CN105861735A - Application of RAP1B in coronary heart disease diagnosis - Google Patents
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Abstract
The invention discloses an application of RAP1B gene and an expression product thereof in preparing a diagnosis tool for coronary heart disease. In the invention, the expression of the RAP1B gene is proved to be different in normal individuals and coronary heart disease patients by a gene chip and RT-PCR experiments; and therefore, the RAP1B gene can be used as a molecular marker for diagnosing coronary heart disease. Furthermore, according to enlarged sample detection, the RAP1B has perfect correlation with coronary heart disease and can be used for preparing a preparation for assisted diagnosis and treatment of coronary heart disease.
Description
Technical field
The present invention relates to biological technical field, more particularly to RAP1B gene purposes in diagnosis of coronary heart disease.
Background technology
Coronary heart disease (coronaryarterydisease, CAD) is the abbreviation of coronary atherosclerotic heart disease,
It is coronary atherosclerosis, makes angiostenosis or obstruction, and (or) because of coronary artery functional change (convulsion
Contraction) cause myocardial ischemia, anoxia or heart disease that is downright bad and that cause.Coronary heart disease is divided into 5 by World Health Organization (WHO)
Big class: silent ischemia (latent coronary heart disease), angina pectoris, myocardial infarction, ischemic heart failure
(ischemic heart desease) and 5 kinds of Clinical types of sudden death.Clinic is usually divided into stable coronary heart disease and acute hat
Shape superior mesenteric artery syndrome.CAD is the one of the main reasons of developed country human mortality at present, in China just in urgency
The acute trend risen, has become as China's population main causes of death, and the feature that age of onset is on the low side occurs,
Serious harm people are healthy and quality of life, and have caused the extensive concern of people.
Study it has proven convenient that CAD is the multigenic disease that inherited genetic factors is caused with environmental factors interaction,
At present its etiological study is concentrated mainly on two aspects: one, continues to find risk factor from macroscopic scale；Two,
The inheritance susceptible mechanism of CAD morbidity is inquired into from microscopic scale.Research shows, including a-lymph toxin gene
(lymphotoxin-alpha, LT-a), proteasome alpha subunit 6 (proteasomesubunitalphatype6,
PSMA6) [8], beta 2-adrenergic receptor (beta2-adrenergicreceptor, β 2AR), Toll-like receptor
8 genes (Toll-likereceptor8, TLR8) and endothelial nitric oxide synthase gene
Multiple genes such as (EndothelialNOSynthase, eNOS) are all relevant to the susceptibility of CAD.Therefore, from
The inheritance susceptible mechanism of molecular genetic Study on Level incidence of coronary heart disease, excavates the new tumor susceptibility gene of CAD, to CAD
Preventing and treating have great importance.
It addition, by contrast normal individual and the comparison of the differential expression of the gene of patients with coronary heart disease or albumen, can
To find coronary heart disease and related gene or the relation of protein factor unconventionality expression, so by check related gene or
The expression of albumen, can diagnose whether patient suffers from coronary heart disease or the possibility suffering from coronary heart disease.
Developing into of high throughput sequencing technologies determines that related gene or albumen provide new with the relation of coronary heart disease
Means.High throughput sequencing technologies is utilized to compare normal individual and patients with coronary heart disease gene or the difference of protein expression profiles,
There is the gene that development is relevant in screening coronary heart disease, it is possible to find the mark of coronary heart disease.
Summary of the invention
It is an object of the invention to provide a kind of molecular marker that can be used for diagnosis of coronary heart disease.Use genetic marker
What thing carried out diagnosis of coronary heart disease has promptness, specificity and susceptiveness, so that patient just can know in ill early stage
Know risk, and for risk height, take to prevent accordingly and remedy measures.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides product the answering in the instrument preparing diagnosis of coronary heart disease of detection RAP1B gene expression
With.
Further, the product of described detection RAP1B gene expression includes detecting RAP1B gene mRNA water
Flat product and/or the product of detection RAP1B protein level.
Further, the product of described detection RAP1B gene expression includes: by RT-PCR, real-time quantitative
PCR, immune detection, in situ hybridization or the gene expression of chip detection RAP1B are with the product of diagnosis of coronary heart disease.
Further, the product of described RT-PCR diagnosis of coronary heart disease at least includes a pair specific amplified RAP1B base
The primer of cause；The product of described real-time quantitative PCR diagnosis of coronary heart disease at least includes a pair specific amplified RAP1B
The primer of gene；The product of described immune detection diagnosis of coronary heart disease includes: be combined with RAP1B protein-specific
Antibody；The product of described in situ hybridization diagnosis of coronary heart disease includes: with the nucleic acid array hybridizing of RAP1B gene
Probe；The product of described chip diagnosis of coronary heart disease includes: protein chip and gene chip；Wherein, albumen
Chip includes the antibody being combined with RAP1B protein-specific, and gene chip includes and the nucleic acid of RAP1B gene
The probe of sequence hybridization.
A pair specific amplified RAP1B base that the product of described real-time quantitative PCR diagnosis of coronary heart disease at least includes
The primer of cause is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of described detection RAP1B gene expression can be the reagent, also of detection RAP1B gene expression
Can be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be the high flux using described reagent
Order-checking platform.
The instrument of described diagnosis of coronary heart disease includes but not limited to that chip, test kit, reagent paper or high-flux sequence are flat
Platform；High-flux sequence platform is the instrument of a kind of special diagnosis of coronary heart disease, along with sending out of high throughput sequencing technologies
Exhibition, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and
The gene expression profile of normal population, the exception easily analyzing which gene is relevant to disease.Therefore, in high pass
Measure the exception purposes that fall within RAP1B gene relevant to coronary heart disease knowing RAP1B gene in sequence, with
Sample is within protection scope of the present invention.
Present invention also offers the instrument of a kind of diagnosis of coronary heart disease, described instrument includes detecting RAP1B gene expression
Reagent；Described reagent includes primer and/or probe, the detection RAP1B egg detecting RAP1B gene mRNA
White antibody.
Described instrument includes but not limited to chip, test kit, reagent paper or high-flux sequence platform.
Wherein, described chip includes gene chip, protein chip；Described gene chip include solid phase carrier with
And it being fixed on the oligonucleotide probe of solid phase carrier, described oligonucleotide probe includes for detecting RAP1B gene
The oligonucleotide probe for RAP1B gene of transcriptional level；Described protein chip include solid phase carrier and
It is fixed on the specific antibody of the RAP1B albumen of solid phase carrier；Described gene chip can be used for detection and includes
RAP1B gene is at the expression of interior multiple genes (such as, relevant to coronary heart disease multiple genes).Institute
State multiple protein that protein chip can be used for detecting including RAP1B albumen (such as relevant to coronary heart disease
Multiple protein) expression.By multiple marks with coronary heart disease are detected simultaneously, can significantly carry
The accuracy rate of high diagnosis of coronary heart disease.
Wherein, described test kit includes gene detecting kit and protein immunization detection kit；Described gene is examined
Test agent box includes the reagent for detecting RAP1B gene transcription level；Described protein immunization detection kit bag
Include the specific antibody of RAP1B albumen.Further, described reagent include use RT-PCR, real-time quantitative PCR,
Reagent required during immune detection, in situ hybridization or chip method detection RAP1B gene expression dose.Excellent
Selection of land, described reagent includes the primer for RAP1B gene and/or probe.Nucleoside according to RAP1B gene
Acid sequence information easily designs the primer and probe that may be used for detecting RAP1B gene expression dose.
Described reagent paper includes the reagent detecting RAP1B gene expression.
Described high-flux sequence platform includes the reagent detecting RAP1B gene expression.
Can be that DNA, RNA, DNA-RNA are embedding with the probe of the nucleic acid array hybridizing of RAP1B gene
Zoarium, PNA or other derivant.The length of described probe does not limit, if complete specific hybrid,
Specific binding with purpose nucleotide sequence, 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, the most whole gene.Owing to different probe length is special to hybridization efficiency, signal
The opposite sex has different impacts, the length of described probe to be typically at least 14 base pairs, the longest is usually no more than
30 base pairs, optimal with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe
Self-complementary sequences is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
Further, the specific antibody of described RAP1B albumen includes monoclonal antibody, polyclonal antibody.Described
The specific antibody of RAP1B albumen include complete antibody molecule, any fragment of antibody or modify (such as,
Chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as described fragment can retain and RAP1B albumen
Binding ability.Well known to a person skilled in the art when the preparation of the antibody of protein level, and this
Invention can use any method to prepare described antibody.
In specific embodiments of the present invention, the primer of described detection RAP1B gene mRNA includes SEQ
Primer pair shown in ID NO.3 and SEQ ID NO.4.
In the context of the present invention, " RAP1B gene " includes RAP1B gene and RAP1B gene
The polynucleotide of any function equivalent.RAP1B gene includes and the most international common core sequence databank
In GeneBank, RAP1B gene (NC_000023.11) DNA sequence has more than 70% homology, and compiles
Code-phase congenerous protein DNA sequence；
Preferably, the coded sequence of RAP1B gene includes any DNA molecular following:
(1) DNA sequence shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) the DNA sequence hybridization that limits and coding identical function protein
DNA sequence；
(3) DNA sequence limited with (1) or (2) has 70%, preferably, more than 90% homology
Property, and coding identical function protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of described RAP1B gene is SEQ ID NO.1
Shown DNA sequence.
In the context of the present invention, RAP1B gene expression product includes RAP1B albumen and RAP1B
The partial peptide of albumen.The partial peptide of described RAP1B albumen contains the functional domain relevant to coronary heart disease.
" RAP1B albumen " includes any function equivalent of RAP1B albumen and RAP1B albumen.Institute
State function equivalent and include RAP1B albumen conservative variation's protein or its active fragment, or its activity is spread out
Biology, allelic variant, natural mutation, induced mutants, energy and RAP1B under high or low stringent condition
The protein coded by DNA of DNA hybridization.
Preferably, RAP1B albumen is the protein with following amino acid sequences:
(1) protein being made up of the aminoacid sequence shown in SEQ ID NO.2 in sequence table；
(2) aminoacid sequence shown in SEQ ID NO.2 is passed through the replacement of one or several amino acid residue
And/or disappearance and/or add and with the aminoacid sequence shown in SEQ ID NO.2 have identical function by
The protein that aminoacid sequence shown in SEQ ID NO.2 is derivative.Replace, lack or add is amino acid whose
Number is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10.
(3) with the aminoacid sequence shown in SEQ ID NO.2, there is at least 80% homology and (be also called sequence
Homogeneity), it is highly preferred that same with the aminoacid sequence at least about 90% to 95% shown in SEQ ID NO.2
Source property, is often the polypeptide of the aminoacid sequence composition of 96%, 97%, 98%, 99% homology.
In specific embodiments of the present invention, described RAP1B albumen has shown in SEQ ID NO.2
The protein of aminoacid sequence.
It is known that, conventionally, in a protein, one or more amino acid whose modifications do not interfere with protein
Function.Those skilled in the art can approve change single amino acids or the aminoacid of little percentage ratio or to aminoacid sequence
Adding individually, lacking, insert, replace of row is conservative modification, and wherein the change generation of protein has similar
The protein of function.It is well known in the art for providing intimate amino acid whose Conservative substitution tables.
It is RAP1B albumen by adding the example of the protein of an aminoacid or multiple Modification of amino acid residues
Fusion protein.Peptide or protein with RAP1B protein fusion is not limited, if the melting of gained
Hop protein retains the biologic activity of RAP1B albumen.
The RAP1B albumen of the present invention also includes repairing the non-conservative of aminoacid sequence shown in SEQ ID NO.2
Decorations, as long as the protein through modifying remains able to retain the biologic activity of RAP1B albumen.At this
In class modifying protein, the amino acid number of sudden change is typically 10 or less, such as 6 or less,
Such as 3 or less.
In the context of the present invention, " diagnosis of coronary heart disease " both includes judging that experimenter has suffered from coronary disease
Sick, also include judging whether experimenter exists the risk suffering from coronary heart disease.
In the context of the present invention, " treatment coronary heart disease " divides from the state change of disease, can include disease
The alleviation of disease, the healing completely of disease.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that RAP1B gene expression is relevant to coronary heart disease, in being organized by detection experimenter
The expression of RAP1B, it can be determined that whether experimenter suffers from coronary heart disease or judge whether experimenter exists trouble
There is the risk of coronary heart disease, thus instruct clinicist to provide prevention scheme or therapeutic scheme to experimenter.
Present invention finds a kind of new molecular marked compound-RAP1B gene, compare traditional detection means, base
Because of diagnosis more in time, more special, sensitiveer, it is possible to realize the early diagnosis of coronary heart disease, thus reduce coronary heart disease
Mortality rate.
Accompanying drawing explanation
Fig. 1 shows the expression utilizing genechip detection RAP1B gene in coronary heart disease；
Fig. 2 show utilize Western blot detect RAP1B albumen expression in coronary heart disease.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are only used for
The bright present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment is logical
Often according to normal condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring HarborLaboratory Press, 1989) condition described in, or according to proposed by manufacturer
Condition.
The collection of embodiment 1 case
Select normal population and suffer from the crowd of coronary heart disease and carry out gene comparison, choosing 6 example patients with coronary heart disease cases
Sample and 5 example Normal group samples, respectively extraction 5-10ml fasting blood.
Embodiment 2 high-flux sequence and analysis
Blood is carried out RNA extraction, RNA extract after agarose gel electrophoresis, can be preliminary from electrophoresis result
Judge that whether up-to-standard the RNA sample extracted is, if may be used for further transcriptome analysis.And then
By the extraction situation of NanoDrop1000 spectrophotometer detection RNA sample, the sample of RNA-seq order-checking
Product require: OD260/OD280 is 1.8-2.2.
Order-checking platform is the HiSeq 2500 high-flux sequence platform of Illumina company, carries out high flux transcript profile
The degree of depth checks order, and after order-checking, we use Fast-QC software that the quality of sequencing data is carried out total evaluation, bag
Include the mass value distribution of base, the position distribution of mass value, G/C content, PCR duplication content, kmer
Frequency etc..When differential genes expression analysis, according to the FPKM value obtained, use internationally recognized
Algorithm EBSeq carries out differential screening.Wherein, during screening, LOG2FC>1 or<-1, FDR<0.05.In order to
Being better understood from the function of difference expression gene, we have carried out Gene Onlogy and letter to difference expression gene
Number path analysis, and difference expression gene is carried out functional annotation and protein interaction analysis of network, in view of
The result that data above is analyzed, in conjunction with document, we have screened difference expression gene RAP1B.
Differential expression in the normal person of embodiment 3RAP1B gene and patients with coronary heart disease
1, sample acquisition: 40 examples (include 20 normal persons and 20 example patients with coronary heart disease), take fasting blood respectively
Liquid 5-10ml.The clinical data of individual of sample includes: height, body weight, blood pressure, blood fat, electrocardiogram etc..
2, the acquisition of RNA in sample blood
The RNA utilizing QINGEN company extracts test kit and carries out the extraction of cell total rna, according to explanation
Book instruction is carried out.
3, reverse transcription
The Reverse Transcriptase kit utilizing TAKARA company carries out the reverse transcription of RNA.
4、QPCR
(1) design of primers
Draw according to the coded sequence design QPCR amplification of RAP1B gene and GAPDH gene in Genbank
Thing, is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
RAP1B gene:
Forward primer is 5 '-CACAGCACAGTCCACATT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-CACACTTATTACCAACAAGAATCA-3 ' (SEQ ID NO.4),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green 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 system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 40s) * 42 circulations.With
SYBR Green, as fluorescent marker, reacts at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument,
Determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
5, statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD
Represent, use SPSS13.0 statistical software to carry out statistical analysis, RAP1B gene expression group and matched group
Between difference use t inspection, it is believed that when P < has statistical significance when 0.05.
6, result
As it is shown in figure 1, by RAP1B gene expression feelings in contrast normal human blood and patients with coronary heart disease blood
Condition finds RAP1B gene expression significantly raised (P < 0.05).
The differential expression of embodiment 4RAP1B albumen
1, object of study is with embodiment 1.
2, blood total protein is extracted
The operation of protein extraction is carried out according to the description of EpiQuik blood/total protein of cell extraction test kit.
3, Western blot detection
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch,
Two anti-hatch, develop the color.
4, statistical procedures
Image J software is used to be analyzed the gray value of protein band, with β-actin as internal reference, will
The gray value of RAP1B protein band is normalized.Result data is all with mean+SD
Mode represents, uses SPSS13.0 statistical software to carry out statistical analysis, and difference between the two uses
T checks, it is believed that when P < has statistical significance when 0.05.
5, result
Result as in figure 2 it is shown, compared with normal individual blood, the table of RAP1B albumen in coronary heart disease blood
The level of reaching dramatically increases, and difference has statistical significance (P < 0.05).
5 one kinds of coronary heart disease gene detecting kits of embodiment
According to the dependency of RAP1B gene expression Yu coronary heart disease, can be by detecting the table of RAP1B gene
The situation of reaching comes whether diagnosis of coronary heart disease occurs, and is thus provided that one is examined based on detection RAP1B gene expression
The test kit of disconnected coronary heart disease, the component in this diagnostic kit is as follows: comprise SYBR Green PCR reactant
System, amplification RAP1B gene and the primer pair of GAPDH gene.Amplification RAP1B gene primer pair: just
Being shown in SEQ ID NO:3 to primer sequence, reverse primer sequences is shown in SEQ ID NO:4；
The primer pair of amplification GAPDH: forward primer sequence is shown in SEQ ID NO:5, reverse primer sequences
Shown in SEQ ID NO:6；SYBR Green PCR reaction system, comprise PCR buffer, dNTPs,
SYBR Green fluorescent dye.PCR buffer components is: 20mM KCl, 2.5mMMgCl2、200mM
(NH4)2SO4。
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It is right to it should be pointed out that,
For those of ordinary skill in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention
Carrying out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (5)
1. the product of detection RAP1B gene expression application in the instrument preparing diagnosis of coronary heart disease.
Application the most according to claim 1, it is characterised in that described product includes: by RT-PCR,
Real-time quantitative PCR, immune detection, in situ hybridization, chip or high-flux sequence detection of platform RAP1B gene
Express the product with diagnosis of coronary heart disease；The product of described RT-PCR diagnosis of coronary heart disease at least include a pair special
The primer of amplification RAP1B gene；The product of described real-time quantitative PCR diagnosis of coronary heart disease at least includes a pair
The primer of specific amplified RAP1B gene；The product of described immune detection diagnosis of coronary heart disease includes: with RAP1B
The antibody that protein-specific combines；The product of described in situ hybridization diagnosis of coronary heart disease includes: with RAP1B base
The probe of the nucleic acid array hybridizing of cause；The product of described chip diagnosis of coronary heart disease includes: protein chip and gene
Chip；Wherein, protein chip includes the antibody being combined with RAP1B protein-specific, gene chip include with
The probe of the nucleic acid array hybridizing of RAP1B gene.
Application the most according to claim 2, it is characterised in that described real-time quantitative PCR diagnoses coronary disease
The primer such as SEQ ID NO.3 and SEQ ID of a pair specific amplified RAP1B gene that sick product at least includes
Shown in NO.4.
4. the instrument of a diagnosis of coronary heart disease, it is characterised in that described instrument includes detecting RAP1B gene table
The reagent reached；Described reagent includes primer and/or probe, the detection RAP1B detecting RAP1B gene mRNA
The antibody of albumen.
Instrument the most according to claim 4, it is characterised in that described detection RAP1B gene mRNA
Primer include the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4.
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|---|---|---|---|---|
| CN110241208A (en) * | 2019-08-02 | 2019-09-17 | 辽宁中医药大学 | Application of the TREM2 as the molecular marker of early diagnosis coronary heart disease |
| CN114650847A (en) * | 2019-09-25 | 2022-06-21 | 犹他大学研究基金会 | Methods and compositions for expressing constitutively active RAP1A from the VMD2 promoter |
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