CN109593771A - A kind of the 1100th bit base mutated gene of mankind MAP2K5 and its detection kit - Google Patents

A kind of the 1100th bit base mutated gene of mankind MAP2K5 and its detection kit Download PDF

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CN109593771A
CN109593771A CN201910095564.0A CN201910095564A CN109593771A CN 109593771 A CN109593771 A CN 109593771A CN 201910095564 A CN201910095564 A CN 201910095564A CN 109593771 A CN109593771 A CN 109593771A
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叶丰
肖林
姜勇
步宏
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West China Hospital of Sichuan University
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Abstract

The invention discloses a kind of mankind's MAP2K5 genes of variation, it is mankind's MAP2K5 mutant gene that the 1100th bit base sports A.The invention also discloses the reagents for detecting aforementioned nucleotide variation to prepare purposes and non-medullary thyroid carcinoma kit for screening in non-medullary thyroid carcinoma screening agent.Kit of the present invention, can be used for the complementary diagnosis of thyroid cancer, and application prospect is excellent.

Description

A kind of the 1100th bit base mutated gene of mankind MAP2K5 and its detection kit
Technical field
The present invention relates to the fields SNP, in particular to SNP relevant to non-medullary thyroid carcinoma.
Background technique
Past 10 years Chinese women thyroid cancer incidence increases about 5 times, becomes the most fast malignant tumour of women speedup. Disease incidence ranked the first in women at less than 30 years old, and full age bracket ranked sixth.Originating from thyroid follicular cells The non-cephaloma (Non-medullary Thyroid Cancer, NMTC) of (Follicular Epithelium Cell) accounts for about The 95% of sum, papillary thyroid carcinoma is the most common type in non-cephaloma；Marrow sample originating from parathyroid gland C cell Cancer (Medullary Thyroid Cancer, MTC) accounts for about 5%.Two types can have heredity.
Although the concept of NMTC is widely accepted, tumor susceptibility gene and corresponding function are not elucidated with and generally yet Approve, so that current still none generally acknowledged tumor susceptibility gene variation can be used as the target spot of NMTC prevention and control or treatment.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of kits for detecting NMTC.
Present invention firstly provides a kind of mankind's MAP2K5 genes of variation, it is the people that the 1100th bit base sports C Class MAP2K5 mutant gene, i.e. c.T1100C (p.M367T) (NM_145160).
The present invention also provides a kind of mankind's MAP2K5 albumen of variation, it is that the 367th amino acids sport threonine Mankind's MAP2K5 variant protein.
The present invention also provides the related reagents of detection the 1100th bit base of mankind MAP2K5 gene variation to prepare non-marrow Purposes in sample screening for thyroid cancer reagent.
Wherein, the screening agent is the screening agent for detecting papillary thyroid carcinoma.
Wherein, the reagent is the related reagent for detecting the 1100th T → C of mankind MAP2K5 gene variation.
Wherein, it is described detection the 1100th T → C of the mankind MAP2K5 gene variation related reagent be sequencing reagent, Snapshot reagent, restriction fragment length polymorphism method reagent or single-strand conformation polymorphism analysis reagent.
Wherein, the sequencing reagent includes the reagent in the 1100th site of PCR amplification MAP2K5 gene.
Preferably, the reagent in amplification the 1100th site of MAP2K5 gene includes following primer pair:
Upstream primer are as follows: 5 '-TCATAATGTGTCCAAGTGAGTC-3 ', downstream primer are as follows: 5 '- TTTACAGTGGAGTGGAAAGAAA-3′。
The present invention also provides a kind of kit for screening of non-medullary thyroid carcinoma, it is characterised in that: it includes optional For detecting the related reagent of the 1100th bit base of mankind MAP2K5 gene variation.
Wherein, the reagent is the related reagent for detecting the 1100th T → C of mankind MAP2K5 gene variation.
Wherein, it is described detection the 1100th T → C of the mankind MAP2K5 gene variation related reagent be sequencing reagent, Snapshot reagent, restriction fragment length polymorphism method reagent or single-strand conformation polymorphism analysis reagent.
Wherein, the sequencing reagent includes the reagent in the 1100th site of PCR amplification MAP2K5 gene.
Preferably, the reagent in amplification the 1100th site of MAP2K5 gene includes following primer pair:
Upstream primer are as follows: 5 '-TCATAATGTGTCCAAGTGAGTC-3 ', downstream primer are as follows: 5 '- TTTACAGTGGAGTGGAAAGAAA-3′。
The present invention also provides a pair of of DNA primers, it is characterised in that: sequence is NO.1~2 the SEQ ID sequence.
The present invention passes through the research to papillary thyroid carcinoma family, it was found that the high-incidence mutated gene with thyroid cancer And specific site, i.e. MAP2K5 c.G961A and MAP2K5 c.T1100C.
Previous site is that the 961st bit base of the gene coding region MAP2K5 by G sports A, corresponding MAP2K5 albumen 321 amino acids are threonine by alanine mutation.The latter site is the 1100th bit base of the gene coding region MAP2K5 by T C is sported, corresponding the 367th amino acids of MAP2K5 albumen sport threonine by methionine.It is tested by further experiment Card, it is found that the mutational site will lead to the change of subsequent cell access, so that the relevant gene of cancer significantly raises, pushes Follicular epithelial cell malignant transformation.
Measuring method measurement of the invention derives from the genomic DNA of people, sample there is no limit, as body fluid (such as blood, Ascites and urine), histocyte (such as hepatic tissue), can prepare genomic DNA by extracting and purifying these samples.
Kit provided by the invention, can effectively screening crowd to be checked to thyroid cancer is suffered from a possibility that, before Scape is good.
Obviously, above content according to the present invention is not taking off according to the ordinary technical knowledge and customary means of this field Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
By the following examples, the specific embodiment of experimental example form remakes further above content of the invention Detailed description.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the present invention The technology that above content is realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is to be mutated sketch map: A, MAP2K5 c.G961A sequencer map, arrow by respondent's Candidate Mutant and MAP2K5 It show mutational site；B, MAP2K5 c.T1100C sequencer map, arrow show mutational site；In C, protein MAP2K5 The function domain information of A321T and M367T；D, the conservative analysis of MAP2K5 A321T and M367T between different plant species.
Fig. 2 is the ERK5 gene, MAP2K5- of FNMTC patient (F1-F4) and SNMTC patient (S1-S4) blood leukocytes The expression of ERK5 approach target gene: A, ERK5 expression；The mRNA thermal map of B, MAP2K5-ERK5 approach target gene；C- H, the detailed expression of target gene FOSB, MEF2, PPARG, JUN, CDK4 and CCND1.
Fig. 3 is in the thyroid gland cancer beside organism and tumor tissues of FNMTC patient (F1-F4) and SNMTC patient (S1-S4) The expression of MAP2K5, ERK5, FRA1, MEF2 gene: A-H, MAP2K5 express feelings between FNMTC patient and SNMTC patient Condition；The N of I-K, ERK5, FRA1 and MEF2 expression in cancer beside organism, abscissa label indicate " normal tissue "；L-N, cancer The T of ERK5, FRA1 and MEF2 expression in tissue, abscissa label indicates " cancerous tissue ".
Fig. 4 is the Ki67 colored graph of the normal follicular epithelium tissue of SNMTC and FNMTC patient and tumor tissues and differentiates figure: A-B, normal tissue dyeing；C-D, cancerous tissue dyeing；E, interpretation of three clinical pathology doctors to coloration result.
Fig. 5 is the figure that TUNEL fluorescent staining detects apoptosis.
Fig. 6 is transgenic cell structure figures: A, GV358 carrier structure；B-C, recombinant vector Sanger sequence verification figure；D The B-CPAP cell line of polygenic locus verifying；The green fluorescence microscopy figure of E-H, WT group, Mu1 group, Mu2 group and GFP group.
Fig. 7 is the ERK5 detection figure of MAP2K5 schematic diagram and transgenic cell: A, MAP2K5 albumen tertiary structure and mutation Site schematic diagram；B, 5 Protein Detection figure of phosphorylated CREB；C, reference protein detection figure；D-F, WT group ERK5 protein subcellular are fixed Bitmap；G-I, Mu1 (A321T) organize ERK5 protein subcellular positioning figure.
Fig. 8 is the MAP2K5-ERK5 access expression of target gene figure of transgenic cell each group.
Specific embodiment
Paraphrase:
The 961st G → A variation of MAP2K5 c.G961A:MAP2K5 gene；
The 321st amino acids of MAP2K5 p.A321T or MAP2K5 A321T:MAP2K5 albumen sport T by A, this change It is different to be caused by MAP2K5 c.G961A；
The 1100th T → C variation of MAP2K5 c.T1100C:MAP2K5 gene；
The 367th amino acids of MAP2K5 p.M367T or MAP2K5 M367T:MAP2K5 albumen sport T by M, this change It is different to be caused by MAP2K5 c.T1100C；
FNMTC patient: the non-medullary thyroid carcinoma in familial follicular cells source；Other no familial diseases, The family patient that at least two first degree relatives are diagnosed as the non-medullary thyroid carcinoma in follicular cells source；
SNMTC patient: the non-medullary thyroid carcinoma in sporadic follicular cells source, MAP2K5 gene are unmutated.
The detection of embodiment mutated gene MAP2K5 c.T1100C
1.DNA is extracted
200 microlitres of whole blood sample are taken, using new hundred bases UPure Blood DNA Extraction Kit (M2002-01) Kit is extracted by new hundred bases UPure Blood DNA Extraction Kit (M2002-01) kit specification.
2.PCR amplification
1) PCR primer:
Upstream primer are as follows: 5 '-TCATAATGTGTCCAAGTGAGTC-3 ' (SEQ ID NO.1),
Downstream primer are as follows: 5 '-TTTACAGTGGAGTGGAAAGAAA-3 ' (SEQ ID NO.2),
Concentration: 100 μm of ol/L.
2) system is prepared:
DNA 2 microlitres
2X Taq PCR MasterMix(KT201) 12.5 microlitres
Upstream 1 microlitre
Downstream 1 microlitre
Aqua sterilisa 9.5 microlitre
3) response procedures:
Step Temperature Time
1 94℃ 3min
2 94℃ 30s
3 60℃ 30s
5 72℃ 1min
6 2 steps are returned, totally 30 times
7 72℃ 5min
3.sanger sequencing
PCR product is subjected to agarose gel electrophoresis, sample presentation is sequenced after identifying primer size, and whether is detection related locus It is mutated.
4. result interpretation
Sequencing result is compared onto reference genome, if discovery No. 15 chromosome the 68040594th is C (reference Sequence should be T), that is, detect MAP2K5 c.T1100C.
The method of the present invention can effectively detect detection the 1100th bit base of MAP2K5 gene by the mutation of T to C.
In order to illustrate MAP2K5 c.T1100C mutation to the facilitation of non-medullary thyroid carcinoma, below with experimental example Mode illustrates.
Experimental example MAP2K5 c.G961A/c.T1100C is mutated the correlation with non-medullary thyroid carcinoma
1. method
1.1 ethics and patient information
This research is ratified through West China Hospital Ethics Committee and executes (2015-108).FNMTC is defined as without other The family patient that familial disease, at least two first degree relatives are diagnosed as the thyroid cancer in follicular cells source.It grinds at this In studying carefully, shares 77 FNMTC patients and meet these standards.From 34 families, (the ratio between women and male are FNMTC patient 3.53:1), it is diagnosed as thyroid papillary carcinoma.
1.2Sanger sequencing and gene frequency inquiry
The DNA of aforementioned patient is extracted, and designs flank primers to expand target region mutational site.According to the side of manufacturer Case, using 3.1 terminator sequencing kit of BigDye (Applied Biosystems), in ABI 3730xL sequenator The PCR product of purifying is sequenced on (Applied Biosystems, USA).
MAP2K5 is searched in Novo-Zhonghua Genomes (promise standing grain-China's genome plan) database The mutated gene frequency in the site c.G961A/c.T1100C.
1.3RT-qPCR measurement
For fresh sample and cell sample, Invitrogen TRIzol reagent (Cat.15596026, Thermo are used Fisher, the U.S.) extract total serum IgE.For the sample of the fixed paraffin embedding of formalin, Qiagen RNeasy is used FFPE kit (Cat.73504, Qiagen Inc, Germany) extracts total serum IgE.For reverse transcription, according to the explanation of manufacturer, Using Qiagen Omniscript RT kit (Cat.205111, Qiagen) by 1mg mRNA in 20ul reaction system It is converted into cDNA.And qPCR is carried out using Bio-Rad Real-Time PCR system (Bio-Rad Inc, USA).Using pair According to the opposite mRNA expression for comparing target gene after gene (GAPDH, ACTIN and GFP) standardization.
1.4 immunohistochemistry
For human sample, immunohistochemical staining is carried out on 4- μ m-thick paraffin section.Glass slide is taken off into stone in dimethylbenzene Wax simultaneously passes through graded ethanol solutions moisturizing again.At room temperature will be interior using 3% hydrogen peroxide ' endogenous peroxidase enzyme closing 15 Minute.Glass slide is washed with distilled water, then glass slide is incubated in micro-wave oven to 15 minutes progress antigen retrievals, then used PBS washing.Then glass slide and the Working dilutions of every kind of monoclonal antibody are incubated overnight at 4 DEG C.It is washed with PBS and carries glass Piece is simultaneously dyed using DAKO EnVision+ system (K5007, Denmark).Using for Ki-67 (1: 200 dilution； Cat.RM-9106-S0, Thermo Fisher) monoclonal antibody and for MEK5 polyclonal antibody (1:200 dilute； Cat.ab210748, Abcam, USA).Finally, by glass slide haematoxylin redyeing.
1.5TUNEL analysis
3 ' the ends-OH DNA are mixed fluorescein -12- by apoptosis detection system Fluorescein (Promega G3250) DUTP acts on detecting the fragmentation DNA of apoptotic cell by terminal deoxynucleotidyl transferase (TdT).By paraffin embedding Tissue is processed into 4- μm of paraffin section and detects for TUNEL apoptotic cell.Slice is dewaxed in dimethylbenzene and passes through gradient second Alcoholic solution moisturizing again.Slice is washed in 0.85%NaCl, is then washed with PBS.It will slice and 100 μ, 120 μ g/ml albumen Enzyme k is incubated 10 minutes at 37 DEG C.It is covered and is sliced with 100 μ l equilibration buffers, then covered with 50 μ lTdT incubation buffers. Negative control is carried out in the case where no TdT.Positive cell is detected by Laser Scanning Confocal Microscope.
1.6 mutation constructions and slow virus preparation
Total serum IgE is extracted using TRIzol reagent (Invitrogen, 15596-026, USA).RNA is resuspended in ultrapure water In, and handled 30 minutes with DNA enzymatic I (Ambion, AM2222) at 37 DEG C, and according to the explanation of manufacturer RNeasy Midi Kit (QIAGEN) carries out RNA purification.It is total from 2.0 μ g using Omniscript Reverse Transcriptase kit (205113, Qiagen) Cause complementary DNA (cDNA) in RNA at random.Normal PCR and over-lap PCR side are carried out using the primer designed for cDNA clone Method.PCR product is cloned into TA carrier (Invitrogen, K451020V, USA；For clone and DNA sequencing) in and carry out Sequencing.To be sequenced correct gene fragment clone to GV358 carrier (Ji Kai, China) Age I site.By by above-mentioned structure It builds body and packaging plasmid cotransfection generates slow virus into HEK293T cell, and 48 hours after transfection, collect HEK293T Cell culture medium is used for next experiment.
1.7 cell culture and transfection
According to illustrate cultivate human thyroid cancerous cell line B-CPAP (DSMZ No.ACR 273).It is measured and is verified by STR Cell identity.Then the slow disease of slow virus and sky by being overexpressed wild type (WT) MAP2K5, MAP2K5 A321T and M367T Poison expression GFP transfects B-CPAP cell line.The puromycin of final concentration of 4 μ g/ml is used to screen the cell of stable virus infection System.Finally, the cell line of four kinds of adenovirus infections is obtained, with stable expression of target gene.
1.8 immunofluorescence dyeing
It will stablize and express MAP2K5, the B-CPAP cell line of MAP2K5 A321T and MAP2K5 M367T are inoculated with and six It is cultivated in orifice plate.ERK5 (lot number: AP070721, Boke, China), phosphorylated CREB 5 (Thr218+Tyr220) (lot number: AE050702, Boke, China), phosphorylated CREB 5 (Ser731+Thr733) (lot number: AC09223656, Boke, China) and phosphorus It is acidified ERK5 (Ser496) (lot number: AC11012356, Boke, China) and is used as the primary antibody of target protein detection.The secondary antibody of label is used In fluorescence detection.Nucleus is dyed with DAPI.Finally, analyzing image by Nikon two-photon Laser Scanning Confocal Microscope.
The detection of 1.9 phosphorylations
In this study by the ERK5 of phosphorylation (Thr218+Tyr220) (lot number: AE050702, Boke, China), The ERK5 (Ser496) of ERK5 (Ser731+Thr733) (lot number: AC09223656, Boke, China) and phosphorylation of phosphorylation (lot number: AC11012356, Boke, China) has checked five phosphorylation sites of ERK5.The anti-secondary antibody of HRP coupling is used In further research, be then incubated for the secondary antibody (anti-rabbit) of horseradish peroxidase conjugation phosphorylation ERK5 (Thr218+ Tyr220), the ERK5 (Ser731+Thr733) of phosphorylation and the ERK5 (Ser496) of phosphorylation or for GAPDH and GFP bis- Anti- (anti-mouse) is incubated for.Made using ECL Plus western blot detection reagent (WBULS0500, Millipore Inc, USA) Protein band visualization, and using 003.02 software of Image Quant TL version 2 in Storm860PhosphorImager It is upper to detect and quantify.
1.10MAP2K5 the computation model of middle A321T and M367T
The natural and catastrophic model A321T and M367T of MAP2K5 residue 146-438 is by Swiss-Model Server (Nucleic Acids Res is generated from the protein structures of people Mek-1 kinases (PDB:3SLS).In July, 2014；42 [network service number: W252-8]).Use PyMOL software (DeLano WL.2002；PyMOL molecular graphics System, DeLano Scientific, Palo Alto, CA.http: //www.pymol.org) generate it is shown herein Model.
1.11 statistical analysis
Use SPSS (version 2 2.0；SPSS Inc., Chicago, USA) it is for statistical analysis.Pass through two groups Student t is examined, and thinks there is significant statistical difference in P < 0.05, the one-way analysis of variance (variance point of multiple groups Analysis) analysis.All tests are bilateral, and P < 0.05 is considered to have statistical significance.
2. result
2.1 are mutated by the MAP2K5 of respondent
MAP2K5 c.G961A (p.A321T) is found in three first degree relatives in one family. MAP2K5 C.T1100C (p.M367T) is found in two first degree relatives in another family.For gene frequency, A321T and Gene frequency of the M367T in FNMTC patient is respectively 0.0390 and 0.0259；And normal Chinese compares (promise standing grain-China Genome plan, n=2200, P < 0.001) both of the aforesaid locus gene frequency be respectively 0 and 0.00022523.It can be seen that MAP2K5 A321T and M367T and FNMTC has extremely significant correlation.
The Sanger sequencing result of MAP2K5 c.G961A (p.A321T) and MAP2K5 c.T1100C (p.M367T) are such as Shown in Figure 1A, B.
Fig. 1 C illustrates position of both of the aforesaid mutational site in MAP2K5.
Fig. 1 D then show the corresponding wild-type base in both of the aforesaid mutational site be it is very conservative, in the mankind, big All there is very high homology in the animals such as mouse, ox.
MAP2K5 after 2.2 mutation activates MAPK-ERK5 signal path
Inventor is detected in MAP2K5 mutation crowd using RT-qPCR, MAP2K5 downstream gene ERK5 and MAP2K5- The expression of the downstream gene of ERK5 approach.
In blood leucocyte, FOSB, MEF2, PPARG, CDK4 and CCND gene of FNMTC crowd ratio SNMTC crowd Expression quantity it is high (Fig. 2).
In parathyroid tissue, either malignant tissue, or health tissues, before FNMTC crowd and SNMTC crowd Stating gene expression does not have marked difference, but the expression of downstream target gene ERK5, FRA1, MEF2 etc. are mutated NMTC people in MAP2K5 Higher (Fig. 3) is expressed in group.
Further, Ki67 dyes (Fig. 4) and TUNEL detection (Fig. 5) display, the cell increasing of SNMTC and FNMTC crowd It grows with level of apoptosis without significant difference.
2.3MAP2K5 is mutated Ser496 the and Ser731+Thr733 phosphorylation for leading to ERK5 albumen
MAP2K5 protein structure schematic diagram and mutational site are as shown in Figure 7 A.
By three kinds of MAP2K5 wild type (WT group), MAP2K5 A321T (Mu1 group) and MAP2K5 M367T (Mu2 group) genes It is integrated into GV358 carrier (band CMV promoter and EGFP label) respectively, by adenovirus, is transferred to human thyroid cancer cell line B- CPAP(DSMZ No.ACC 273)；Unloaded GV358 carrier is transferred to B-CPAP as control (GFP group) (figure by adenovirus 6)。
Then the phosphorylation site for using commercialized antibody test (passing through protein immunoblot) ERK5, corresponds to phosphorus Polyadenylation sites have Ser496, Ser731+Thr733 and Thr218+Tyr220 respectively.For be overexpressed GFP, WT, Mu1 and B-CPAP, GAPDH and the GFP of Mu2 is both used as referring to ensure that endogenous and foreign protein total amount is about phase same level (Fig. 7 B And C).
As a result, it has been found that being overexpressed the total of the significant raising downstream ERK5 of B-CPAP of WT, Mu1 and Mu2 using GFP as reference It measures (Fig. 7 C).Further detect ERK5 phosphorylation site: Ser496, Ser731+Thr733 and Thr218+Tyr220.With GFP It is compared with WT group, the significant increase (Fig. 7 B) of the phosphorylation level of the ERK5 of the Ser496 of Mu1 and Mu2 group.In addition, with GFP and WT Group is compared, the significant increase (Fig. 7 B) of ERK5 Ser731+Thr733 phosphorylation level in Mu1 group.
The ERK5 transporte to cells core of 2.4 phosphorylations regulates and controls downstream gene expression
Inventor detects the ERK5 subcellular localization in above-mentioned transgenic cell using immunofluorescence dyeing.Hair Existing, in MAP2K5 overexpression group, p-ERK5 Ser731+Thr733 is located in entire cytoplasm (Fig. 7 D-7F)；However, In MAPK2K5 A321T overexpression group, downstream p-ERK5 Ser731+Thr733 is only located in nucleus (Fig. 7 G-7I).Show MAPK2K5 A321T mutation leads to ERK5 transporte to cells core.
Inventor further analyzes the expression of target gene of above-mentioned transgenic cell.Most of MAPK-ERK5 letters as the result is shown Number conductive targets, which are marked in A321T and M367T group, to raise, such as ERK5, FOSB, MEF2, CDK4, CCND1 and FRA1, wherein JUN and PPARG only raises (Fig. 8) in a group.
Experimental example the result shows that, MAP2K5 mutation lead to ERK5 phosphorylation, further cause ERK5 transporte to cells core In, and then regulate and control downstream gene, so that the relevant gene of cancer significantly raises, push follicular epithelial cell pernicious Transformation, causes the illness probability of thyroid cancer to rise.
To sum up, it is related to thyroid cancer by the variation of T to C to illustrate the 1100th bit base of MAP2K5 gene by the present invention Property；It is detected using the tissue of kit of the invention to people, if detecting the mutation, that is, can determine whether tested person group There are the risks of thyroid cancer.The present invention can be used for the auxiliary diagnosis of thyroid cancer.
SEQUENCE LISTING
<110>Huaxi Hospital Attached to Sichuan Univ
<120>a kind of the 1100th bit base mutated gene of mankind MAP2K5 and its detection kit
<130> GY026-2018P012679CC
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
tcataatgtg tccaagtgag tc 22
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
tttacagtgg agtggaaaga aa 22

Claims (13)

1. a kind of mankind's MAP2K5 gene of variation, it is characterised in that: it is the mankind MAP2K5 that the 1100th bit base sports A Mutant gene.
2. a kind of mankind's MAP2K5 albumen of variation, it is characterised in that: it is the mankind that the 367th amino acids sport threonine MAP2K5 variant protein.
3. the related reagent of detection the 1100th bit base of mankind MAP2K5 gene variation is preparing non-medullary thyroid carcinoma screening examination Purposes in agent.
4. purposes according to claim 3, it is characterised in that: the screening agent is the sieve for detecting papillary thyroid carcinoma Look into reagent.
5. purposes according to claim 3, it is characterised in that: the reagent is detection mankind MAP2K5 gene the 1100th The related reagent of T → C variation.
6. purposes according to claim 3 or 4, it is characterised in that: described the 1100th T of detection mankind MAP2K5 gene → The related reagent of C variation is sequencing reagent, Snapshot reagent, restriction fragment length polymorphism method reagent or list Chain conformation polymorphism analysis reagent.
7. purposes according to claim 6, it is characterised in that: the sequencing reagent includes PCR amplification MAP2K5 gene The reagent in the 1100th site.
8. purposes according to claim 7, it is characterised in that: the reagent packet in amplification the 1100th site of MAP2K5 gene Include following primer pair:
Upstream primer are as follows: 5 '-TCATAATGTGTCCAAGTGAGTC-3 ', downstream primer are as follows: 5 '- TTTACAGTGGAGTGGAAAGAAA-3′。
9. a kind of kit for screening of non-medullary thyroid carcinoma, it is characterised in that: it includes optional for detecting the mankind The related reagent of the 1100th bit base of MAP2K5 gene variation.
10. kit according to claim 9, it is characterised in that: the reagent is detection mankind MAP2K5 gene the The related reagent of 1100 T → C variation.
11. kit according to claim 9 or 10, it is characterised in that: the detection mankind MAP2K5 gene the 1100th The related reagent of position T → C variation is sequencing reagent, Snapshot reagent, restriction fragment length polymorphism method reagent Or single-strand conformation polymorphism analysis reagent.
12. kit according to claim 11, it is characterised in that: the sequencing reagent includes PCR amplification MAP2K5 The reagent in the 1100th site of gene.
13. according to right want 12 described in kit, it is characterised in that: it is described amplification the 1100th site of MAP2K5 gene reagent Including following primer pair:
Upstream primer are as follows: 5 '-TCATAATGTGTCCAAGTGAGTC-3 ', downstream primer are as follows: 5 '- TTTACAGTGGAGTGGAAAGAAA-3′。
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