CN105255927A - KIAA1217-RET fusion gene - Google Patents

Abstract

The invention discloses a KIAA1217-RET fusion gene. The KIAA1217-RET fusion gene is formed by exons from number one to number eleven of a KIAA1217 gene and exons from number eight to number nineteen of an RET gene in a fusion mode, and the nucleotide sequence is shown in figure 1. Disclose of the fusion gene is favorable for further study of drive genetic modification not found in thyroid cancer, and the clinical pathological classification and treatment strategies of patients suffering from papillary thyroid carcinoma are optimized.

Description

A kind of KIAA1217-RET fusion gene

Technical field

The invention belongs to gene engineering field, specifically refer to a kind of KIAA1217-RET fusion gene.

Background technology

The sickness rate of thyroid carcinoma has increased more than three times in nearly 30 years, and its histological types and gene expression profile are also along with the time changes to some extent.In all thyroid carcinomas, except medullary carcinoma, it is all the follicular cells originating from the unicellular epithelial lining of composition Tiroidina.Thyroid papillary carcinoma (PapillaryThyroidCarcinoma, PTC) is modal histological type in thyroid malignancy, gains the name, account for greatly 80% in all thyroid malignancies because of its mammillate histological structure.PTC is recoverable often, and its 5 years survival rates are about 95%.But some PTC can dedifferente and become aggressive, fatefulue thyroid carcinoma.Current primary treatment scheme has the endocrine therapy of surgical operation, Tiroidina anti-tsh hormone and radioiodine therapy (utilizing thyroid follicular cells to have the principle of high-affinity to iodine).

Gene variation on RET/RAS/BRAF/MAPK signal path, has now confirmed to play very keying action in the pathogenesis of papillary carcinoma.About have the gene alteration of 70% to occur on this path in all papillary carcinomas, and seldom there is juxtaposition in RET/RAS/BRAF gene variation, point out the activation of an effector molecule in this path to be namely enough to celliferous vicious transformation.In this path, the most general gene variation is the V600E sudden change of BRAF gene, and its incidence accounts for 40% of papillary carcinoma.Also the thyroid papillary carcinoma and AKAP9-BRAF fusion that report and find that existence is caused by BRAF chromosome rearrangement is had.This rearrangements seldom appears in sporadic papillary carcinoma, and is more more common in by radiation-induced thyroid malignancy.In thyroid papillary carcinoma, about have the point mutation that 10% exists RAS gene (N-RAS, H-RAS, K-RAS), and great majority occur in the papillary carcinoma of folliculus hypotype.The RET protooncogene activation that chromosome rearrangement is correlated with is another more common gene alteration in PTC.

RET proto-oncogene is positioned at 10 chromosomal 1 districts 1 and is with 2 subzones (i.e. 10q11.2), and its coding generates the tyrosine kinase receptor albumen of cross-cell membrane.This albumen is made up of three structural domains: ligand binding domain (comprise four cadherin sample repeating structures and be rich in halfcystine structure), hydrophobic transmembrane and comprise born of the same parents' internal area of tyrosine kinase domain.Its part belongs to the neutrophilia factor family of glia cell line-derived, include neurturin (NRTN), persephin (PSPN) and artemin (ARTN), with cause the Dimerized of tyrosine kinase receptor after ligand binding, the Tyrosylprotein kinase of key position in autophosphorylation born of the same parents internal area, activates associated downstream signal path.

In parathyroid tissue, RET proto-oncogene high expression level is in parafollicular cell (i.e. C cell), and its point mutation can cause developing of medullary thyroid carcinoma.In thyroid follicular cells, RET proto-oncogene can be activated because of chromosome rearrangement, causes 3 ' end of 5 ' of some uncorrelated genes end and RET gene to merge, produces RET/PTC and reset.It is also that papillary carcinoma the most common resets carcinogenic type that three kinds of RET/PTC of identified discovery the earliest reset.Wherein, RET/PTC1 and RET/PTC3 is occurred to merge by H4 gene (also known as D10S170 or CCDC6) and NCOA4 gene (also known as RFG or ELE1) and RET gene respectively and is formed.RET/PTC1 and RET/PTC3 is formed by the chromosomal same arm transposition of same bar, and upstream and downstream two genes namely occurring to merge all are positioned at No. 10 karyomit(e).Unlike, RET/PTC2 is No. 10 and No. 17 karyomit(e) generation transposition, causes PRKAR1A and RET to be formed and merges.In the last few years, the RET/PTC fusion of a series of novel type was in the news and was found in PTC case, and wherein the overwhelming majority is formed by interchromosomal translocation.

Although found some gene alterations relevant to PTC, but still the thyroid carcinoma mechanism of some is not bright.Therefore, also need to study it further.

Summary of the invention

The object of the invention is the shortcoming and defect existed to overcome prior art, and a kind of new RET fusion and KIAA1217-RET are provided, this fusion gene contribute to probing into further in thyroid carcinoma do not find drive gene alteration, optimize patients with papillary thyroid carcinoma clinical pathology classification and Strategy of Diagnosis.

For achieving the above object, technical scheme of the present invention is that the 1-11 exon of this KIAA1217-RET fusion gene by KIAA1217 gene merges mutually with the 8-19 exon of RET gene and form, and its nucleotide sequence is as shown in SEQIDNO:1.

Further setting is that the KIAA1217-RET fusion rotein expressed by KIAA1217-RET fusion gene is positioned in nucleus, and reduces MAPK pathway activity.KIAA1217-RET fusion rotein expressed by KIAA1217-RET fusion gene forms self dimer by coiled-coiled structure, and the kinases territory activating RET albumen in KIAA1217-RET fusion rotein is active.

Further setting is a peptide species, it is characterized in that: it is encoded by described KIAA1217-RET fusion gene and obtains.

Advantage of the present invention is:

The aminoacid sequence of KIAA1217-RET proteins encoded finds that it is positioned nucleus, and around be positioned tumour cell with the immunofluorescence experiment of RET antibody nuclear membrane with verifying its disperse.The ability of RET protein activation MAPK signal path is detected with immunoblot experiment.Due to disperse be positioned nucleus, cause the reduced capability of RET protein activation MAPK signal path in kytoplasm.Fusion gene activates Tyrosylprotein kinase promotes mosaic gene expression by merging the highly active promotor of upstream gene, and utilizes the activation of the Dimerized and kinase activity of dimer functional domain mediating ligand dependent/non-dependent.Use COILS and Paircoil2 software discovery KIAA1217-RET fusion rotein to there is coiled-coiled structure (CC structure), the kinases territory activating RET albumen in KIAA1217-RET fusion rotein is active.

Below in conjunction with specification drawings and specific embodiments, the present invention is described further.

Accompanying drawing explanation

The transcript profile sequenced genes sequence of Fig. 1 KIAA1217-RET fusion gene of the present invention merges figure, and the middle vertical line of Fig. 1 is the tie point position of fusion gene;

Integrity (RIN) the qualification figure of Fig. 2 R56T;

Integrity (RIN) qualification of Fig. 3 R56N;

The based composition situation map of Fig. 4 R56T;

The base mass value distribution situation figure of Fig. 5 R56T;

The comparison statistics figure of Fig. 6 R56T;

The comparison statistics figure of Fig. 7 R56N;

The order-checking randomness evaluation graph of Fig. 8 R56T;

The order-checking randomness evaluation graph of Fig. 9 R56N;

The gene coverage statistical graph of Figure 10 R56T;

The gene coverage statistical graph of Figure 11 R56N;

Figure 122 0 patient (comprising cancer and cancer beside organism) fusion gene Circos that transcript profile checks order schemes;

The fusion gene Circos of Figure 13 R56T transcript profile order-checking schemes (wherein KIAA1217-RET fusion is noted by red collimation mark);

The gene coordinate diagram of Figure 14 KIAA1217

The gene coordinate diagram of Figure 15 RET;

The molecular biology structure iron of Figure 16 KIAA1217-RET fusion gene;

Figure 17 selects the preliminary experiment figure of the best primer of KIAA1217-RET fusion gene;

The forward Sanger sequencer map of Figure 18 KIAA1217-RET fusion gene;

The reverse Sanger sequencer map of Figure 19 KIAA1217-RET fusion gene;

The WesternBlot figure of Figure 20 KIAA1217-RET fusion gene;

The result figure of Figure 21 NNCN algorithm predicts fusion rotein location;

The result figure of Figure 22 k-NN algorithm predicts fusion rotein location;

The KIAA1217-RET fusion rotein location map of Figure 23 band green fluorescent label;

The horizontal WesternBlot figure of MAPK path of Figure 24 R56 tumour and normal specimen

The rna expression level view of CyclinD1, MMP1 and MMP9 in Figure 25 R56 sample;

Figure 26 uses COILS (left side) and Paircoil2 (right side) software whether new fusion rotein to be existed to the prognostic chart of coiled-coiled structure.

Embodiment

Below by embodiment, the present invention is specifically described; only be used to further illustrate the present invention; can not be interpreted as limiting the scope of the present invention, the technician in this field can make some nonessential improvement and adjustment according to the content of foregoing invention to the present invention.

(1), the acquisition of KIAA1217-RET fusion gene and screening

1 Papillary Thyroid Carcinoma Specimen origin

1.1 samples enter group standard

Sample is all diagnosed as the corrective surgery Operated Specimens of thyroid papillary carcinoma from the court's surgical oncology, and does not all accept other medical interventional (as chemicotherapy) before all 20 routine operation in patients.Before specimen collection, Yi Huo Hospital Ethical Committee ratifies, and signs Informed Consent Form with patient and family members thereof in the preoperative.Neoplasm staging is with reference to the thyroid papillary carcinoma criteria for classification of american cancer joint committee (AJCC).Each tumor tissues sample has corresponding healthy tissues sample, i.e. Carcinoma side normal tissue (if tumour is one-sided focus, then Carcinoma side normal tissue is from the uninvolved gland leaf texture of offside).Each tumor specimen is furnished with a HE stained of mating with its FFPE paraffin mass.

Making a definite diagnosis of 1.2 thyroid papillary carcinomas

The fresh specimens of tumour and its corresponding healthy tissues is embedded in optimum Cutting temperature (OCT) mixture, then makes tissue slice for pathological diagnosis.High age and service seniority pathologist assesses the HE section of sample, and confirms that tumor specimen meets in the diagnosis of thyroid papillary carcinoma and the healthy tissues of pairing not containing tumour cell in histopathology.Be wherein that tumour cell average abundance is greater than 60% to the requirement meeting tumor specimen, and necrotic tissue composition is less than 20%.If folliculus hypotype structure reaches 99%, be then classified as thyroid follicle hypotype papillary carcinoma.If the cell proportion with high cell characteristic is greater than 50%, be namely defined as Tiroidina height cell subsets papillary carcinoma.

1.3 clinical datas are collected

The clinical data of 20 routine patients with papillary thyroid carcinoma is all from the electronic medical record system of the court's surgical oncology.Obtain clinical pathology and demographic data as follows: age during diagnosis, obtain tissue sample time, sex, nationality, whether have malignant tumour medical history, knub position, tumor size, AJCC thyroid papillary carcinoma classify (T primary tumo(u)r size, N regional nodes involvement, M distant metastasis situation), whether whether clinical stages, tumour are multifocal, tumor histology's hypotype and have radiation to radiate history.

2 experiment reagents and instrument consumptive material

2.1 experiment reagent

2.2 instrument consumptive materials

Experimental technique

1RNA-Seq high-flux sequence detects the track fusion level difference into group patients with papillary thyroid carcinoma

The extraction of 1.1 total tissue RNA and integrity (RIN) qualification

(1) first guarantee that the tissue block weight for extracting RNA is not more than 50mg;

(2) tissue block of suitable size is put into 1.5mlEP pipe, and add 700ulQIAzol lysate, make its abundant cracking and mixing with tissue grinder;

(3) homogenate room temperature is positioned over 5min on worktable;

(4) in homogenate, add 140ul chloroform, cover the cap of EP pipe, high vibration 15s;

(5) homogenate room temperature is placed in 2-3min on worktable;

(6) 12000xg, 4 DEG C of centrifugal 15min of low-temperature and high-speed;

(7) moved in new EP pipe by the supernatant liquor after centrifugal, and add about 525ul dehydrated alcohol, fully piping and druming is mixed;

(8) getting 700ul mixed solution moves in the RNeasyMini centrifugal column of 2ml, and the centrifugal 15s of >=8000xg room temperature, abandons centrifugate;

(9) remaining liq in mixed solution is moved in the RNeasyMini centrifugal column of 2ml in the lump, repeat the 8th step;

(10) in RNeasyMini centrifugal column, add 700ulRWT, the centrifugal 15s of >=8000xg room temperature, abandons centrifugate;

(11) in RNeasyMini centrifugal column, add 500ulRPE, the centrifugal 15s of >=8000xg room temperature, abandons centrifugate;

(12) again in RNeasyMini centrifugal column, add 500ulRPE, the centrifugal 2min of >=8000xg room temperature, abandons centrifugate;

(13) RNeasyMini centrifugal column is transferred in new 2ml collection tube, at full speed centrifugal 1min;

(14) RNeasyMini centrifugal column being moved in new 1.5mEP pipe, adding 40ul through removing the nucleic acid water of RNA ferment treatment, the centrifugal 1min of >=8000xg, so that the RNA that wash-out extracts;

(15) with Agilent2100, Quality Control is carried out to the RNA extracted: leading indicator comprises the ratio of RNA concentration, RIN value and 28S/18S.

1.2RNA-Seq high-flux sequence

Collect the RNA extracted to deliver to Hua Da gene (Guangzhou) company and be two generation NGS and check order, concise and to the point step is as follows.From the total serum IgE extracted, there is with the Beads enrichment with Oligo (dT) mRNA of polyA structure.With special lysate, mRNA is broken into short small segment.With these small segments as masterplate, random hexamers reverse transcription is utilized to synthesize the Article 1 chain of cDNA.Use the Article 2 chain of the tube-nursery cDNA such as damping fluid, dNTPs, RNaseH and DNA polymerase i more respectively.With the small segment that QIAQuickPCRextractionkit kits interrupts, and with its sticky end of EB damping fluid polishing and add polyA tail.Connect upper sequence measuring joints to subsequently these small segments.According to the result of agarose gel electrophoresis, choose suitable small segment and do pcr amplification.Finally, the HiSeq2000 high-flux sequence instrument cDNA library established being put into Illumina company checks order, and produces the reads data that 2x90 is paired.

1.3 bioinformatic analysis

After removing sequence measuring joints data in original reads and inferior quality reads, Tophat (v2.0.8) software is used the reads and human genomic sequence (hg19) that meet Quality Control to be compared, software parameter employing default form.Utilize Cufflinks (v2.0.2) software to assess gene expression abundance, its abundance unit is the reads number coming from the every kilobase length of certain gene in RPKM and every 1,000,000 reads.Detection fusion gene adopts Soapfuse (v1.2.4) software.

The expression level of fusion gene in the patients with papillary thyroid carcinoma tissue sample of 2RT-PCR experimental verification order-checking

2.1 fusion gene design of primers and synthesis

The specific primer design principle of fusion gene is as follows:

The Auele Specific Primer synthesis of fusion gene: synthesized by Li Fei biotechnology (Shanghai) Co., Ltd.

The specific primer sequence of table 1KIAA1217-RET fusion gene

2.2 total serum IgE reverse transcriptions

(1) sex change of RNA: added in the EP pipe of 200ul by the RNA of extraction, put into PCR instrument, is set to 70 DEG C of 10min, after the time arrives, EP pipe is placed in cooled on ice;

(2) configure reaction solution, adopt the ReverTra of Japanese TOYOBO company qPCRRTKit test kit, concrete reaction system is as following table 2;

Table 2RNA reverse transcription reaction system (cumulative volume 20ul)

(3) PCR programming: first 16 DEG C of 5min, then 42 DEG C of 30min, last 98 DEG C of 5min.

2.3PCR amplification

(1) configure reaction solution, use sky with the 2xTaqPCRMasterMixKit test kit of company, concrete reaction system is as following table 3;

Table 3PCR reaction system (cumulative volume 50ul)

(2) PCR programming: first 94 DEG C of 3min, then 94 DEG C of 30sec, 57 DEG C of 30sec and 72 DEG C 1min of 30 circulations, last 72 DEG C of 5min.

2.4PCR product agarose gel electrophoresis runs glue

(1) glue: the sepharose preparing different size according to each difference of running glue sample number, dissolves agarose with 0.5XTAE electrophoretic buffer, makes its concentration reach 2%.Mixed solution is put into microwave oven heat, allow mixed solution fully dissolve, when fluid temperature is down to 50-60 DEG C, drip GoldView (100ml gel adds 5ul) again and mix.Finally mixing liquid is poured in previously prepd glue mould, treat that temperature is down to room temperature and is made sepharose block.

(2) experimental procedure: the DNAmarker of 5ul and PCR primer are added in the hole of sepharose block respectively, sepharose is put into the electrophoresis chamber filling 0.5XTAE electrophoretic buffer, regulate electrophoresis apparatus to constant voltage 120V, electrophoresis 20min, finally puts into gel image analyser gel and carries out ultraviolet exposure and take pictures.

2.5 order-checking

Electrophoresis is run in glue the sample meeting object size strip and get 30-40ul packing, deliver to Li Fei biotechnology (Shanghai) Co., Ltd. in the lump and do generation Sanger order-checking.Sequencing data Chromas (v2.4.1) software carries out analyzing and arranging.

2.6 immunoblot experiment detection fusion albumen

(1) tissue protein extracts: the tissue block of suitable size is put into 1.5mlEP pipe, and adds 500ulRIPA lysate (by force), make its abundant cracking and mixing with tissue grinder.By centrifugal for homogenate 12000rpm 3min, get supernatant liquor and move in new 1.5mlEP pipe.

(2) Tissue protein concentration measures: it is 50ul (5ul sample+45ul distilled water) that configuration measures the system of concentration, adds in the cuvette that prior distilled water cleans up, then add 45ul distilled water and fully blow and beat by 5ul testing sample.Blank then replaces sample with the RIPA of 5ul.Detect the concentration of each sample with protein nucleic acid determinator, often surveyed and once cleaned cuvette 3 times with distilled water.

(3) WesternBlot experimental procedure:

1) in advance with liquid detergent soak sheet glass spend the night, then with distilled water flushing, dry and assemble and build rear leakage detection;

2) will treat that well heater that the sample of loading is placed in 99 DEG C heats up sex change 5min, generally often the protein mass of pipe applied sample amount is 40ug;

3) join glue scheme and refer to table 4 and 5.With 1ml rifle head, separation gel mixing liquid is added in sheet glass along U-shaped groove, note avoiding bubble, and use distilled water sealing.Horizontal rest is light-illuminating 30min on worktable, according to join in sebific duct remain mixing liquid solidify situation, remove the distilled water of sealing, distilled water cleans 3 times again, then filter paper blots.With 1ml rifle head, concentrated glue mixing liquid is added in sheet glass along U-shaped groove, note avoiding bubble.Slowly inserted in glue by comb after encapsulating, light coagulates 30min;

4), after gelling to be concentrated admittedly, slowly extract comb, add sample and the Marker of appropriate amount as required.First electrophoresis 80V40min in concentrated glue, to enter after separation gel regulating voltage to 140V until Marker, terminates electrophoresis run glue when the minimum band of Marker runs the most lower edge to separation gel;

5) according to target protein molecular weight, the gel after electrophoresis is cut into suitable size, and cuts the 0.2mm nitrocellulose membrane of corresponding size by gel size." sandwich " structure of blank, sponge pad, filter paper, film, glue, filter paper, sponge pad, blackboard composition is put into electric turn trough, and ice compress electricity turns 300mA90min;

6), after electricity turns end, take out tunica fibrosa 5% skim-milk room temperature shaker and close 3h, PBST embathes 10min*3 time afterwards;

7) soak rabbit anti-human RET primary antibodie (CST, 1:1000) 1ml on tunica fibrosa, and to place in wet box 4 DEG C and spend the night, PBST embathes 10min*3 time afterwards;

8) anti-(CST, 1:5000) 1ml of soak goat antirabbit two on tunica fibrosa, and place room temperature 2h in wet box, PBST embathes 10min*3 time afterwards;

9) darkroom exposure or the exposure of Odyssey scanner.

The biological function research of 3 fusion roteins

The organoid horizontal location of 3.1 Bioinformatics Prediction fusion roteins

PSORTII software is used to predict the organoid location of merging the new albumen formed, NNCN algorithm (Reinhardt ' smethodforCytplasmic/Nucleardiscrimination) and k-NN algorithm (k-nearestneighboralgorithm) is adopted together to predict location respectively, the consistence of assessment two kinds of algorithms, helps the design and implementation instructing next step experimental program.

The accuracy of 3.2 immunofluorescence experiment verifying software prediction location

(1) fresh specimens of getting R56 cooks frozen section, with adhesivity slide glass paster;

(2) with after methyl alcohol room temperature fixing section 5min, then 3 times are embathed with PBS;

(3) with after 0.3%Triton room temperature penetrating section 15min, then 3 times are embathed with PBS;

(4), after closing section 15min by lowlenthal serum liquid chamber temperature, slide glass is dried gently;

(5) soak rabbit anti-human RET primary antibodie (CST, 1:200) 100ul in section, and to place in wet box 4 DEG C and spend the night;

(6) take out section room temperature to be placed on worktable after 10min, then embathe section 5 times with PBS shaking table, each 1min;

(7) anti-(Alexa of soak goat antirabbit fluorescence two in section 488,1:500) 100ul, and be placed in room temperature lucifuge 30min on worktable;

(8) embathe section 5 times, each 1min with PBS shaking table, note lucifuge;

(9) after redying section 4min by DAPI (the green skies) room temperature, then embathe 3 times with PBS, note lucifuge equally;

(10) after slide glass being dried gently, with anti-cancellation mountant (the green skies) mounting;

(11) take pictures with fluorescence microscope biopsy tissues staining conditions.

The phosphorylation level of 3.3 immunoblot experiment detection fusion albumen MAPK signal paths

(1) tissue protein extracts: the tissue block of suitable size is put into 1.5mlEP pipe, and adds 500ulRIPA lysate (by force), make its abundant cracking and mixing with tissue grinder.By centrifugal for homogenate 12000rpm 3min, get supernatant liquor and move in new 1.5mlEP pipe.

(2) Tissue protein concentration measures: it is 50ul (5ul sample+45ul distilled water) that configuration measures the system of concentration, adds in the cuvette that prior distilled water cleans up, then add 45ul distilled water and fully blow and beat by 5ul testing sample.Blank then replaces sample with the RIPA of 5ul.Detect the concentration of each sample with protein nucleic acid determinator, often surveyed and once cleaned cuvette 3 times with distilled water.

(3) WesternBlot experimental procedure:

1) in advance with liquid detergent soak sheet glass spend the night, then with distilled water flushing, dry and assemble and build rear leakage detection;

2) will treat that well heater that the sample of loading is placed in 99 DEG C heats up sex change 5min, generally often the protein mass of pipe applied sample amount is 40ug;

3) join glue scheme and refer to table 4 and 5.With 1ml rifle head, separation gel mixing liquid is added in sheet glass along U-shaped groove, note avoiding bubble, and use distilled water sealing.Horizontal rest is light-illuminating 30min on worktable, according to join in sebific duct remain mixing liquid solidify situation, remove the distilled water of sealing, distilled water cleans 3 times again, then filter paper blots.With 1ml rifle head, concentrated glue mixing liquid is added in sheet glass along U-shaped groove, note avoiding bubble.Slowly inserted in glue by comb after encapsulating, light coagulates 30min;

4), after gelling to be concentrated admittedly, slowly extract comb, add sample and the Marker of appropriate amount as required.First electrophoresis 80V40min in concentrated glue, to enter after separation gel regulating voltage to 140V until Marker, terminates electrophoresis run glue when the minimum band of Marker runs the most lower edge to separation gel;

5) according to target protein molecular weight, the gel after electrophoresis is cut into suitable size, and cuts the 0.2mm nitrocellulose membrane of corresponding size by gel size." sandwich " structure of blank, sponge pad, filter paper, film, glue, filter paper, sponge pad, blackboard composition is put into electric turn trough, and ice compress electricity turns 300mA90min;

6), after electricity turns end, take out tunica fibrosa 5% skim-milk room temperature shaker and close 3h, PBST embathes 10min*3 time afterwards;

7) soak rabbit anti-human ERK and p-ERK primary antibodie (CST, 1:1000) 1ml on tunica fibrosa, and to place in wet box 4 DEG C and spend the night, PBST embathes 10min*3 time afterwards;

8) anti-(CST, 1:5000) 1ml of soak goat antirabbit two on tunica fibrosa, and place room temperature 2h in wet box, PBST embathes 10min*3 time afterwards;

9) darkroom exposure or the exposure of Odyssey scanner.

Table 412% separation gel prepares system

Table 54% concentrates glue and prepares system

3.4RealTime detects the expression level of phenotypic correlation molecule

(1) design of primers of phenotypic correlation molecule and synthesis

The specific primer design principle of phenotypic correlation molecule and internal reference is as follows:

The Auele Specific Primer synthesis of phenotypic correlation molecule and internal reference: synthesized by Li Fei biotechnology (Shanghai) Co., Ltd.

The specific primer sequence of table 6 phenotypic correlation molecule and internal reference

(2) total serum IgE reverse transcription

The total serum IgE reverse transcription of reaction system and same 2.2 fusion genes of experimental procedure.

(3) Real-timeqPCR amplification

1) configure reaction solution, adopt Japanese TOYOBO company qPCRMasterMixkit, concrete reaction system is as following table 7;

Table 7PCR reaction system (cumulative volume 20ul)

2) PCR programming: first 95 DEG C of 2min, then 95 DEG C of 15sec, 60 DEG C of 1min of 40 circulations, last 72 DEG C of 5min, choose melting curve programs option simultaneously.

(4) interpretation of result

Use relative quantification method to carry out statistical study, record the Ct value of respective sample goal gene and B-Actin reference gene respectively, both difference △ Ct, then relative expression levels's difference of the goal gene of comparison of tumor tissue and corresponding cancer beside organism.

3.5 prediction of fusion rotein conformation and Mechanism Discussions

Adopt COILS and Paircoil2 software whether to there is coiled-coil domain to the new albumen merging formation respectively to predict, the consistence of assessment two kinds of softwares, help the mechanism explaining fusion rotein performance biological effect further.Wherein the length of window of COILS software when the coil region that scanning peptide chain is possible is 14,21 and 28, and the length of window of Paircoil2 software is 28, and the P value that its inspection may exist coiled-coil domain is set in 0.025.

(2), data detection and analysis

1 checks order into the clinical pathology of group patient and demographic data

Mean age when this enters to organize patient diagnosis is 35.5 years old, and wherein 20% patient age is not less than 45 years old.Enter in group data to have 35% male sex and 65% women.The clinical stage of associated patient comprises three phases, is 85% I phase respectively, 5% III phase and 10% IV phase.The mean length of tumour maximum diameter is 22.4mm, and scope is from 8mm to 45mm.Enter to organize in patient to have 15% patient to be bilateral thyroid papillary carcinoma when diagnosing, all patients are all without the past malignant tumour medical history.Most patient is without known neck or Systemic radiation history and radiation history, and 5% shows it is not clear whether there was radiation history.Clinical pathology and the demographic data of 20 routine patients refer to table 1.

The clinical pathology of all patients of table 1 and demographic data

2RNA-Seq high-flux sequence detects the track fusion level difference into group patients with papillary thyroid carcinoma

Integrity (RIN) qualification of 2.1 total tissue RNA

With Agilent2100, Quality Control is carried out to the RNA extracted: leading indicator comprises the ratio of concentration, RIN and 28S/18S.20 pairs of selected RNA sample all meet the requirement of RNA-Seq order-checking.As there is the R56T sample that KIAA1217-RET merges, its censorship order-checking sample concentration is 384ng/uL, volume 31uL, total amount 11.9ug, RIN=7.4,28S/18S=2.1 (Fig. 2).Corresponding R56N then concentration is 304ng/uL, volume 28uL, total amount 8.51ug, RIN=6.7,28S/18S=1.8 (Fig. 3).

The quality evaluation of 2.2RNA-Seq high-flux sequence

Analysis of quality control: respectively from the based composition situation of reads, base mass value distribution situation, assess with five sequencing qualities of aspect to RNA-Seq such as the gene coverage situation with reference to gene (group) comparison situation, the stochastic condition that checks order and each sample.Such as R56T sample, the sample based composition equilibrium (Fig. 4) of its tumor tissues, each base accounts for 25%; The base mass value overwhelming majority is greater than Q20 (Fig. 5).Reference gene (group) the comparison situation of its tumour and cancer beside organism refers to Fig. 6 and 7, its separately reads be distributed to 3 ' end from 5 ' end more equably and namely check order randomness better (Fig. 8 and 9), the ratio of its each self-contained 90% ~ 100% coverage is higher (Figure 10 and 11).

2.3 bioinformatic analysis

After sequencing quality control filters, use Tophat (v2.0.8) software satisfactory reads and human genomic sequence (hg19) to be compared, software parameter adopts default form.Utilize Cufflinks (v2.0.2) software to detect gene expression abundance, its abundance unit is RPKM.Detection fusion gene adopts Soapfuse (v1.2.4) software.The fusion gene (Figure 12) of some amount is all there is in the tumour of each patient and normal specimen.Wherein find 20 to order-checking sample in find a sample there is the RET fusion sequence reads (Fig. 1) never reported, namely R56T KIAA1217-RET fusion (Figure 13).The gene coordinate (Figure 14 and 15) as shown below that the upstream and downstream gene that new RET merges is original on chromosome.New RET fusion mRNA is made up of the 1-11 exon of KIAA1217 and the 8-19 exon of RET, and concrete structure refers to Figure 16.

The expression level of fusion gene and the existence of immunoblotting checking fusion rotein in the patients with papillary thyroid carcinoma tissue sample of 3RT-PCR checking order-checking

For the tie point sequence of KIAA1217-RET fusion gene, PrimerPremier5 software is used to synthesize four pairs of primers according to the correlation principle of design of primers.Find that the specificity of the 2nd pair of primer is best by preliminary experiment, the band the most clear (Figure 17) of glue is run in gel electrophoresis, therefore selected 2nd pair of primer (i.e. FP:AGAAGTTGTGCGAGTTGGA, RP:AGTTCCTGGTGATCCCTTT) is as the experiment primer of further enlarged sample amount checking KIAA1217-RET fusion gene occurrence frequency.Product Song Lifei biotechnology (Shanghai) Co., Ltd. of 2nd pair of primer PCR is done Sanger order-checking simultaneously, there is the gold standard of KIAA1217-RET fusion gene sequence using generation sequencing result as checking, Figure 18 and 19 is respectively forward order-checking and the backward sequencing figure of KIAA1217-RET fusion gene sequence.The immunoblot experiment anti-human RET primary antibodie of rabbit (its for epitope hold at the C of RET albumen) removes detection fusion albumen, and its result as shown in figure 20.Namely this fusion gene can translate fusion rotein really, and molecular weight of albumen also meets prediction size (150kDa), and occurs over just in corresponding R56 tumor tissues sample.

The biological function research of 4 fusion roteins

The organoid horizontal location of 4.1 Bioinformatics Prediction fusion roteins

PSORTII software is used to predict the organoid location of merging the new albumen formed, (possibility of 94.1% is at nucleus to adopt NNCN algorithm respectively, Figure 21) (possibility of 69.6% is at nucleus with k-NN, Figure 22) algorithm together predicts location, find that the consistence of two kinds of algorithms is better, all predict that its fusion rotein has larger may being positioned in nucleus, help the design and implementation instructing next step experimental program.

4.2 use the organoid of immunohistofluorescence's technical identification fusion rotein to locate

Find the perinuclear new fusion rotein of RET (Figure 23, position shown in arrow) really having band green fluorescent label of DAPI dyeing, its green fluorescent label position is different from the after birth kytoplasm position of RET protein localization originally.

The phosphorylation level of the MAPK signal path that 4.3 immune-blotting method fusion roteins are correlated with

According to software prediction and immunohistofluorescence's technical identification, KIAA1217-RET fusion rotein location is transferred to nucleus from the after birth kytoplasm of original RET albumen, namely in R56T sample without BRAF mutain and the RET albumen being positioned after birth kytoplasm, corresponding classical MAPK pathway activity should weaken thereupon.Based on this imagination contrived experiment, the height of crucial molecules ERK phosphorylation activity on WesternBlot technology for detection MAPK path is used to assess the power of MAPK pathway activity.As shown in figure 24, the p-ERK level of R56T is starkly lower than R56N, and the MAPK pathway activity of the design before demonstrating and R56T sample weakens.

4.4Real-timeqPCR detects the expression level of phenotypic correlation molecule

The expression level of Real-timeqPCR technology to phenotypic correlation marker molecule is used to detect: propagation aspect (Ki-67, PCNA, AKT), cycle aspect (CyclinDs, CDK4/6, p27), apoptosis aspect (p53, caspase-3, BCL-2) and transfer aspect (MMPs).The rna expression level that found that CyclinD1, MMP1 and MMP9 of R56T comparatively R56N significantly raises, as shown in figure 25.

4.5 prediction of fusion rotein conformation and Mechanism Discussions

Adopt COILS and Paircoil2 software whether to there is coiled-coil domain to the new albumen merging formation respectively to predict, the consistence of assessment two kinds of softwares, help the mechanism explaining fusion rotein performance biological effect further.Wherein COILS software prediction exists the amino acid of coiled-coil domain is then 646-677AA at 648-685AA, Paircoil2 software, and two software prediction consistence better (Figure 26).Namely KIAA1217-RET fusion rotein easily forms self dimer by coiled-coiled structure, thus the kinases territory activating RET albumen in KIAA1217-RET fusion rotein is active.

SEQUENCELISTING

<110> Wenzhou Medical University

<120> KIAA1217-RET fusion gene

<160>2

<170>PatentInversion3.3

<210>1

<211>5158

<212>DNA

<213> artificial sequence

<221>MISC_FEATURE

<223> polynucleotide

<400>1

agagcctcggtggtttgcagcagtggaaccaggcaggcccagttgtgggtaggagaggcc60

gtcacctgttgaggcctcccccccacacccccgcatcgccctgccctggcagagcccagc120

ccccagtccccggagagcgcgcctgaggacggacggacggacggacggacagacctaggg180

acggagggccaggggcaggggagatccaagaggccccgcgctggaatgcagttttctcgg240

gcgagggagactttgcaccggagtggaaaatagtttggggtggggtttcgcaccgtcccc300

tcctccccagccccgggccccctcccaggcgctttctgggagcttttagaactgcgctct360

gaagtttccagagagcgaggagcttttgcggcaggcagagacaatggaagaaaatgaaag420

ccagaaatgtgagccgtgccttccttactcagcagacagaagacagatgcaggaacaagg480

caaaggcaatctgcatgtaacatcaccagaagatgcagaatgccgcagaaccaaggaacg540

cctttctaatggaaacagtcgtggttcagtttccaagtcttcccgcaatatcccaaggag600

acacaccctaggggggccccgaagttccaaggaaatactgggaatgcaaacatctgagat660

ggatcggaagagagaagcgttcctagaacatctgaagcagaagtacccccaccacgcctc720

tgcaatcatgggtcaccaagagaggctgagagaccagacaaggagccccaaactgtctca780

cagtcctcaaccacccagtctgggtgacccggtcgagcatttatcagagacgtccgctga840

ttctttggaagccatgtctgagggggatgctccaacccctttttccagaggcagccggac900

tcgtgcgagccttcctgtggtgaggtcaaccaaccagacgaaagaaagatctctgggggt960

tctctatctccagtatggagatgaaaccaagcagctcaggatgccgaatgaaatcacaag1020

tgcagacacaatccgtgctctcttcgtaagtgcctttccacagcagctcaccatgaaaat1080

gctggaatcgcccagtgtcgccatttacatcaaagatgaaagcagaaatgtctattatga1140

attaaatgatgtaaggaacattcaagacagatcactcctcaaagtgtacaacaaggatcc1200

tgcacatgcgtttaatcacacaccaaaaactatgaatggagacatgaggatgcagagaga1260

acttgtttatgcaagaggagatggccctggggcccctcgccccggatctactgctcatcc1320

accccatgcgattccaaattccccaccgtctactccagtgccccattccatgcccccctc1380

cccgtccagaattccttatgggggcacccgctccatggttgttcctggcaatgccaccat1440

ccccagggacagaatctccagcctgccagtctccagacccatctctccaagcccaagcgc1500

cattttagaaagaagagatgtcaagcctgatgaagacatgagtggcaaaaacattgcaat1560

gtacagaaatgagggtttctatgctgatccttacctttatcacgagggacggatgagcat1620

agcctcatcccatggtggacacccactggatgtccccgaccacatcattgcatatcaccg1680

caccgccatccggtcagcgagtgcttattgtaacccctcaatgcaagcggaaatgcatat1740

ggaacaatcactgtacagacagaaatcaaggaaatatccggatagccatttgcctacact1800

gggctccaaaacaccccctgcctctcctcacagagtcagtgacctgaggatgatagacat1860

gcacgctcactataatgcccacggcccccctcacaccatgcagccagaccgggcctctcc1920

gagccgccaggcctttaaaaaggagccaggcaccttggtgtatatagaaaagccacggag1980

cgctgcaggattatccagccttgtagacctcggccctcctctaatggagaagcaagtttt2040

tgcctacagcacggcgacaatacccaaagacagagagaccagagagaggatgcaagccat2100

ggagaaacagattgccagtttaactggccttgttcagtctgcgctttttaaagggcccat2160

tacaagttatagcaaagatgcgtctagcgagaaaatgatgaaaaccacagccaacaggaa2220

ccacacagatagtgcaggaacgccccatgtgtctggtgggaagatgctcagtgctctgga2280

gtccacggtgcctcccagccagcctccacctgtgggcacctcagccatccacatgagcct2340

gcttgagatgaggcggagcgtggcggaactcaggctccagctccagcagatgcggcagct2400

ccagctgcagaaccaggagttgctgagggcaatgatgaagaaggccgagctggaaatcag2460

tggcaaagtgatggaaacaatgaagagactggaggatcccgtgcagcgacagcgcgtcct2520

agtggagcaagagagacaaaaatatcttcatgaggaagagaagatcgtcaagaagttgtg2580

cgagttggaagactttgttgaagacttgaagaaggactccacggcagccagccgattggt2640

tactctgaaagacgtggaagacggggctttcctcctgcgtcaagtgggagaggctgtagc2700

taccctgaaagatgtggccgaggaggcgggctgccccctgtcctgtgcagtcagcaagag2760

acggctggagtgtgaggagtgtggcggcctgggctccccaacaggcaggtgtgagtggag2820

gcaaggagatggcaaagggatcaccaggaacttctccacctgctctcccagcaccaagac2880

ctgccccgacggccactgcgatgttgtggagacccaagacatcaacatttgccctcagga2940

ctgcctccggggcagcattgttgggggacacgagcctggggagccccgggggattaaagc3000

tggctatggcacctgcaactgcttccctgaggaggagaagtgcttctgcgagcccgaaga3060

catccaggatccactgtgcgacgagctgtgccgcacggtgatcgcagccgctgtcctctt3120

ctccttcatcgtctcggtgctgctgtctgccttctgcatccactgctaccacaagtttgc3180

ccacaagccacccatctcctcagctgagatgaccttccggaggcccgcccaggccttccc3240

ggtcagctactcctcttccggtgcccgccggccctcgctggactccatggagaaccaggt3300

ctccgtggatgccttcaagatcctggaggatccaaagtgggaattccctcggaagaactt3360

ggttcttggaaaaactctaggagaaggcgaatttggaaaagtggtcaaggcaacggcctt3420

ccatctgaaaggcagagcagggtacaccacggtggccgtgaagatgctgaaagagaacgc3480

ctccccgagtgagctgcgagacctgctgtcagagttcaacgtcctgaagcaggtcaacca3540

cccacatgtcatcaaattgtatggggcctgcagccaggatggcccgctcctcctcatcgt3600

ggagtacgccaaatacggctccctgcggggcttcctccgcgagagccgcaaagtggggcc3660

tggctacctgggcagtggaggcagccgcaactccagctccctggaccacccggatgagcg3720

ggccctcaccatgggcgacctcatctcatttgcctggcagatctcacaggggatgcagta3780

tctggccgagatgaagctcgttcatcgggacttggcagccagaaacatcctggtagctga3840

ggggcggaagatgaagatttcggatttcggcttgtcccgagatgtttatgaagaggattc3900

ctacgtgaagaggagccagggtcggattccagttaaatggatggcaattgaatccctttt3960

tgatcatatctacaccacgcaaagtgatgtatggtcttttggtgtcctgctgtgggagat4020

cgtgaccctagggggaaacccctatcctgggattcctcctgagcggctcttcaaccttct4080

gaagaccggccaccggatggagaggccagacaactgcagcgaggagatgtaccgcctgat4140

gctgcaatgctggaagcaggagccggacaaaaggccggtgtttgcggacatcagcaaaga4200

cctggagaagatgatggttaagaggagagactacttggaccttgcggcgtccactccatc4260

tgactccctgatttatgacgacggcctctcagaggaggagacaccgctggtggactgtaa4320

taatgcccccctccctcgagccctcccttccacatggattgaaaacaaactctatggtag4380

aatttcccatgcatttactagattctagcaccgctgtcccctttgcactatccttcctct4440

ctgtgatgctttttaaaaatgtttctggtctgaacaaaaccaaagtctgctctgaacctt4500

tttatttgtaaatgtctgactttgcatccagtttacatttaggcattattgcaactatgt4560

ttttctaaaaggatgtgaaaataagtgtaattaccacattgcccagcaacttaggatggt4620

agaggaaaaaacagatcagggcggaactctcaggggagaccaagaacaggttgaataagg4680

cgcttctggggtgggaatcaagtcatagtacttctactttaactaagtggataaatatac4740

aaatctggggaggtattcagttgagaaaggagccaccagcaccactcagcctgcactggg4800

agcacagccaggttcccccagacccctcctgggcaggcaggtgcctctcagaggccaccc4860

ggcactggcgagcagccactggccaagcctcagccccagtcccagccacatgtcctccat4920

caggggtagcgaggttgcaggagctggctggccctgggaggacgcacccccactgctgtt4980

ttcacatcctttcccttacccaccttcaggacggttgtcacttatgaagtcagtgctaaa5040

gctggagcagttgctttttgaaagaacatggtctgtggtgctgtggtcttacaatggaca5100

gtaaatatggttcttgccaaaactccttcttttgtctttgattaaatactagaaattt5158

<210>2

<211>1334

<212>PRT

<213> is artificial

<221>MISC_FEATURE

<223> polypeptide

<400>2

MetGluGluAsnGluSerGlnLysCysGluProCysLeuProTyrSer

151015

AlaAspArgArgGlnMetGlnGluGlnGlyLysGlyAsnLeuHisVal

202530

ThrSerProGluAspAlaGluCysArgArgThrLysGluArgLeuSer

354045

AsnGlyAsnSerArgGlySerValSerLysSerSerArgAsnIlePro

505560

ArgArgHisThrLeuGlyGlyProArgSerSerLysGluIleLeuGly

65707580

MetGlnThrSerGluMetAspArgLysArgGluAlaPheLeuGluHis

859095

LeuLysGlnLysTyrProHisHisAlaSerAlaIleMetGlyHisGln

100105110

GluArgLeuArgAspGlnThrArgSerProLysLeuSerHisSerPro

115120125

GlnProProSerLeuGlyAspProValGluHisLeuSerGluThrSer

130135140

AlaAspSerLeuGluAlaMetSerGluGlyAspAlaProThrProPhe

145150155160

SerArgGlySerArgThrArgAlaSerLeuProValValArgSerThr

165170175

AsnGlnThrLysGluArgSerLeuGlyValLeuTyrLeuGlnTyrGly

180185190

AspGluThrLysGlnLeuArgMetProAsnGluIleThrSerAlaAsp

195200205

ThrIleArgAlaLeuPheValSerAlaPheProGlnGlnLeuThrMet

210215220

LysMetLeuGluSerProSerValAlaIleTyrIleLysAspGluSer

225230235240

ArgAsnValTyrTyrGluLeuAsnAspValArgAsnIleGlnAspArg

245250255

SerLeuLeuLysValTyrAsnLysAspProAlaHisAlaPheAsnHis

260265270

ThrProLysThrMetAsnGlyAspMetArgMetGlnArgGluLeuVal

275280285

TyrAlaArgGlyAspGlyProGlyAlaProArgProGlySerThrAla

290295300

HisProProHisAlaIleProAsnSerProProSerThrProValPro

305310315320

HisSerMetProProSerProSerArgIleProTyrGlyGlyThrArg

325330335

SerMetValValProGlyAsnAlaThrIleProArgAspArgIleSer

340345350

SerLeuProValSerArgProIleSerProSerProSerAlaIleLeu

355360365

GluArgArgAspValLysProAspGluAspMetSerGlyLysAsnIle

370375380

AlaMetTyrArgAsnGluGlyPheTyrAlaAspProTyrLeuTyrHis

385390395400

GluGlyArgMetSerIleAlaSerSerHisGlyGlyHisProLeuAsp

405410415

ValProAspHisIleIleAlaTyrHisArgThrAlaIleArgSerAla

420425430

SerAlaTyrCysAsnProSerMetGlnAlaGluMetHisMetGluGln

435440445

SerLeuTyrArgGlnLysSerArgLysTyrProAspSerHisLeuPro

450455460

ThrLeuGlySerLysThrProProAlaSerProHisArgValSerAsp

465470475480

LeuArgMetIleAspMetHisAlaHisTyrAsnAlaHisGlyProPro

485490495

HisThrMetGlnProAspArgAlaSerProSerArgGlnAlaPheLys

500505510

LysGluProGlyThrLeuValTyrIleGluLysProArgSerAlaAla

515520525

GlyLeuSerSerLeuValAspLeuGlyProProLeuMetGluLysGln

530535540

ValPheAlaTyrSerThrAlaThrIleProLysAspArgGluThrArg

545550555560

GluArgMetGlnAlaMetGluLysGlnIleAlaSerLeuThrGlyLeu

565570575

ValGlnSerAlaLeuPheLysGlyProIleThrSerTyrSerLysAsp

580585590

AlaSerSerGluLysMetMetLysThrThrAlaAsnArgAsnHisThr

595600605

AspSerAlaGlyThrProHisValSerGlyGlyLysMetLeuSerAla

610615620

LeuGluSerThrValProProSerGlnProProProValGlyThrSer

625630635640

AlaIleHisMetSerLeuLeuGluMetArgArgSerValAlaGluLeu

645650655

ArgLeuGlnLeuGlnGlnMetArgGlnLeuGlnLeuGlnAsnGlnGlu

660665670

LeuLeuArgAlaMetMetLysLysAlaGluLeuGluIleSerGlyLys

675680685

ValMetGluThrMetLysArgLeuGluAspProValGlnArgGlnArg

690695700

ValLeuValGluGlnGluArgGlnLysTyrLeuHisGluGluGluLys

705710715720

IleValLysLysLeuCysGluLeuGluAspPheValGluAspLeuLys

725730735

LysAspSerThrAlaAlaSerArgLeuValThrLeuLysAspValGlu

740745750

AspGlyAlaPheLeuLeuArgGlnValGlyGluAlaValAlaThrLeu

755760765

LysAspValAlaGluGluAlaGlyCysProLeuSerCysAlaValSer

770775780

LysArgArgLeuGluCysGluGluCysGlyGlyLeuGlySerProThr

785790795800

GlyArgCysGluTrpArgGlnGlyAspGlyLysGlyIleThrArgAsn

805810815

PheSerThrCysSerProSerThrLysThrCysProAspGlyHisCys

820825830

AspValValGluThrGlnAspIleAsnIleCysProGlnAspCysLeu

835840845

ArgGlySerIleValGlyGlyHisGluProGlyGluProArgGlyIle

850855860

LysAlaGlyTyrGlyThrCysAsnCysPheProGluGluGluLysCys

865870875880

PheCysGluProGluAspIleGlnAspProLeuCysAspGluLeuCys

885890895

ArgThrValIleAlaAlaAlaValLeuPheSerPheIleValSerVal

900905910

LeuLeuSerAlaPheCysIleHisCysTyrHisLysPheAlaHisLys

915920925

ProProIleSerSerAlaGluMetThrPheArgArgProAlaGlnAla

930935940

PheProValSerTyrSerSerSerGlyAlaArgArgProSerLeuAsp

945950955960

SerMetGluAsnGlnValSerValAspAlaPheLysIleLeuGluAsp

965970975

ProLysTrpGluPheProArgLysAsnLeuValLeuGlyLysThrLeu

980985990

GlyGluGlyGluPheGlyLysValValLysAlaThrAlaPheHisLeu

99510001005

LysGlyArgAlaGlyTyrThrThrValAlaValLysMetLeuLys

101010151020

GluAsnAlaSerProSerGluLeuArgAspLeuLeuSerGluPhe

102510301035

AsnValLeuLysGlnValAsnHisProHisValIleLysLeuTyr

104010451050

GlyAlaCysSerGlnAspGlyProLeuLeuLeuIleValGluTyr

105510601065

AlaLysTyrGlySerLeuArgGlyPheLeuArgGluSerArgLys

107010751080

ValGlyProGlyTyrLeuGlySerGlyGlySerArgAsnSerSer

108510901095

SerLeuAspHisProAspGluArgAlaLeuThrMetGlyAspLeu

110011051110

IleSerPheAlaTrpGlnIleSerGlnGlyMetGlnTyrLeuAla

111511201125

GluMetLysLeuValHisArgAspLeuAlaAlaArgAsnIleLeu

113011351140

ValAlaGluGlyArgLysMetLysIleSerAspPheGlyLeuSer

114511501155

ArgAspValTyrGluGluAspSerTyrValLysArgSerGlnGly

116011651170

ArgIleProValLysTrpMetAlaIleGluSerLeuPheAspHis

117511801185

IleTyrThrThrGlnSerAspValTrpSerPheGlyValLeuLeu

119011951200

TrpGluIleValThrLeuGlyGlyAsnProTyrProGlyIlePro

120512101215

ProGluArgLeuPheAsnLeuLeuLysThrGlyHisArgMetGlu

122012251230

ArgProAspAsnCysSerGluGluMetTyrArgLeuMetLeuGln

123512401245

CysTrpLysGlnGluProAspLysArgProValPheAlaAspIle

125012551260

SerLysAspLeuGluLysMetMetValLysArgArgAspTyrLeu

126512701275

AspLeuAlaAlaSerThrProSerAspSerLeuIleTyrAspAsp

128012851290

GlyLeuSerGluGluGluThrProLeuValAspCysAsnAsnAla

129513001305

ProLeuProArgAlaLeuProSerThrTrpIleGluAsnLysLeu

131013151320

TyrGlyArgIleSerHisAlaPheThrArgPhe

13251330

Claims (4)

1. a KIAA1217-RET fusion gene, it is characterized in that: this KIAA1217-RET fusion gene to be merged mutually with the 8-19 exon of RET gene by the 1-11 exon of KIAA1217 gene and forms, and its nucleotide sequence is as shown in SEQIDNO:1.

2. a kind of KIAA1217-RET fusion gene according to claim 1, is characterized in that: the KIAA1217-RET fusion rotein expressed by KIAA1217-RET fusion gene is positioned nucleus nuclear membrane, and reduces MAPK pathway activity.

3. a kind of KIAA1217-RET fusion gene according to claim 1, it is characterized in that: the KIAA1217-RET fusion rotein expressed by KIAA1217-RET fusion gene forms self dimer by coiled-coiled structure, the kinases territory activating RET albumen in KIAA1217-RET fusion rotein is active.

4. a peptide species, is characterized in that: it is encoded by KIAA1217-RET fusion gene according to claim 1 and produces.