in 25%-49%)；Strong positive (+++, it is positive thin Born of the same parents' sum is 50% or more).According to above method using integral comprehensive metering.Calculation formula are as follows: (+) * 1+ (++) * 2+ (+++) * 3；Total value is (+) less than 1；Total value is that 1-1.5 person is (++)；It is (+++) that total value, which is greater than 1.5,；It is seen at random under mirror Examine 5-10 HPF.

2. experimental result

As a result as depicted in figs. 1 and 2, wherein Fig. 1 is that organization chip detects PARK2 gene in human esophageal carcinoma and positive often feeding Expression comparison diagram in tubing；Fig. 2 is that organization chip detects PARK2 albumen in the human esophageal carcinoma of different neoplasm stagings Expression comparison diagram.The results show that the protein level of PARK2 gene is aobvious in human esophageal carcinoma compared with normal esophageal tissue Writing reduces.PARK2 expression is related to cancer of the esophagus TNM stage, and neoplasm staging is higher, and PARK2 expression is lower.

Embodiment 2 analyzes expression of the PARK2 gene in TCGA database

1. data collection

The PARK2 that 185 human esophageal carcinomas and 11 cancer beside organisms are collected from TCGA database expresses modal data, analysis Expression of the PARK2 in human esophageal carcinoma and cancer beside organism；Draw box figure.

2. result

The expression of PARK2 as shown in figure 3, compared to the control group expression of the PARK2 in human esophageal carcinoma significantly lower.

The research discovery PARK2 gene of the above-mentioned two embodiment of the present invention is normal group by patient with esophageal carcinoma cancerous tissue and cancer Expression is had differences in knitting, PARK2 is significantly reduced compared with cancer beside organism in the expression of human esophageal carcinoma, and the table of PARK2 Up to cancer of the esophagus neoplasm staging negative correlation, in addition, by comparing the above differential expression of TCGA database cross validation.PARK2 It is a kind of E3 ubiquitin ligase of RING-BETWEEN-RING (RBR) family, present invention firstly discovers that PARK2 gene expression It is related to the cancer of the esophagus, pass through the expression of PARK2 in detection subject esophageal tissue, it can be determined that whether subject suffers from oesophagus Cancer or judge subject whether there is with the cancer of the esophagus risk.

Explanation of the embodiment 3 about PARK2 gene and its expression product

On the basis of above-mentioned two embodiment research discovery, inventor is developed with PARK2 gene or its expression product It is specific as shown in embodiment 4- embodiment 13 for the detection kit of core, genetic chip, protein chip and pharmaceutical preparation etc..

1. about PARK2 gene

The coded sequence of PARK2 gene is DNA sequence dna shown in SEQ ID NO.1.In the context of the invention, " PARK2 base Cause " includes the polynucleotides of any functional equivalent of PARK2 gene and PARK2 gene.PARK2 gene include at present PARK2 gene (Gene ID:5071) DNA sequence dna has 70% or more homology in Pubmed database, and encodes identical function The DNA sequence dna of protein.

The coded sequence of PARK2 gene includes any of the following DNA molecular:

(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table；

(2) hybridize under strict conditions with the DNA sequence dna that (1) limits and encode the DNA sequence dna of identical function protein；

(3) DNA sequence dna limited with (1) or (2) has 70% homology, preferably 90% or more homology, and encodes The DNA molecular of identical function protein.

2. about PARK2 gene expression product

PARK2 gene expression product includes the partial peptide of PARK2 albumen and PARK2 albumen." PARK2 albumen " includes Any functional equivalent of PARK2 albumen and PARK2 albumen.The functional equivalent includes PARK2 albumen conservative variation Protein or its active fragment or its reactive derivative, allelic variant, natural mutation, induced mutants, high or low Stringent condition under can be with the encoded protein of DNA of the DNA hybridization of PARK2.The partial peptide of the PARK2 albumen contain with The relevant functional domain of the cancer of the esophagus.

PARK2 albumen is the protein with following amino acid sequences:

(1) protein that the amino acid sequence shown in SEQ ID NO.2 in sequence table forms relates in the embodiment of the present invention And to PARK2 albumen be the protein with the amino acid sequence；

(2) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked Lose and/or addition and with the ammonia with the same function as shown in SEQ ID NO.2 of amino acid sequence shown in SEQ ID NO.2 Protein derived from base acid sequence.The number for the amino acid for replacing, lacking or adding is usually 1-50, preferably 1-30 It is a, more preferably 1-20, most preferably 1-10.

(3) there is at least 80% homology (also known as sequence identity) with amino acid sequence shown in SEQ ID NO.2, It is highly preferred that the homology with amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, Chang Wei 96%, 97%, 98%, the polypeptide that the amino acid sequence of 99% homology is constituted.

It is known in the art that the modification of one or more amino acid will not influence the function of protein in a protein.It is right It is to protect in the amino acid for changing single amino acids or small percentage or to individual additions, missing, insertion, the replacement of amino acid sequence Modification is kept, wherein the change of protein generates the protein with identity function.The conservative of intimate amino acid is provided to replace It changes and belongs to techniques well known.Such as it is obtained by the protein of one amino acid of addition or more amino acid modification The fusion protein of PARK2 albumen；For the peptide or protein with PARK2 protein fusion, there is no limit as long as resulting fusion The biological activity of albumen reservation PARK2 albumen.Similar relevant technological improvement belong to disclosure of the invention range and/ Or protection scope.

PARK2 albumen of the invention further includes the non-conservative modification to amino acid sequence shown in SEQ ID NO.2, as long as Protein by modification still is able to retain the biological activity of PARK2 albumen.It is mutated in such modification protein Amino acid number be usually 10 perhaps less such as 6 perhaps less such as 3 or less.

Inventor is developed based on the research discovery of embodiment 1 and embodiment 2 using PARK2 gene or its expression product as core Detection kit, genetic chip, protein chip and pharmaceutical preparation of the heart etc., it is specific as follows to state shown in embodiment 4 to embodiment 13.

Cancer of the esophagus detection, diagnostic preparation are RT-PCR kit in 4 the present embodiment of embodiment.

The kit includes the primer of following sequence:

SEQ ID NO.3:RT-PCR-PARK2-F:5 '-CACACCCCACCTCTGACAAG-3 '；

SEQ ID NO.4:RT-PCR-PARK2-R:5 '-CTAAGCAAATCACGTGGCGG-3 '；

SEQ ID NO.5:PCR-GAPDH-F:5 '-GAGAAGGCTGGGGCTCATTT-3 '；

SEQ ID NO.6:PCR-GAPDH-R:5 '-AGTGATGGCATGGACTGTGG-3 '.

Cancer of the esophagus detection, diagnosis or preparation are real-time quantitative PCR kit in 5 the present embodiment of embodiment.

The kit includes the primer of following sequence:

SEQ ID NO.7:PCR-PARK2-F:5 '-GTGTTTGTCAGGTTCAACTCCA-3 '；

SEQ ID NO.8:PCR-PARK2-R:5 '-GAAAATCACACGCAACTGGTC-3 '；

SEQ ID NO.5:PCR-GAPDH-F:5 '-GAGAAGGCTGGGGCTCATTT-3 '；

SEQ ID NO.6:PCR-GAPDH-R:5 '-AGTGATGGCATGGACTGTGG-3 '.

Cancer of the esophagus detection, diagnostic preparation are in situ hybridization kit in 6 the present embodiment of embodiment.In kit comprising with The probe of the nucleic acid array hybridizing of PARK2 gene.

Include the probe with the nucleic acid array hybridizing of PARK2 gene in the in situ hybridization kit.

It can be DNA, RNA, DNA-RNA chimera, PNA or other with the probe of the nucleic acid array hybridizing of PARK2 gene Derivative.There is no limit any as long as completing specific hybrid, specifically binding with purpose nucleotide sequence for the length of probe Length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of probe can be grown To 60,80,100,150,300 base-pairs or longer or even whole genes.Due to different probe lengths to hybridization efficiency, Signal specificity has different influences, and the length of the probe is typically at least 14 base-pairs, and longest is usually no more than 30 Base-pair, the length complementary with purpose nucleotide sequence are best with 15-25 base-pair.The probe self-complementary sequences are best Less than 4 base-pairs, in order to avoid influence hybridization efficiency.

Probe sequence used is as follows in the present embodiment:

SEQ ID NO.9:GCAGAGACCGTGGAGAAAAG.

Cancer of the esophagus detection, diagnostic preparation are genetic chip in 7 the present embodiment of embodiment.It include solid phase carrier in genetic chip And it is fixed on the PARK2 oligonucleotide probe of solid phase carrier.

It include solid phase carrier and the PARK2 oligonucleotide probe for being fixed on solid phase carrier in genetic chip.Wherein solid phase Carrier is slide, is purchased from VWR International company, sequence oligonucleotide probe are as follows: SEQ ID NO.9: GCAGAGACCGTGGAGAAAAG。

Cancer of the esophagus detection, the immunity detection reagent that diagnostic preparation is PARK2 albumen in 8 the present embodiment of embodiment.

Include antibody (the Santa Cruz article No. sc-32282 in conjunction with PARK2 protein-specific in kit；Abcam Article No. ab15494；Invitroge article No. PA1-751.).

Antibody can be monoclonal antibody, polyclonal antibody.The specific antibody of albumen includes complete antibody molecule, resists Any segment of body or modification, for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc., as long as the segment can retain with The binding ability of PARK2 albumen.When preparation for the antibody of protein level well known to a person skilled in the art, and The present invention may use any method to prepare the antibody.

Cancer of the esophagus detection, diagnostic preparation are protein chip in 9 the present embodiment of embodiment

Protein chip includes solid phase carrier and the PARK2 protein antibodies for being fixed on solid phase carrier.Wherein solid phase carrier is Slide is purchased from VWR International company ((Santa Cruz article No. sc-32282；Abcam article No. ab15494； Invitroge article No. PA1-751.).

The application of 10 PARK2 gene of embodiment or its expression product in the drug of the preparation treatment cancer of the esophagus

The reagent in the drug of the cancer of the esophagus comprising promoting PARK2 gene expression is treated in the present embodiment.

This reagent may is that the reagent containing PARK2 gene, and the carrier for carrying PARK2 gene is formed by reagent, take Host cell with PARK2 gene is formed by reagent, or the reagent containing PARK2 protein.

The application of 11 PARK2 gene of embodiment or its expression product in the drug of the preparation treatment cancer of the esophagus

The reagent in the drug of the cancer of the esophagus comprising promoting PARK2 gene expression product stability is treated in the present embodiment.Tool Body is PARK2 protein stabiliser.

The application of 12 PARK2 gene of embodiment or its expression product in the drug of the preparation treatment cancer of the esophagus

It is treated in the present embodiment in the drug of the cancer of the esophagus comprising improving the active reagent of PARK2 gene expression product.

The application of 13 PARK2 gene of embodiment or its expression product in the drug of the preparation treatment cancer of the esophagus

The reagent comprising enhancing PARK2 gene expression product function in the drug of the cancer of the esophagus is treated in the present embodiment.

It can also include pharmaceutically acceptable load in the drug of the treatment cancer of the esophagus in above-described embodiment 10- embodiment 13 Body.This kind of carrier includes but is not limited to: diluent, excipient such as water etc.；Filler such as starch, sucrose etc.；Adhesive is such as fine Tie up plain derivative, alginates, gelatin and polyvinylpyrrolidone；Wetting agent such as glycerol；Disintegrating agent such as agar, calcium carbonate and carbonic acid Hydrogen sodium；Sorbefacient quaternary ammonium compound；Surfactant such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubrication Agent such as talcum powder, calcium stearate and magnesium, polyethylene glycol etc..

The drug of the above-mentioned treatment cancer of the esophagus, which imports tissue or the mode of cell, can be divided into external or intracorporal side Formula.Vitro formats include importing the drug containing PARK2 gene or the drug containing PARK2 protein in cell, then will Cell transplantation is fed back in vivo.Internal mode includes directly by the drug containing PARK2 gene or containing PARK2 protein Infusion of medicine in-vivo tissue in.Drug of the invention can also with the drug combination of the other treatment cancer of the esophagus, it is a variety of medication combined Use the success rate that can mention treatment significantly.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.

Sequence table

<110>Xinxiang College of Medical Science

<120>a kind of cancer of the esophagus molecular marker and its application

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1398

<212> DNA

<213> PARK2(PARK2)

<400> 1

atgatagtgt ttgtcaggtt caactccagc catggtttcc cagtggaggt cgattctgac 60

accagcatct tccagctcaa ggaggtggtt gctaagcgac agggggttcc ggctgaccag 120

ttgcgtgtga ttttcgcagg gaaggagctg aggaatgact ggactgtgca gaattgtgac 180

ctggatcagc agagcattgt tcacattgtg cagagaccgt ggagaaaagg tcaagaaatg 240

aatgcaactg gaggcgacga ccccagaaac gcggcgggag gctgtgagcg ggagccccag 300

agcttgactc gggtggacct cagcagctca gtcctcccag gagactctgt ggggctggct 360

gtcattctgc acactgacag caggaaggac tcaccaccag ctggaagtcc agcaggtaga 420

tcaatctaca acagctttta tgtgtattgc aaaggcccct gtcaaagagt gcagccggga 480

aaactcaggg tacagtgcag cacctgcagg caggcaacgc tcaccttgac ccagggtcca 540

tcttgctggg atgatgtttt aattccaaac cggatgagtg gtgaatgcca atccccacac 600

tgccctggga ctagtgcaga atttttcttt aaatgtggag cacaccccac ctctgacaag 660

gaaacatcag tagctttgca cctgatcgca acaaatagtc ggaacatcac ttgcattacg 720

tgcacagacg tcaggagccc cgtcctggtt ttccagtgca actcccgcca cgtgatttgc 780

ttagactgtt tccacttata ctgtgtgaca agactcaatg atcggcagtt tgttcacgac 840

cctcaacttg gctactccct gccttgtgtg gctggctgtc ccaactcctt gattaaagag 900

ctccatcact tcaggattct gggagaagag cagtacaacc ggtaccagca gtatggtgca 960

gaggagtgtg tcctgcagat ggggggcgtg ttatgccccc gccctggctg tggagcgggg 1020

ctgctgccgg agcctgacca gaggaaagtc acctgcgaag ggggcaatgg cctgggctgt 1080

gggtttgcct tctgccggga atgtaaagaa gcgtaccatg aaggggagtg cagtgccgta 1140

tttgaagcct caggaacaac tactcaggcc tacagagtcg atgaaagagc cgccgagcag 1200

gctcgttggg aagcagcctc caaagaaacc atcaagaaaa ccaccaagcc ctgtccccgc 1260

tgccatgtac cagtggaaaa aaatggaggc tgcatgcaca tgaagtgtcc gcagccccag 1320

tgcaggctcg agtggtgctg gaactgtggc tgcgagtgga accgcgtctg catgggggac 1380

cactggttcg acgtgtag 1398

<210> 2

<211> 465

<212> PRT

<213> PARK2(PARK2)

<400> 2

Met Ile Val Phe Val Arg Phe Asn Ser Ser His Gly Phe Pro Val Glu

1 5 10 15

Val Asp Ser Asp Thr Ser Ile Phe Gln Leu Lys Glu Val Val Ala Lys

20 25 30

Arg Gln Gly Val Pro Ala Asp Gln Leu Arg Val Ile Phe Ala Gly Lys

35 40 45

Glu Leu Arg Asn Asp Trp Thr Val Gln Asn Cys Asp Leu Asp Gln Gln

50 55 60

Ser Ile Val His Ile Val Gln Arg Pro Trp Arg Lys Gly Gln Glu Met

65 70 75 80

Asn Ala Thr Gly Gly Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys Glu

85 90 95

Arg Glu Pro Gln Ser Leu Thr Arg Val Asp Leu Ser Ser Ser Val Leu

100 105 110

Pro Gly Asp Ser Val Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg

115 120 125

Lys Asp Ser Pro Pro Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn

130 135 140

Ser Phe Tyr Val Tyr Cys Lys Gly Pro Cys Gln Arg Val Gln Pro Gly

145 150 155 160

Lys Leu Arg Val Gln Cys Ser Thr Cys Arg Gln Ala Thr Leu Thr Leu

165 170 175

Thr Gln Gly Pro Ser Cys Trp Asp Asp Val Leu Ile Pro Asn Arg Met

180 185 190

Ser Gly Glu Cys Gln Ser Pro His Cys Pro Gly Thr Ser Ala Glu Phe

195 200 205

Phe Phe Lys Cys Gly Ala His Pro Thr Ser Asp Lys Glu Thr Ser Val

210 215 220

Ala Leu His Leu Ile Ala Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr

225 230 235 240

Cys Thr Asp Val Arg Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg

245 250 255

His Val Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr Arg Leu

260 265 270

Asn Asp Arg Gln Phe Val His Asp Pro Gln Leu Gly Tyr Ser Leu Pro

275 280 285

Cys Val Ala Gly Cys Pro Asn Ser Leu Ile Lys Glu Leu His His Phe

290 295 300

Arg Ile Leu Gly Glu Glu Gln Tyr Asn Arg Tyr Gln Gln Tyr Gly Ala

305 310 315 320

Glu Glu Cys Val Leu Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly

325 330 335

Cys Gly Ala Gly Leu Leu Pro Glu Pro Asp Gln Arg Lys Val Thr Cys

340 345 350

Glu Gly Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe Cys Arg Glu Cys

355 360 365

Lys Glu Ala Tyr His Glu Gly Glu Cys Ser Ala Val Phe Glu Ala Ser

370 375 380

Gly Thr Thr Thr Gln Ala Tyr Arg Val Asp Glu Arg Ala Ala Glu Gln

385 390 395 400

Ala Arg Trp Glu Ala Ala Ser Lys Glu Thr Ile Lys Lys Thr Thr Lys

405 410 415

Pro Cys Pro Arg Cys His Val Pro Val Glu Lys Asn Gly Gly Cys Met

420 425 430

His Met Lys Cys Pro Gln Pro Gln Cys Arg Leu Glu Trp Cys Trp Asn

435 440 445

Cys Gly Cys Glu Trp Asn Arg Val Cys Met Gly Asp His Trp Phe Asp

450 455 460

Val

465

<210> 3

<211> 20

<212> DNA

<213>artificial sequence (RT-PCR-PARK2-F)

<400> 3

cacaccccac ctctgacaag 20

<210> 4

<211> 20

<212> DNA

<213>artificial sequence (RT-PCR-PARK2-R)

<400> 4

ctaagcaaat cacgtggcgg 20

<210> 5

<211> 20

<212> DNA

<213>artificial sequence (PCR-GAPDH-F)

<400> 5

gagaaggctg gggctcattt 20

<210> 6

<211> 20

<212> DNA

<213>artificial sequence (PCR-GAPDH-R)

<400> 6

agtgatggca tggactgtgg 20

<210> 7

<211> 22

<212> DNA

<213>artificial sequence (PCR-PARK2-F)

<400> 7

gtgtttgtca ggttcaactc ca 22

<210> 8

<211> 21

<212> DNA

<213>artificial sequence (PCR-PARK2-R)

<400> 8

gaaaatcaca cgcaactggt c 21

<210> 9

<211> 20

<212> DNA

<213>artificial sequence (probe)

<400> 9

gcagagaccg tggagaaaag 20

Claims

1. a kind of cancer of the esophagus molecular marker, it is characterised in that: the molecular marker includes PARK2 gene and/or PARK2 base The expression product of cause.

2. a kind of cancer of the esophagus molecular marker according to claim 1, it is characterised in that: the coding of the PARK2 gene Sequence is DNA sequence dna shown in SEQ ID NO.1；Or hybridizes with the SEQ ID NO.1 DNA sequence dna limited and encode identical function The DNA sequence dna of protein；Or there is 70% or more homology with two class DNA sequence dnas of restriction, and encode identical function protein DNA molecular.

3. a kind of cancer of the esophagus molecular marker according to claim 1, it is characterised in that: the expression of the PARK2 gene Product includes PARK2mRNA and/or PARK2 albumen.

4. a kind of cancer of the esophagus molecular marker according to claim 3, it is characterised in that: the PARK2 albumen includes The functional equivalent of PARK2 albumen and/or PARK2 albumen.

5. a kind of cancer of the esophagus molecular marker according to claim 4, it is characterised in that: the amino of the PARK2 albumen Acid sequence is sequence shown in SEQ ID NO.2；Or amino acid sequence shown in SEQ ID NO.2 is passed through into several amino acid Derived from the substitution and/or deletion and/or addition of residue, or the amino acid sequence as shown in SEQ ID NO.2, and with SEQ ID The amino acid sequence with the same function of amino acid sequence shown in NO.2.

6. a kind of cancer of the esophagus molecular marker a method as claimed in any one of claims 1 to 5 is in preparation cancer of the esophagus detection correlation RT-PCR examination Agent box or real-time quantitative PCR kit or in situ hybridization kit or immunity detection reagent or genetic chip or protein chip or Treat the application in the drug of the cancer of the esophagus.

7. a kind of application of cancer of the esophagus molecular marker according to claim 6, it is characterised in that: the RT-PCR reagent The testing product of box or real-time quantitative PCR kit or in situ hybridization kit or high-flux sequence detection of platform is PARK2 base Because of the product of mRNA level in-site expression.

8. a kind of application of cancer of the esophagus molecular marker according to claim 7, it is characterised in that: the RT-PCR reagent Box or real-time quantitative PCR kit include the primer of specific amplified PARK2 gene, the primer of the specific amplified PARK2 gene Sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.

9. a kind of application of cancer of the esophagus molecular marker according to claim 6, it is characterised in that: the in situ hybridization examination Agent box includes the probe hybridized with PARK2 gene nucleic acid sequence；The genetic chip includes miscellaneous with the nucleic acid sequence of PARK2 gene The probe of friendship；The protein chip includes the antibody in conjunction with PARK2 protein-specific.

10. a kind of application of cancer of the esophagus molecular marker according to claim 6, it is characterised in that: the treatment oesophagus The drug of cancer includes PARK2 gene and/or the promotor of its expression product；The promotor is to promote PARK2 gene expression Reagent or promote the reagent of PARK2 gene expression product stability or improve the active reagent of PARK2 gene expression product or Enhance the reagent of PARK2 gene expression product function；The reagent for promoting PARK2 gene expression is that PARK2 gene is promoted to turn The reagent of record promotes the reagent of PARK2 gene translation or promotes the reagent of PARK2 protein content or contain PARK2 gene Reagent or carry PARK2 gene carrier be formed by reagent or carry PARK2 gene host cell be formed by examination Agent or reagent containing PARK2 protein.