CN105567852A - IQCE gene and application of expression product thereof in diagnosis of diabetes mellitus type I - Google Patents
IQCE gene and application of expression product thereof in diagnosis of diabetes mellitus type I Download PDFInfo
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Abstract
The invention discloses an IQCE gene and application of an expression product thereof in the diagnosis of diabetes mellitus type I. The IQCE gene presents high expression in diabetes mellitus type I, and whether a patient suffers from diabetes mellitus type I or the probability that the patient suffers from diabetes mellitus type I is judged through detecting the expression level of the IQCE gene. The IQCE gene disclosed by the invention can greatly improve the sensitivity and specificity of the diagnosis of diabetes mellitus type I, and an early diagnosis means is provided for diabetes mellitus type I.
Description
Technical field
The present invention relates to biological technical field, relate to IQCE gene and the purposes of expression product in type i diabetes diagnosis thereof particularly.
Background technology
The metabolic disease of diabetes to be one group with hyperglycemia be feature.Hyperglycemia be then due to defect of insulin secretion or its biological action impaired, or both have concurrently and cause.Long-standing hyperglycemia during diabetes, causes various tissue, particularly eye, kidney, heart, blood vessel, neural chronic lesion, dysfunction.Diabetes are divided into Four types according to nosetiology, i.e. the diabetes of type i diabetes, type ii diabetes, gestational diabetes and specific type.
The cause of disease of diabetes and pathogenesis are comparatively complicated, not yet completely clear so far.Think that type i diabetes is a kind of under nature-nurture interacts at present, a kind of disease being main pathogenesis with the specificity beta Cell of islet autoimmune destruction of T cell mediation.Type i diabetes has polygenic inheritance background, and susceptibility is very complicated, disease be several genes interact, interactional result.
Use antibody screening and diagnosis type i diabetes at present clinically.As long as described specific antibody exists, As time goes on, overt diabetes tend develops.Although antibody screening can detect the increase level of type i diabetes risk, described risk to inform based on population selection whether individual patient disease exists the risk suffering from diabetes.Therefore, a kind of method is needed to realize Individual Diagnosis clinically.
In recent years, along with increasingly deep to the research of type i diabetes genes involved, although reported some to develop relevant gene to the generation of type i diabetes, but be not applied to clinically, therefore find that a species specific type i diabetes marker is applied to clinical diagnosis and has great importance.
Summary of the invention
In order to make up the deficiencies in the prior art, the object of the present invention is to provide a kind of molecular marker-IQCE gene that can be used for type i diabetes early diagnosis.Compare the diagnostic method of traditional type i diabetes, use molecular marker to diagnose type i diabetes to have promptness, specificity and susceptibility, make patient just can know risk in early days in disease, for risk height, take corresponding prevention and therapy measure.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides a kind of IQCE gene and the application of expression product in the product of preparation diagnosis type i diabetes thereof.
Further, the diagnostic products mentioned above comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection IQCE gene and expression product thereof to diagnose the product of type i diabetes.
Further, described RT-PCR diagnoses the product of type i diabetes at least to comprise the primer of a pair specific amplified IQCE gene; The product of described real-time quantitative PCR diagnosis type i diabetes at least comprises the primer of a pair specific amplified IQCE gene; The product of described immunodetection diagnosis type i diabetes comprises: the antibody be combined with IQCE protein-specific; The product of described in situ hybridization diagnosis type i diabetes comprises: with the probe of the nucleic acid array hybridizing of IQCE gene; The product of described chip diagnosis type i diabetes comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with IQCE protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of IQCE gene.
Further, described diagnostic products comprises chip and/or test kit; Wherein said chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit and protein immunization detection kit.Described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase IQCE gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
The invention provides a kind of product diagnosing type i diabetes, described product can diagnose type i diabetes by the expression level detecting IQCE gene.
Further, product recited above comprises chip or test kit.Wherein, described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe on solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for IQCE gene for detecting IQCE gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of the IQCE albumen on solid phase carrier; Described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting IQCE gene transcription level; Described protein immunization detection kit comprises the specific antibody of IQCE albumen.Wherein, described gene chip can be used for detecting the expression level of the multiple genes (such as, relevant to type i diabetes multiple genes) comprising IQCE gene.Described protein chip can be used for detecting the expression level of the multiple protein (such as relevant to type i diabetes multiple protein) comprising IQCE albumen.By being detected by multiple mark with type i diabetes simultaneously, the accuracy rate of type i diabetes diagnosis greatly can be improved.
Further, above, described reagent comprises the reagent used needed in RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip method detection IQCE gene expression dose process.Preferably, described reagent comprises primer for IQCE gene and/or probe.
Further, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase IQCE gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.The primer pair sequence of described amplification IQCE gene is as follows: forward primer sequence is 5 '-AACTCCAGACCGATATGAAG-3 '; Reverse primer sequences is 5 '-GCACCTCCTCGTAGTATG-3 '; Described house-keeping gene is GAPDH, and the primer sequence of amplification GAPDH is to as follows: forward primer sequence is 5 '-CTCTGGTAAAGTGGATATTGT-3 '; Reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
Further, described gene detecting kit also comprises M-MLV reverse transcription system, and described reverse transcription system comprises T and repeats oligonucleotide OligodT, reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.Described gene detecting kit can be used for detecting the expression level of the multiple genes (such as, relevant to type i diabetes multiple genes) comprising IQCE gene.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of IQCE gene in the present invention.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
The specific antibody of the albumen of IQCE described in the present invention comprises monoclonal antibody, polyclonal antibody.The specific antibody of described IQCE albumen comprises complete antibody molecule, any fragment of antibody or modification, such as, and chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.As long as described fragment can retain the binding ability with IQCE albumen.Be well known to a person skilled in the art for detecting the preparation of the antibody of protein level, and the present invention can use any method to prepare described antibody, as described in fragment or recombinant DNA technology can be utilized to synthesize by chemical method de novo synthesis.
In the context of the present invention, " IQCE gene " comprises the polynucleotide of any function equivalent of people IQCE gene and people IQCE gene.IQCE gene comprises and has more than 70% homology with IQCE gene (NC_000007.14) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of IQCE gene comprises any one DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1;
(2) DNA sequence dna limited with (1) is under strict conditions hybridized and identical function protein DNA sequence of encoding;
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiment of the invention scheme, the encoding sequence of described IQCE gene is the DNA sequence dna shown in SEQIDNO.1.
IQCE gene of the present invention can be natural or synthetic, or uses the vector-transfected cell can expressing the DNA fragmentation of IQCE to obtain.Described in described carrier, carrier comprises virus vector, carrier for expression of eukaryon.
Virus vector can be any suitable carrier, includes but not limited to retroviral vector, adenovirus carrier, adeno-associated virus (AAV) carrier, simplexvirus (such as hsv, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any suitable expression vector, include but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEFBos expression vector, pTet expression vector, pTRE expression vector or the carrier through transforming on the basis of known expression vector, such as pBin438, pCAMBIA1301 etc.
In the context of the present invention, IQCE gene expression product comprises the partial peptide of people IQCE albumen and people IQCE albumen.The partial peptide of described IQCE albumen contains the functional domain relevant to type i diabetes.
" IQCE albumen " comprises any function equivalent of IQCE albumen and IQCE albumen.Described function equivalent comprises IQCE albumen conservative variation's protein or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of people IQCE under high or low stringent condition.
Preferably, IQCE albumen is the protein with following amino acid sequences:
(1) protein be made up of the aminoacid sequence shown in SEQ ID NO.2;
(2) aminoacid sequence shown in SEQIDNO.2 had the protein derivative by the aminoacid sequence shown in SEQIDNO.2 of identical function with the aminoacid sequence shown in SEQIDNO.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.The amino acid whose number replacing, lack or add is generally 1-50, preferably 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), preferably, with the aminoacid sequence shown in SEQIDNO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence form polypeptide.
In specific embodiment of the invention scheme, described IQCE albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
Usually, it is known that in a protein one or more amino acid whose modification can not affect the function of protein.Those skilled in the art can approve the amino acid that changes single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, wherein the change of protein produces the protein with identity function, provides intimate amino acid whose Conservative substitution tables to be well known in the art.
By adding the fusion rotein that the example of the protein of an amino acid or multiple Modification of amino acid residues is IQCE albumen.Peptide or protein with IQCE protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of IQCE albumen.
IQCE albumen of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying still can retain the biologic activity of IQCE albumen.The amino acid number suddenlyd change in this type of modifying protein normally 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " diagnosis type i diabetes " had both comprised and had judged whether experimenter has suffered from type i diabetes, also comprised and judge whether experimenter exists the risk suffering from type i diabetes.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention IQCE genetic expression is relevant to type i diabetes, by detecting the expression of IQCE in experimenter's blood, can judge whether experimenter suffers from type i diabetes or judge whether experimenter exists the risk suffering from type i diabetes, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-IQCE gene of type i diabetes, compare traditional detection means, gene diagnosis more in time, more special, sensitiveer, the early diagnosis of type i diabetes can be realized, thus improve the life quality of type i diabetes.
Accompanying drawing explanation
Fig. 1 display utilizes QPCR to detect the expression of IQCE gene in type i diabetes patient.
Concrete embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1 screens the gene marker relevant to type i diabetes
1, sample collection
The routine normal human blood of each collection 10 and type i diabetes blood samples of patients sample, obtaining all by the agreement of Ethics Committee of above-mentioned all samples.
2, the preparation of RNA sample and mass analysis
The preparation of 2.1RNA sample
Use the RNA of Promega company to extract test kit and extract total serum IgE.Concrete steps are as follows:
1) get 1ml and be collected in whole blood in the test tube of heparin or EDTA process, put in sterile centrifugation tube;
2) collect hemocyte: 3000rpm (400g) centrifugal 5min, siphon away supernatant from sample top carefully;
3) add 1ml hemocyte lysate, careful suction is put 4-5 time, resuspended throw out;
4) the centrifugal 5min of 3000rpm;
5) repeating step 3), 4) twice (totally three times);
6) avoid cell precipitation thing, carefully siphon away nearly all supernatant, only retain 100 μ l supernatant liquors; Check that BME has been added in RNA lysate, then add 175 μ lRNA lysates in cell, inhale and put resuspended and lysing cell;
7) add 350 μ lRNA diluents, put upside down mixing 3-4 time;
8) 70 DEG C of water-bath 3min are placed in;
9) the centrifugal 10min of 13000g under room temperature;
10) limpid lysate is transferred in a sterile centrifugation tube;
11) in cleared lysate, add 200 μ l95% ethanol, inhale with liquid-transfering gun and put 3-4 time with mixing; This mixture is transferred in centrifugal column assembly, the centrifugal 1min of 13000g;
12) centrifugal column is taken down from centrifugal column assembly, discard the liquid in collection tube, centrifugal column is installed onto on collection tube, check that RNAWashSolution uses alcohol dilution, add 600 μ lRNA washingss in centrifugal column assembly, the centrifugal 1min of 13000g;
13) discard the liquid in collection tube, centrifugal column is installed onto on collection tube, the DNase mixtures incubated of the fresh preparation of 50 μ l is directly added on the film in centrifugal column;
14) incubated at room temperature 15min, adds 200 μ lDNA enzyme stop buffers (confirming to add ethanol), the centrifugal 1min of 13000g in centrifugal column;
15) 600 μ lRNA washingss (adding ethanol) are added, 13000g, centrifugal 1min;
16) empty collection tube, in centrifugal column, add 250 μ lRNA washingss (adding ethanol), high speed centrifugation 2min;
17) centrifugal column is transferred on wash-out pipe from collection tube, on film, add 100 μ l nuclease free water, put centrifugal column assembly into whizzer and make the lid facing outwards of wash-out pipe, the centrifugal 1min of 13000g, abandon centrifugal column, build the wash-out pipe filling RNA, be stored in-70 DEG C.
The mass analysis (NanoDrop1000 spectrophotometer) of 2.2RNA sample
NanoDrop1000 spectrophotometer detects RNA sample, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.
The mass analysis (AgilentTechnologies2100Bioanalyzer) of 2.3RNA sample
The RNA of said extracted is carried out agarose gel electrophoresis, AgilentTechnologies2100Bioanalyzer detects RNA sample quality, observation 28SrRNA and 18SrRNA master tape obviously, nothing is degraded, RNA Perfection Index is qualified, concentration meets the requirements of the requirement meeting RNA-seq order-checking cDNA library structure, may be used for library construction and order-checking.
3, high-throughput transcript profile order-checking
The 3.1RNA-seq section of reading is located
First the low-quality section of reading is removed and obtain the clean section of reading, then utilize TopHatv1.3.1 will clean fragment to mate with reference to genome (hg19) with UCSCH.sapiens, the index built in advance of H.sapiensUCSChg19 version is downloaded from TopHat homepage, and as reference genome, when utilizing TopHat to mate with genome, each section of reading (defaulting to 20) is allowed to have multiple coupling site, maximum 2 mispairing.TopHat sets up possible shearing site storehouse according to exon region and GT-AG shear signal, will not navigate to the genomic section of reading navigate on genome according to these shearing site storehouses.We use the system default parameter of TopHat method.
3.2 transcript abundance assessments
What match reads segment file by Cufflinksv1.0.3 process, and RNA-seq segment number is carried out the relative abundance of standardized calculation transcript by Cufflinksv1.0.3.FPKM value refers in each 1,000,000 sequenced fragments the segment number matching the long exon region of specific gene 1kb.The fiducial interval of FPKM estimated value is calculated by Bayesian inference method.The GTF comment file of the reference that Cufflinks uses downloads (Homo_sapiens.GRCh37.63.gtf) from Ensembl database.
The detection of 3.3 difference expression genes
By the EnsemblGTF file of download be transferred to Cuffdiff by the source document that TopHat mates, Cuffdiff uses original matching files again to estimate the gene expression abundance of the transcript listed in GTF file, and checkout discrepancy is expressed.In Cuffidff exports, only have q value < 0.01, test display is successfully more just considered to differential expression.
4, result
RNA-seq result shows, and the expression amount of IQCE gene in type i diabetes blood samples of patients is significantly higher than the expression amount of normal people.
The differential expression of embodiment 2QPCR sequence verification IQCE gene
1, IQCE gene is selected to carry out large sample QPCR checking according to the detected result of high-flux sequence.According to the sample collection way selection type i diabetes blood samples of patients in embodiment 1 and each 70 examples of normal human blood.
2, RNA extraction step is with embodiment 1.
3, reverse transcription: use the Reverse Transcription box of TAKARA company to operate.Concrete steps are as follows:
(1) get total serum IgE 2 μ g and carry out reverse transcription, add Oligo (dT) 2 μ l, fully mix; 70 DEG C of water-baths; Ice bath 2-3min immediately after 5min;
(2) build 25 μ l reaction systems, comprising 5 × RT Buffer 5 μ l, dNTP (2.5mM) 5 μ l, RNasin40U/ μ l, M-MLV200U/ μ l, mend nuclease free water to 25 μ l;
After (3) 42 DEG C of water-bath 60min, 95 DEG C of water-bath 5min are with deactivation M-MLV;
(4)-20 DEG C store for future use.
4, QPCR amplification
(1) design of primers
According to the encoding sequence design QPCR amplimer of IQCE gene and GAPDH gene in Genebank, synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
IQCE gene:
Forward primer is 5 '-AACTCCAGACCGATATGAAG-3 ' (SEQIDNO.3);
Reverse primer is 5 '-GCACCTCCTCGTAGTATG-3 ' (SEQIDNO.4).
GAPDH gene:
Forward primer is 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQIDNO.5);
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQIDNO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBRGreen polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction | 12.5μl |
| System template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction conditions: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 45 circulations.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler quantitative real time PCR Instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
5, statistical method
Experiment employing repeats experiment for 3 times, result data is all represent in the mode of mean+SD, use SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
6, result
As shown in Figure 1, compared with normal human blood, the up-regulated of IQCE gene in type i diabetes blood samples of patients, difference has statistical significance (P<0.05) to result, consistent with RNA-sep result.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.
Claims (10)
- The application in the product of preparation diagnosis type i diabetes of 1.IQCE gene and expression product thereof.
- 2. application according to claim 1, is characterized in that, described product comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection IQCE gene and expression product thereof to diagnose the product of type i diabetes.
- 3. application according to claim 2, is characterized in that, described RT-PCR diagnoses the product of type i diabetes at least to comprise the primer of a pair specific amplified IQCE gene; The product of described real-time quantitative PCR diagnosis type i diabetes at least comprises the primer of a pair specific amplified IQCE gene; The product of described immunodetection diagnosis type i diabetes comprises: the antibody be combined with IQCE protein-specific; The product of described in situ hybridization diagnosis type i diabetes comprises: with the probe of the nucleic acid array hybridizing of IQCE gene; The product of described chip diagnosis type i diabetes comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with IQCE protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of IQCE gene.
- 4. the application according to any one of claim 1-3, is characterized in that, described product comprises chip and/or test kit; Wherein said chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit and protein immunization detection kit.
- 5. application according to claim 4, is characterized in that, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase IQCE gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
- 6. diagnose a product for type i diabetes, it is characterized in that, described product can diagnose type i diabetes by the expression level detecting IQCE gene.
- 7. product according to claim 6, is characterized in that, described product comprises chip or test kit; Wherein, described chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit and protein immunization detection kit.
- 8. product according to claim 7, is characterized in that, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase IQCE gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
- 9. product according to claim 8, is characterized in that, the primer pair sequence of described amplification IQCE gene is as follows: forward primer sequence is 5 '-AACTCCAGACCGATATGAAG-3 '; Reverse primer sequences is 5 '-GCACCTCCTCGTAGTATG-3 '; Described house-keeping gene is GAPDH, and the primer sequence of amplification GAPDH is to as follows: forward primer sequence is 5 '-CTCTGGTAAAGTGGATATTGT-3 '; Reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
- 10. product according to claim 8 or claim 9, it is characterized in that, described gene detecting kit also comprises M-MLV reverse transcription system, and described reverse transcription system comprises T and repeats oligonucleotide OligodT, reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
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