CN105755152A - Application of JAM3 gene to preparation of colorectal cancer diagnosis kit and kit - Google Patents

Application of JAM3 gene to preparation of colorectal cancer diagnosis kit and kit Download PDF

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CN105755152A
CN105755152A CN201610300705.4A CN201610300705A CN105755152A CN 105755152 A CN105755152 A CN 105755152A CN 201610300705 A CN201610300705 A CN 201610300705A CN 105755152 A CN105755152 A CN 105755152A
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gene
jam3
test kit
kit
colorectal cancer
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张云
周丹
安汉祥
汤玮玮
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Xiamen Institute of Rare Earth Materials
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    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/57419—Specifically defined cancers of colon
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    • G01—MEASURING; TESTING
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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Abstract

The invention provides application of a colorectal cancer diagnosis molecular marker, namely, a JAM3 gene which is good in specificity and sensitivity in preparing a colorectal cancer diagnosis kit, and the colorectal cancer diagnosis kit. The application has the main benefits that a novel molecular marker of colorectal cancer is discovered, application of the JAM3 gene to preparation of the colorectal cancer diagnosis kit and the colorectal cancer diagnosis kit are provided, and compared with a conventional detection method, the molecular marker is relatively timely and specific in diagnosis, so that the five-year survival rate of patients suffering from colorectal cancer can be increased, the death rate is reduced, and wide application prospects can be achieved.

Description

JAM3 gene preparation diagnosis of colorectal carcinoma test kit in application and test kit
(1) technical field
The present invention relates to the application in preparation diagnosis of colorectal carcinoma test kit of the JAM3 gene and a kind of diagnosis of colorectal carcinoma test kit.
(2) background technology
Colorectal cancer (colorectalcancer, CRC) is one of modal malignant tumor of digestive tract, and worldwide, sickness rate occupy malignant tumor the 3rd, and mortality rate occupies the 4th, there are about 1,200,000 new cases, serious threat human health every year.Colorectal cancer has obvious Regional Distribution difference in the world, and wherein Asia sickness rate is higher, especially China, main with age, family history, unsound diet and living habit etc. risk factor relevant.Major part colorectal cancer patients has been in the tumour progression phase when making a definite diagnosis, loses the therapic opportunity of the best, causes that five year survival rate is less than 20%, and prognosis is poor.Therefore, explore the appraisal procedure of colorectal cancer quick diagnosis, improve treatment level, improve survival rate, be focus and the difficult point of current research.
The method being used for diagnosis of colorectal carcinoma at present clinically includes: optical check and morphological examination, and conventional inspection method mainly has Excreta examination of feces ocoult blood, Excreta immunologic test, the inspection of barium agent double contrast radiograph, elastic sigmoidoscopy, colonoscopy and CT Colonography.But, above-mentioned detection method still suffers from certain limitation, as loaded down with trivial details in examination of feces ocoult blood separation process, is subject to the interference such as antibacterial, food and intestinal mucus；The requirement that colonoscopy checks equipment and testing staff's technology is higher, and misdiagnosis rate is bigger.
Along with the pathogenetic research of colorectal cancer is more and more extensive, molecular biomarker importance wherein is more and more obvious.These labels have become the important target spot of diagnosis of colorectal carcinoma and treatment.JAM3 belongs to one of connection adhesion protein molecule families member, connect adhesion protein closely related with various kinds of cell activity, including the formation etc. participating in epithelial cell and the close-connected composition of endotheliocyte, the migration of leukocyte, new vessels, there are two immunoglobulin like domain.Epithelial compact siro spinning technology has participated in cancer to be developed, but JAM3 occurs developing effect also indefinite in colorectal cancer.
(3) summary of the invention
Present invention aim at the application providing a kind of high specificity, highly sensitive colorectal cancer gene diagnosis molecular marker JAM3 gene in preparation diagnosis of colorectal carcinoma test kit and a kind of diagnosis of colorectal carcinoma test kit.
The technical solution used in the present invention is:
The application in preparation diagnosis of colorectal carcinoma test kit of the JAM3 gene.
JAM3 gene includes the polynucleotide of any function equivalent of people's JAM3 gene, precursor, hypotype and people's JAM3 gene.JAM3 gene include with JAM3 gene (NC_000011.10) DNA sequence at present international common core sequence databank GeneBank have more than 70% homology, and coding identical function protein DNA sequence.
The coded sequence of JAM3 gene includes any one DNA molecular following:
(1) DNA sequence shown in SEQ ID NO.1；
(2) the DNA sequence hybridization limited with (1) under strict conditions and coding identical function protein DNA sequence；
(3) DNA sequence limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA sequence.
Preferably, described JAM3 gene order is such as shown in SEQIDNo.1:
The invention still further relates to the JAM3 gene expression product (i.e. JAM3 albumen) application in preparation diagnosis of colorectal carcinoma test kit.
JAM3 albumen includes all function equivalents of JAM3 albumen and JAM3 albumen.Described function equivalent includes JAM3 albumen conservative variant protein matter or its active fragment, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of people's JAM3 gene under condition high or low forbidding.
JAM3 albumen can be the protein with following amino acid sequences:
(1) protein that the aminoacid sequence shown in SEQ ID NO.2 forms；
(2) aminoacid sequence shown in SEQIDNO.2 through the replacement of one or more amino acid residues and/or disappearance and/or is added and has the protein having the aminoacid sequence shown in SEQIDNO.2 derivative of identical function with the aminoacid sequence shown in SEQIDNO.2.The aminoacid number replaced, lack or add is at least above 1.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology, it is preferable that there is the polypeptide of the Amino acid profile of the homology of at least 90% with the aminoacid sequence shown in SEQIDNO.2.
nullPreferably，Described JAM3 gene expression product aminoacid sequence is such as shown in SEQIDNo.2: MALRRPPRLRLCARLPDFFLLLLFRGCLIGAVNLKSSNRTPVVQEFESVELSCIIT DSQTSDPRIEWKKIQDEQTTYVFFDNKIQVKPVTPVCRVPKAVPVGKMATLHCQES EGHPRPHYSWYRNDVPLPTDSRANPRFRNSSFHLNSETGTLVFTAVHKDDSGQYYC IASNDAGSARCEEQEMEVYDLNIGGIIGGVLVVLAVLALITLGICCAYRRGYFINN KQDGESYKNPGKPDGVNYIRTDEEGDFRHKSSFVI.
The JAM3 albumen of the present invention also includes the non-conservative modification to aminoacid sequence shown in SEQIDNO.2, as long as the protein after modifying still has the biologic activity of JAM3 albumen.The modification of aminoacid sequence is modified after may originate from spontaneous mutation or heredity, it is possible to artificial induction produces.
Generally, in a protein one or more amino acid whose modifications without influence on the function of protein.Those skilled in the art can approve that change includes replacing, lack or adding, and the aminoacid of single amino acids or little percentage ratio is conservative modification, and the protein of generation has identity function.It is well known in the art for providing intimate amino acid whose Conservative substitution tables.
The invention still further relates to a kind of diagnosis of colorectal carcinoma test kit, described test kit diagnoses by detecting the expression of JAM3 gene and expression product thereof, described test kit specifically includes that the specific primer (RT-PCR diagnostic kit or quantitative fluorescent PCR diagnostic kit) of amplification JAM3 gene, or the antibody being combined with JAM3 protein-specific (immune detection diagnostic kit), or the probe (in situ hybridization diagnostic kit) with the nucleic acid array hybridizing of JAM3 gene, and detection reagent (corresponding PCR reaction or immune detection, reagent required in situ hybridization and expression detection).Described diagnosis of colorectal carcinoma comprises and judges whether experimenter has suffered from colorectal cancer, also comprises and judges whether experimenter exists the risk suffering from colorectal cancer.
The specific antibody of described JAM3 albumen includes monoclonal and polyclonal antibody.The specific antibody of described JAM3 albumen includes complete antibody molecule, any fragment of antibody, variant or modification.As long as described fragment can retain and the binding ability of JAM3 albumen.Antibody for detecting protein level is prepared by known, and the present invention can use any method to prepare described antibody.
The probe of the described nucleic acid array hybridizing with JAM3 gene can be DNA, RNA, DNA-RNA chimera, PNA or other derivants.Described probe length does not limit, it is possible to complete specific hybrid and specific binding with purpose nucleotide sequence.Described probe length scope is more than 10 bases.
Preferably, described test kit is gene detecting kit and protein immunization detection kit.
When described test kit is gene detecting kit, described gene detecting kit mainly includes specific primer and the PCR reaction reagent of amplification JAM3 gene；
The specific primer sequence of described amplification JAM3 gene is as follows:
Forward primer: 5 '-CTGCTGTTCACAAGGACGAC-3 '；
Downstream primer: 5 '-CAGATGCCCAACGTGATCAG-3 '.
Described test kit may also include the specific primer of amplification endogenous control gene GAPDH:
Forward primer: 5 '-ACCACAGTCCATGCCATCAC-3 '；
Downstream primer: 5 '-TCCACCACCCTGTTGCTGTA-3 '.
Described test kit may also include JAM3 gene standard substance, and described JAM3 gene standard substance sequence is such as shown in SEQIDNo.1.Using JAM3 gene standard substance as comparison, JAM3 gene expression amount in sample to be tested can be carried out detection by quantitative.
Described gene detecting kit PCR reaction reagent is the required reagent of Standard PCR reaction, including dNTP, Mg2+, Taq enzyme (thermal starting enzyme), PCR buffer, SYBRGreenII etc..
Present invention firstly discovers that JAM3 gene expression and colorectal cancer are closely related, by detecting the expression of JAM3 in experimenter's colorectal carcinoma, may determine that whether experimenter has suffered from colorectal cancer or whether there is the risk suffering from colorectal cancer, thus provide prevention or therapeutic scheme to clinicist.
The beneficial effects are mainly as follows: present invention finds the molecular marker JAM3 gene that a kind of colorectal cancer is new, provide its application in preparation diagnosis of colorectal carcinoma test kit, and one diagnosis of colorectal carcinoma test kit, compared with traditional detection means, the diagnosis of molecular marker more in time, more special, thus improving 5 years survival rates of colorectal cancer patients, reducing mortality rate, having a extensive future.
(4) accompanying drawing explanation
Fig. 1 is the amplification curve representative graph with volume increase thing dissolving peak of JAM3 gene；A is in 27 pairs of Colorectal Carcinomas and corresponding distant place normal bowel tissue, utilizes the amplification curve of fluorescence quantitative PCR detection JAM3 gene；B is the representative graph that amplified production dissolves peak.
Fig. 2 is JAM3 gene mRNA expression (2 in 27 pairs of Colorectal Carcinomas and corresponding distant place normal bowel tissue-△CtValue compares, * * P < 0.01).
Fig. 3 is the JAM3 gene mRNA expression (2 at normal intestinal epithelial cell line NCM460 and colorectal cancer cell system (SW480, SW620, HCT116, HT29)-△CtValue compares, * * * P < 0.001).
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited to that:
Embodiment 1: fluorescence quantitative PCR method detection JAM3 gene is at the differential expression of Colorectal Carcinoma and corresponding distant place normal bowel tissue
1, the 27 distant place normal bowel tissue samples to Colorectal Carcinoma and correspondence are collected, all Colorectal Carcinoma specimen provide by the attached First Hospital sample storehouse of Xiamen University, each patient's specimen all has clear and definite idagnostic logout, and by the agreement of Ethics Committee, frozen in liquid nitrogen after making a collection of specimens.
2, the preparation of RNA sample
(1) pre-treatment of sample: take out sample from liquid nitrogen container, put samples in the aseptic EP pipe of 2ml, add 1mlTrizolRNA extracting solution, overnight with 0.1%DEPC water soaking and puts in Potter-Elvehjem Tissue Grinders and is fully ground after shredding with the shears sterilized.
(2) extracting: add the chloroform of 500 μ L, acutely reverse mixing 15～30s, put in 4 DEG C of refrigerated centrifuges centrifugal, and 12000rpm is centrifuged 4min.Sample is classified into three layers: upper strata is that to contain RNA, intermediate layer and lower floor be the organic facies containing albumen or other impurity to inorganic aqueous phase.
(3) precipitation: careful take out upper strata aqueous phase, adds in the aseptic EP pipe containing 750 μ L isopropanols and 150 μ L5%LiCl, acutely reverse mixing 15～30s, puts in 4 DEG C of refrigerated centrifuges centrifugal, and 12000rpm is from 5min.
(4) washing: abandon supernatant, adds 1mL75% ethanol (preparation of DEPC water), puts in 4 DEG C of refrigerated centrifuges centrifugal, and 12000rpm, from 1min, repeats this step after abandoning supernatant.
(5) dry: to blot the ethanol in pipe, dry RNA precipitate 2～3min under room temperature.
(6) dissolve: add 20 μ LDEPC water dissolution RNA precipitate.
(7) concentration is measured: use the Nanodrop2000 UV spectrophotometer measuring 27 concentration to RNA sample, as follows:
3, RNA reverse transcription obtains cDNA
Adopting Takara company Reverse Transcription box (article No. RR047A) to carry out cDNA reverse transcription, experimental implementation is undertaken by product description, and concrete operations are as follows:
(1) in 200 μ LPCR pipes, the reactant liquor of DNA, total system 10 μ L are removed in preparation, comprise gDNAEraser1 μ L, 5 × gDNAEraserBuffer2 μ L, RNAx μ L, RNaseFreedH2The volume of O7-x μ L, RNA is determined by concentration, x=1 μ g/RNA concentration.
(2) PCR pipe removing DNA reactant liquor that will be equipped with preparing puts into PCR instrument (Bio-rad, T100TM) in, hatch 2min for 42 DEG C.
(3) preparing inverse transcription reaction liquid in 200 new μ LPCR pipes, system is 10 μ L, comprises 5 × PrimeSriptBuffer4 μ L, PrimeSriptRTEnzymeMixI1 μ L, RTPrimerMix1 μ L, RNaseFreedH2O4μL。
(4) 10 μ L solution first two steps obtained are mixed to get 20 μ L systems, PCR instrument (Bio-rad, T100TM) 37 DEG C hatch 5min, hatch 5s for 85 DEG C, obtain cDNA, the RNaseFreedH of reverse transcription2O is diluted to 200 μ L.
4, fluorescence quantitative PCR detection:
(1) in eight connecting legs, often pipe preparation fluorescence quantitative PCR reaction solution, cumulative volume 20 μ L, SYBRPremixExTaqII10 μ L, JAM3 forward primer 0.8 μ L, JAM3 downstream primer 0.8 μ L, ROXDyeII0.4 μ L, cDNA template 2 μ L, RNaseFreedH2O6μL。
(2) putting in PCR detector (ABIViiA7) after capping, program setting is: 50 DEG C of 2min, 95 DEG C of 10min, then 45 circulations: 95 DEG C of 15s, 61 DEG C of 60s.
5, data analysis: experiment all completes for 3 times according to repetition, adopts relative quantification 2-△CtThe method of (△ Ct=sample JAM3Ct value-sample GAPDHCt value) carries out statistical analysis, and GAPDH is as reference gene, and data separate GraphPad5.0 software is analyzed, and all statistical results all think there is significant difference with P < 0.05.
6, result: fluorescent quantitative PCR curve representative graph display amplification curve flex point is clear, overall collimation is better, it was shown that the amplification efficiency of each reaction tube is close (Figure 1A)；Sample amplified production solubility curve representative graph display solubility curve is substantially unimodal, illustrates that amplified production only has one, for specific amplification (Figure 1B)；Compared with corresponding distant place normal bowel tissue, JAM3 gene expresses notable downward (P < 0.01) (Fig. 2) in colorectal cancer cancerous tissue.
Embodiment 2: fluorescence quantitative PCR method detection JAM3 gene is at colorectal cancer cell system and normal intestinal epithelial cell line differential expression
1. cultivating Human colorectal cancer cells system (SW480, SW620, HCT116 and HT29) and people normal intestinal epithelial cell line NCM460, concrete cultural method is as follows: people normal bowel cell line NCM460 with containing 10% viviparity Ox blood serum and penicillin, each 100U/mL of streptomycin DMEM culture medium at 37 DEG C and 5%CO2Incubator in cultivate, within every 2～3 days, change culture fluid and also go down to posterity.People intestinal cancer SW480 adopts L15 (containing 10% hyclone), and people intestinal cancer HCT116, SW620 and HT29 cell line adopt DMEM (containing 10% hyclone) at 37 DEG C and 5%CO2Incubator in cultivate.
2. when cell degrees of fusion in 60mm culture dish reaches 80%, discard culture fluid, add the aseptic PBS of 1mL, repeating 1 time after discarding PBS, add 1mLTrizolRNA extracting solution, left and right rocks culture dish gently, TrizolRNA extracting solution is made all not have bottom culture dish, adopt 1mL liquid-transfering gun to blow and beat adherent cell gently so that cell is suspended in TrizolRNA extracting solution completely, the TrizolRNA extracting solution containing cell is collected to EP pipe aseptic for 1.5mL.
3.RNA extraction, reverse transcription, quantitative fluorescent PCR and data analysing method are with embodiment 1.
4. result: compared with normal intestinal epithelial cell line NCM460, the expression of colorectal cancer cell system (SW480, SW620, HCT116 and HT29) JAM3 gene mRNA is all remarkably decreased (P < 0.001) (Fig. 3).
The introduction of above-described embodiment is only used for understanding method and the core content thereof of the present invention.Of particular note, for a person skilled in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention is improved and modifies, but these improve and modify also will belong to the protection domain of the claims in the present invention.

Claims (9)

  1. The application in preparation diagnosis of colorectal carcinoma test kit of the 1.JAM3 gene.
  2. 2. apply as claimed in claim 1, it is characterised in that described JAM3 gene order is such as shown in SEQIDNo.1.
  3. The application in preparation diagnosis of colorectal carcinoma test kit of the 3.JAM3 gene expression product.
  4. 4. apply as claimed in claim 3, it is characterised in that described JAM3 gene expression product aminoacid sequence is such as shown in SEQIDNo.2.
  5. 5. a diagnosis of colorectal carcinoma test kit, described test kit diagnoses by detecting the expression of JAM3 gene and expression product thereof, described test kit specifically includes that the specific primer of amplification JAM3 gene, or the antibody being combined with JAM3 protein-specific, or with the probe of the nucleic acid array hybridizing of JAM3 gene, and detection reagent.
  6. 6. test kit as claimed in claim 5, it is characterised in that described test kit is gene detecting kit or protein immunization detection kit.
  7. 7. test kit as claimed in claim 6, it is characterised in that described test kit is gene detecting kit, described gene detecting kit mainly includes specific primer and the detection reagent of amplification JAM3 gene；
    The specific primer sequence of described amplification JAM3 gene is as follows:
    Forward primer: 5 '-CTGCTGTTCACAAGGACGAC-3 '；
    Downstream primer: 5 '-CAGATGCCCAACGTGATCAG-3 '.
  8. 8. test kit as claimed in claim 7, it is characterised in that described test kit also includes the specific primer of amplification endogenous control gene GAPDH:
    Forward primer: 5 '-ACCACAGTCCATGCCATCAC-3 '；
    Downstream primer: 5 '-TCCACCACCCTGTTGCTGTA-3 '.
  9. 9. test kit as claimed in claim 8, it is characterised in that described test kit also includes JAM3 gene standard substance, and described JAM3 gene standard substance sequence is such as shown in SEQIDNo.1.
CN201610300705.4A 2016-05-09 2016-05-09 Application of JAM3 gene to preparation of colorectal cancer diagnosis kit and kit Pending CN105755152A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434951A (en) * 2016-10-27 2017-02-22 刘鹏飞 Reagent system and reagent kit for detecting colorectal cancer spindle apparatus assembly checking point and application
CN109975547A (en) * 2018-02-28 2019-07-05 中山大学 Application of the RANTES detection reagent in terms of preparing diagnosis of colorectal carcinoma agent
CN113710800A (en) * 2019-04-18 2021-11-26 学校法人庆应义塾 Method and kit for detecting carcinogenic risk of colorectal cancer

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CN105200147A (en) * 2015-10-27 2015-12-30 山东大学齐鲁医院 Kit for screening early-stage cervical carcinoma based on HPV-DNA detection and DNA methylation

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CN105200147A (en) * 2015-10-27 2015-12-30 山东大学齐鲁医院 Kit for screening early-stage cervical carcinoma based on HPV-DNA detection and DNA methylation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434951A (en) * 2016-10-27 2017-02-22 刘鹏飞 Reagent system and reagent kit for detecting colorectal cancer spindle apparatus assembly checking point and application
CN106434951B (en) * 2016-10-27 2020-01-24 刘鹏飞 Reagent system and kit for detecting spindle assembly inspection point of colorectal cancer and application of reagent system and kit
CN109975547A (en) * 2018-02-28 2019-07-05 中山大学 Application of the RANTES detection reagent in terms of preparing diagnosis of colorectal carcinoma agent
CN113710800A (en) * 2019-04-18 2021-11-26 学校法人庆应义塾 Method and kit for detecting carcinogenic risk of colorectal cancer

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