CN106636324A - Application of nucleic acid and protein thereof to diagnosis of rheumatoid arthritis risk of subject and kit - Google Patents

Abstract

The invention relates to application of a nucleic acid to diagnosing whether a subject has the risk of suffering from rheumatoid arthritis and/or whether the subject is suffering from rheumatoid arthritis, and a kit used in diagnosis. The sequence of the nucleic acid comprises at least one selected from a group consisting of any sequence segments of ifi202b, ifi203, ifi204, ifi205 and Mndal genes, or at least one selected from a group consisting of sequences originated from human beings, homologous to and/or consistent with the above sequences and having same functions as the above sequences; and preferably, the sequence of the nucleic acid comprises any sequence segment selected from a sequence as shown in SEQ ID No. 28.

Description

A kind of application of nucleic acid and its albumen in diagnosis main body rheumatoid arthritis risk And kit

Technical field

The application is related to the application and its kit of nucleic acid and its albumen in diagnosis main body rheumatoid arthritis risk, Main body is more particularly to diagnosed using ifi202b, ifi203, ifi204 and ifi205 gene or its protein with the presence or absence of suffering from Rheumatoid arthritis risk and/or with rheumatoid arthritis.

Background technology

Rheumatoid arthritis (rheumatoid arthritis, be abbreviated as RA) is a kind of common frdquently encountered disease.It is right at present The drug therapy of the rheumatoid arthritis for having been formed is also without important breakthrough.Morbidity early stage or front period of disease treatment effect It is really best.Especially some immunological regulation class medicines are best in morbidity early stage curative effect.Therefore to the prediction of Susceptible population in rheumatoid It is particularly important in the arthritic preventing and treating of property.Although at present some are in progress in terms of gene remembers genic prediction, class wind Diseases caused by dampness be by it is extremely complex it is multifactor cause, predictive genes aspect is badly in need of improving to improve prediction effect.

According to the knowledge in terms of current hereditary pathology, before the pathological change of phenotype occurs in terms of body physiological biochemistry Change occurs first.The change of some gene expressions is the most direct early stage change of some diseases.Therefore find and cause a disease closely Related gene is the key of Accurate Prediction disease.

In recent years, irreversible destruction of joint the starting earlier than conventional prediction in rheumatoid arthritis has been illustrated, and And therefore it has been recommended that start actively using the biologic product for showing high destruction of joint inhibition effect from early stage, this increases class wind The importance of the early diagnosis of wet arthritis.

Traditional diagnosis of rheumatoid arthritis is mainly carried out by symptom.But in recent years, with contained in patients serum Material receive publicity for the diagnostic method of mark.As such material, it is known to rheumatoid factor (rheumatoid Factor) (for the autoantibody of denaturation IgG), the citrullinated peptide antibody of antioxidant cyclic (antiCCP antibody) etc..

However, in report so far, it is 50-70% that the sensitivity of rheumatoid factor is 75-80%, specificity, The sensitivity of antiCCP antibody is 50-75%, specificity is 85-95%.Therefore, the sensitivity of diagnosis of rheumatoid arthritis and The methods such as specificity still have much room for improvement.

The content of the invention

In order to overcome disadvantages mentioned above of the prior art, inventor is through further investigation, it is proposed that one kind by determine with The transcriptional level for suffering from the closely related gene of rheumatoid arthritis diagnose subject whether there is with rheumatoid close Save the risk of inflammation and/or whether suffer from rheumatoid arthritis.Wherein with rheumatoid arthritis is suffered from, closely related gene is Ifi202b, ifi203, ifi204 and ifi205, this is also to propose to use ifi202b, ifi203, ifi204 and ifi205 first As mark to subject is with the presence or absence of the risk with rheumatoid arthritis and/or whether suffers from rheumatoid joint Inflammation is diagnosed.The present invention has the advantages that following prominent：1) can be to from living tissue detection sample, blood, blood plasma, serum Quantitative analysis is carried out with the RNA of the related gene (such as ifi202b, ifi203, ifi204 and ifi205) in the cell of lymph liquid, For example can be carried out by chip method and/or fluorescence quantitative PCR method quantitative；2) can be with replication；3) water is transcribed between individuality Significant difference on flat, easily measurement；4) RNA quantitative determinations of the invention are more more sensitive than protein detection method.

Therefore, an object of the present invention there are provided a kind of nucleic acid and prepare for diagnosing main body with the presence or absence of suffering from Application in the risk of rheumatoid arthritis and/or the whether kit with rheumatoid arthritis, the sequence of the nucleic acid Row include any in any one section of sequence, the sequence as shown in SEQ ID No.2 in the sequence as shown in SEQ ID No.1 In any one section of sequence and the sequence as shown in SEQ ID No.23 in one section of sequence, the sequence as shown in SEQ ID No.3 Any one section of sequence at least one；Or the sequence of the nucleic acid is included from people, and with as shown in SEQ ID No.1 Sequence in any one section of sequence, any one section of sequence in the sequence as shown in SEQ ID No.2, such as SEQ ID No.3 Any one section of sequence and such as SEQ ID in any one section of sequence in shown sequence, the sequence as shown in SEQ ID No.4 Any one section of sequence homology in sequence shown in No.23 and/or with sequence identity, and in function identical sequence extremely Few one kind.Any one section of sequence, such as SEQ in the wherein above-mentioned sequence from people and the sequence as shown in SEQ ID No.1 Any one section of sequence in any one section of sequence in sequence shown in ID No.2, the sequence as shown in SEQ ID No.3 and such as Any one section of sequence in sequence shown in SEQ ID No.23 have more than 50%, more than 55%, more than 60%, 65% with Above, more than 70%, more than 75% or more than 80% uniformity.Specifically, through sequence alignment analysis, people source cDNA MNDA (myeloid cell nuclear differentiation antigen) with the present invention in mouse source ifi204, Ifi205 and Mndal is ortholog, and its sequence is as shown in SEQ ID No.28.It is therefore preferable that the sequence of the nucleic acid Including any one section of sequence in the sequence as shown in SEQ ID No.28.

The risk of rheumatoid arthritis and/or the diagnosis of rheumatoid arthritis are carried out using the nucleic acid of the present invention, can To carry out the inspection of the transcriptional level of related gene by the mode of biochip (being also called microarray, or micro-array chip) Survey, it is also possible to the detection of the transcriptional level of related gene is carried out by reverse transcription quantitative fluorescent PCR.That is, using this Two methods can reach the purpose of the transcriptional level height of detection said gene.When the transcriptional level of the related gene of detection Higher than normal level (as the level of control), then illustrate that subject is risky and/or just in afflicted with rheumatoid arthritis； When the transcriptional level of the related gene of detection is less than normal level (as the level of control), then it is considered that subject is basic Go up without risk and/or there is no afflicted with rheumatoid arthritis.

In the present invention, the sequence of the nucleic acid can be single-stranded, or double-strand, those skilled in the art can be with Determine that the sequence of the nucleic acid is specially single-stranded or double-strand according to actual conditions.The single chain lengths of the sequence of the nucleic acid can be with For 40-300nt.When by way of biochip to be detected, the single chain lengths of the sequence of the nucleic acid can be 40- 200nt, the single chain lengths of preferred nucleic acid sequence are 50-100nt.If being represented from double chain form, the sequence of the nucleic acid The length of row can be 40-200bp, and the length of preferred nucleic acid sequence is 50-100bp.

In one embodiment, sequence of the sequence of the nucleic acid as shown in SEQ ID Nos.5 and/or 6.

In one embodiment, the sequence of the nucleic acid is cDNA sequence and/or RNA sequence.

When by way of reverse transcription quantitative fluorescent PCR to be detected, the single chain lengths of the sequence of the nucleic acid can Think 80-300nt, the single chain lengths of preferred nucleic acid sequence are 80-200nt, and more preferably the single chain lengths of nucleotide sequence are 80- 110nt.If being represented from double chain form, the length of the sequence of the nucleic acid can be 80-300bp, preferred nucleic acid sequence The length of row is 80-200bp, and more preferably the length of nucleotide sequence is 80-110bp.

In one embodiment, in sequence of the sequence of the nucleic acid as shown in SEQ ID Nos.7-10 and 24 extremely Few one kind.

In one embodiment, the sequence such as SEQ ID of amplification upstream and downstream primer of sequence as shown in SEQ ID No.7 Shown in No.11 and SEQ ID No.12, the sequence of the probe of sequence is as shown in SEQ ID No.13 as shown in SEQ ID No.7； Amplification as shown in SEQ ID No.8 the upstream and downstream primer of sequence sequence as shown in SEQ ID No.14 and SEQ ID No.15, The sequence of the probe of sequence is as shown in SEQ ID No.16 as shown in SEQ ID No.8；Amplification sequence as shown in SEQ ID No.9 Upstream and downstream primer sequence as shown in SEQ ID No.17 and SEQ ID No.18, the spy of sequence as shown in SEQ ID No.19 The sequence of pin is as shown in SEQ ID No.19；The sequence such as SEQ of amplification upstream and downstream primer of sequence as shown in SEQ ID No.10 Shown in ID No.20 and SEQ ID No.21, the sequence of the probe of the sequence such as SEQ ID No.22 institutes as shown in SEQ ID No.10 Show；The sequence such as SEQ ID No.25 and SEQ ID No.26 institutes of amplification upstream and downstream primer of sequence as shown in SEQ ID No.24 Show, the sequence of the probe of sequence is as shown in SEQ ID No.27 as shown in SEQ ID No.24.

In one embodiment, primer therein is double-stranded DNA and/or single stranded DNA；Probe therein is single stranded DNA, Such as cDNA.

The second object of the present invention there are provided a kind of for diagnosing main body with the presence or absence of with rheumatoid arthritis Risk and/or the whether kit with rheumatoid arthritis, include as above any one nucleic acid in the kit Sequence.Wherein the nucleotide sequence can be according to being determined using the detection principle of biochip, such as specific nucleotide sequence can Think sequence as shown in SEQ ID Nos.5 and/or 6 etc.；Can also be according to the detection principle of reverse transcription quantitative fluorescent PCR To determine, such as specific nucleotide sequence can be at least one in the sequence as shown in SEQ ID Nos.7-10 and 24 etc. Deng.

It is above-mentioned for selected by biochip test except including in the kit when using biochip to detect Nucleotide sequence outside, also including the chip that can fix the nucleotide sequence, preferably the material of the chip is selected from nylon membrane, glass One kind in glass piece, plastics, silica gel chip and miniature magnetic bead；And also optionally include from from described in the kit The reagent and/or chip hybridization reagent used by RNA is extracted in the detected sample of main body；It is preferred that the detected sample is work At least one in tissue detection sample, blood, blood plasma, serum and lymph liquid；More preferably described living tissue detection sample is next From the living tissue detection sample of spleen

The specific reagent for extracting RNA can include：The Trizol of Life Technoligies companies of the U.S. The Rneasy kits of Reagents and Qiagen companies.

Chip hybridization reagent can include：Illumina companies of the U.S.TotalPrep^TM RNA Amplification kits and HumanHT-12v4Expression chips.

It is as above glimmering for reverse transcription except including in the kit when the detection using reverse transcription quantitative fluorescent PCR Outside the nucleotide sequence of Fluorescent Quantitative PCR, also including the reagent carried out used by quantitative fluorescent PCR, and also appoint in the kit Selection of land includes that the reagent and/or reverse transcription quantitative fluorescent PCR that extract from the detected sample from the main body used by RNA are used Reagent；It is preferred that the detected sample is at least one in living tissue detection sample, blood, blood plasma, serum and lymph liquid；More It is preferred that the living tissue detection sample is the living tissue detection test sample product from spleen, but clinically it is difficult to obtain, so excellent Choosing uses blood examination.

Specific reverse transcription fluorescence quantitative kit includes：The TaqMan of Life Technoligies companies of the U.S.^TM Gene expression probes, TaqMan^TMReal-timepcr master mixes andReverse Transcription Reagents kits.

In the present invention, term " subject " or " main body " refer to mammal, such as mouse (including rat and mouse) And people.But in general, " main body " in the present invention refers to people.Known in those skilled in the art, for studying the class of people The model of rheumatic arthritis is generally the mouse model with idiopathic arthritis used in the present invention.Therefore, the present invention In the result of study of idiopathic arthritis of mouse can be used in the rheumatoid arthritis of people, i.e., research knot of the invention Fruit can be used for diagnosing people with the presence or absence of the risk with rheumatoid arthritis and/or whether with rheumatoid arthritis, Simply both differences in sequence.

Term in the present invention is typically the general term of this area, and those skilled in the art is according to the normal of this area Rule technology can clearly determine its implication.In the case where being otherwise noted, then the explanation in the present invention is defined.

In the present invention, the title of mouse subbreed can be using writing a Chinese character in simplified form for partial information be saved, while also not to size The mother that writes makes a distinction.For example BALB is equal to Balb.

Description of the drawings

Fig. 1：Compared with the transcriptional level of DBA/1 wild-type mices, the base of horizontal down-regulation is transcribed in BALB.D1-1 mouse Cause.Ordinate should be the multiple of gene upregulation or downward in figure, and what abscissa was indicated is the title of gene.

Fig. 2：Compared with the transcriptional level of DBA/1 wild-type mices, the base that transcriptional level is raised in BALB.D1-1 mouse Cause.Ordinate should be the multiple of gene upregulation or downward in figure, and what abscissa was indicated is the title of gene.

Fig. 3：GeneNetwork show the gene transcription level for identifying not with II1rn and other il-1 families Gene transcription level direct correlation.Because the portion gene title in the network is covered by grid line, therefore, the present invention is right The Gene Name being wherein blanked is identified using arrow.

Fig. 4：Compared with the expression of DBA-/- mouse, the gene that expression is lowered in BALB.D1-1 mouse.Figure Middle ordinate should be the multiple of gene upregulation or downward, and what abscissa showed is the title of gene.

Fig. 5：Compared with the expression of DBA-/- mouse, the gene that expression is raised in BALB.D1-1 mouse.Figure Middle ordinate should be the multiple of gene upregulation or downward, and what abscissa showed is the title of gene.

Fig. 6：The data analysis of GeneNetwork shows that the gene expression dose for identifying and II1rn family genes are expressed Level is without being closely connected.

Fig. 7：Compared with the expression of BALB/c mouse, the gene that expression is raised in BALB.D1-1 mouse.Figure Middle ordinate should be the multiple of gene upregulation or downward, and what abscissa showed is the title of gene.

Fig. 8：Compared with the expression of BALB/c mouse, the gene that expression is lowered in BALB.D1-1 mouse.Figure Middle ordinate should be the multiple of gene upregulation or downward, and what abscissa showed is the title of gene.

Fig. 9：GeneNetwork shows, the gene expression dose for identifying not with Il1rap or other interleukin 1s Family gene expression is directly contacted.

Figure 10：Down-regulated gene in BALB.D- type congenic line mices.

It is probe/Gene Name on trunnion axis.Ordinate left side of the digital is differential transcription numerical value.

In Fig. 10, BALB.D-1 types congeric strains transcriptional level decline, therefore it become than sensitive strain BALB/c-/- more Opposing arthritis.By with BALB/c-/- compare, ifi204 genetic transcriptions are reduced more than 13 times, at the same time ifi203 genes Reduce by more than 33 times.Therefore, when the transcriptional level of ifi genes is reduced, arthritic neurological susceptibility is also reduced (Figure 10).

Figure 11：Idiotype network based on spleen cdna express spectra.

Figure 12：Idiotype network based on T (auxiliary) cellular gene expression.

Figure 13：Idiotype network based on T (regulation and control) cellular gene expression.

Figure 14：The chromosome 1 that ifi clusters are located in QTL genomic regions is shown from the Genome Atlas of Ensembl databases On, QTL is non-mhc gene seat.Mhc gene seat is located on chromosome 17.It is different chromosomes.

Figure 15：Not comparing the genetic transcription of determination BALB.D-1 type congeric strains ifi by polyphyly reduces.

The transcriptional level of 12 genes of congeric strains group is with other four groups of comparisons.

Ordinate left side of the digital is multiple change.Abscissa is the title of probe/gene.Four homology Yong not different face Color marker.Congeric strains BALB.D-1 and BALB-/- and BALB/c systems compare, gene transcription level is lowered, but same DBA-/- and DBA/1 compares, without significant difference.

As shown by data, the expression of ifi202b, ifi203, ifi204 and ifi205 gene is (dry thin in spleen and blood Born of the same parents) between have very strong positive correlation.

Figure 16：Balb.D1-1-/phenotypic the order of severity of-homology.Balb.D1-1-/- congeric strains order of severity is reduced.

Figure 17：Balb.D1-1-/phenotypic the incidence of disease of-homology.Balb.D1-1-/- congeric strains incidence of disease is reduced.

Wherein, as shown in figure 22, with DBA/1-/- compare, the transcriptional level of congeric strains DBA.B-1IFI gene strengthens. Ensuing two figures show that ifi gene transcription level increases cause to reduce arthritis opposing.

Figure 18：With DBA/1-/- compare, the phenotypic incidence of disease of DBA.B-1-/- homology increases.

Figure 19：DBA.B-1 congenic line mices equally increase relative to the horizontal order of severity of arthritic of DBA/1 systems mouse Plus.

Summarize data above, ifi202b, ifi203, ifi204 and ifi205 gene transcription level and arthritis are into positive Close.Ifi202b, ifi203, ifi204 and ifi205 gene transcription level can be detected by blood sample.Therefore, this Bright patent is to detect ifi gene transcription levels to check to arthritic neurological susceptibility by using blood sample.

Figure 20：Illustrate that transcriptional level of the ifi204 genes in blood and splenocyte is in notable positive correlation.Ifi204 genes It is 2.39e-0.3 that 0.724, P values are transcribed between spleen and haemocyte, shows obvious positive correlation.

As Figure 10 and Figure 15 shows, Balb.D1-1-/- congeric strains IFI gene transcription level is reduced.We are with ensuing Two charts are bright, and because IFI genetic transcriptions are reduced, Balb.D1-1-/- congeric strains increase arthritis in other words to arthritis opposing Occurring degree is also reduced.

Figure 21：In congeric strains ifi202b, ifi203, ifi204 and ifi205 gene transcription level with other four groups in phase Answer the comparison of the transcriptional level of gene.

Ordinate is multiple change.Relatively system another name claims to be marked on following axle.Three probes are with different colors Mark.The same DBA- of congeric strains (DBA.B-1)/- compare with DBA/1 systems, the transcriptional level of gene is raised, but same BALB-/- and BALB/c systems compare indifference.The genome area of ifi gene of the congeric strains (DBA.B-1) comprising rheumatoid arthritis, because This is susceptible to suffer from arthritis.DBA-/- it is that a kind of arthritic system of opposing is other.Therefore when the transcriptional level of ifi genes increases, by with DBA-/- compare, congeric strains (DBA.B-1) become to be susceptible to suffer from rheumatoid arthritis.With opposing be other DBA-/- compare, ifi204 bases Increase to 46 times (Figure 22) because transcriptional level increased 327 times and ifi205 gene transcription levels.The notable increasing of rna level Plus, the technology of the present invention is made easily using the transcriptional differences with easily identification.

Figure 22：Illustrate that transcriptional level of the ifi203 genes in blood and splenocyte is in notable positive correlation.Ifi203 genes It is 5.99e-0.4 that 0.776, P values are transcribed between spleen and haemocyte, shows obvious positive correlation.

Specific embodiment

With reference to embodiment in detail the application is described in detail, but the application is not limited to these embodiments.

If no special instructions, the raw material and catalyst in embodiments herein is bought by commercial sources.

Embodiment 1

The downward of ifi gene expression doses is set to cause to spontaneous joint by lowering the expression of congenic line mice Tcrb genes Inflammation opposing strengthens.

Animal used as test

BALB/c systems mouse produces spontaneous in default of IL-1 (interleukin 1) receptor antagonist protein (Il-1ra) Arthritis disease (SAD), and DBA/1IL1rn^-/-It is that mouse has this defect but not fall ill.Previously, we located one certainly The property sent out No. 1 chromosome QTL (quantitative trait locus) of arthritis, then develops a kind of BALB.D1-1^-/-Congenic line mice.This Congenic line mice is planted with BALB/c^-/-Include from DBA/1 for background opposing^-/-The QTL genetic fragment relevant with Antiarthritic.

Material and method

From BALB.D1-1 congeric strains and four, other are other (BALB/c wild types, DBA/1, DBA/1IL1rn^-/-Deficiency With BALB/c IL1rn^-/-The spleen of mouse obtains full-length genome express spectra.Then the similarities and differences of congeric strains and four parents are compared. Potential Disease-causing gene is filtered out on the basis of differential expression level and gene function.

As a result

In congeric strains and three parent systems BALB/c, DBA/1, and DBA/1^-/-With the presence of a considerable amount of genes in mouse Differential expression.But only little difference expression gene by congeric strains and BALB/c-/- be that comparison between mouse is true It is fixed.These difference expression genes are essentially from φt cell receptor β chains (Tcrb) and interferon activator protein (ifi) gene.These bases Because having differential expression similarly in congeric strains and BALB/c systems mouse.However, in congeric strains expression with DBA/1 And DBA/1^-/-Expression in mouse is similar.The expression of Tcrb-j genes is in positive with two genes of ifi gene families Close.

Conclusion

Ifi gene expression doses are made to lower so as to strengthen to spontaneity by lowering the expression of congenic line mice Tcrb genes Arthritic opposing.

Embodiment 2

Materials and methods

Congeric strains BALB.D1-1, BALB/c and DBA/1 wild-type mices, BALB/c and DBA/1Il1rn gene knockout types 4 months female mices, using Illumina platforms generate genetic transcription data.BALB.D1-1 systems mouse is in BALB/c On genome background, comprising on the first chromosome of DBA/1 between two DNA markers of D1Mit55 and D1Mit209 Region of DNA domain.

Genotyping

Insertion congeric strains BALB.D1-1 murine genes type was confirmed before RNA extractings and microarray analysis：Bored a hole by ear Obtain the tissue for extracting genomic DNA.Generally process is DNA to be extracted from tissue and is entered by polymerase chain reaction (PCR) Row microsatellite marker is expanded.PCR primer Mega-Ge dual high flux vertical electrophoresis system (C.B.S.Scientific, Del Mar, CA) carry out acrylamide gel electrophoresis analysis.

RNA is extracted

The extracting of spleen rna is carried out using Trizol reagents (Invitrogen, CA), total serum IgE passes through RNeasy MinElute Cleanup Kit (Qiagen, CA) kits are purified, and the quality and integrality of RNA is biological using Agilent Analyzer is analyzed^[1]。

Microarray procedures

Initial amount adopts 200ng high-quality total serum IgEs, and RIN (RNA integrality scores) numerical value is more than 7, uses TotalPrep^TMRNA Amplification Kit (Ambion) kits obtain cDNA and cRNA.According to the explanation of manufacturer Multiple step procedures in book, five independent samples each take the cRNA samples of 1.5ug with Illumina companies mouse-6v1.1 Expression magnetic bead chip hybrid is overnight.Then cleaning-drying scans chip with BeadArray Reader (Illumina, CA), uses BeadStudio 2.3.41 (Illumina, CA) generate initial data^[2]。

The analysis of microarray data

Initial data uses BeadStudio software quantile method standardization processings.Four groups are compared.And with regard to murine genes Express spectra by the same BALB/c- of BALB.D1-1/- be, BALB/c wild types, DBA/1-/- be and DBA/1 wild-type mices enter respectively Row compares.Show that Diff fractions are to be converted P values by quantile method, and based on reference to signal between group and control group The difference of averaging provides P value directionality.It is assumed that P values are 0.05, Diff fractions=± 13；It is assumed that P values are 0.01, Diff point Number=± 22；Setting P values are 0.001, Diff fractions=± 33；Diff values are set as ± 10 at initial analysis by us, to guarantee Potential candidate gene will not be lost^[2]。

Candidate gene, path analysis and structure

In order to determine candidate gene and build path/connection, in QTL regions and Il1rn, including Il1a, Il1b, Il1r1, Correlated expression is established between the candidate gene of Il1r2.On path analysis, GeneNetwork websites (network address is taken full advantage ofhttp://www.genenetwork.org/webqtl/main.py) gene expression profile available data.Use from spleen and obtained Obtain RIL (RI) gene expression data from C57BL/6j (B6) and DBA/2j (D2)^[3-4].Data be by The generation of Affymetrix GeneChip murine genes 1.0ST arrays (http://www.genenetwork.org/webqtl/ main.pyFormID=sharinginfo＆GN_AccessionId=283) generate.In the case of multiple probes, choosing Select wherein expression highest to be analyzed.GeneNetwork is built using GeneNetwork application tools.Based on correlation The intersection chart and diagram that matrix is combined is building GeneNetwork.Network is commonly used to show multigroup interaction.Spring model sets Meter (diminished strength) is used for the diagram method of all figure samples.We follow strong correlation, correlation, irrelevance be standard Condition.When R values equal or exceed ± 0.7, it is considered as strong positive correlation or negative correlation.When R values are 0.5 to 0.69 or -0.5 To between -0.69, it is considered as the presence of very weak correlation.When R values are 0 to ± 0.5, as not arriving weak correlation^[2].Use PGMapper initial analysis arthritis gene associations^[5]。

As a result

The full genome express spectra of the other mouse of inhomogeneity system has similitude

BALB.D1-1 congeric strains mark the base of both sides comprising D1Mit55 and D1Mit209 in the DBA/1 under BALB/c backgrounds Because of pack section^[1].It is similitude that other mouse full-length genome is expressed that BALB.D1-1 systems are studied first with other four.Illumina Mouse -6v1.1 expression magnetic bead chips are comprising 30774 probes (46632 fragments) for representing 29940 murine genes.Calculate BALB.D1-1 systems and the R values (correlation) that other four is other probe expression.Although other four is other R values showing The express spectra of BALB.D1-1 is not significantly correlated with this four, but has slight difference.Because BALB.D1-1 systems lack white The receptor antagonist protein of interleukin 1 (IL-1RA), relative to wild type BALB/c and DBA/1 systems not, its express spectra closer to Knock out DBA^-/-And BALB/c^-/-Be other, due to inserting DBA/1 fragment, the behavior of BALB.D1-1 systems is similar to DBA^-/-, show The same DBA of genome of opposing idiopathic arthritis is shown^-/-It is similar.However, because BALB.D1-1 is in BALB/c backgrounds, extremely Few related to DBA/1, R values are 0.976964.

BALB.D1-1 and DBA/1 wild type gene express spectras

Compared with the expression of DBA/1 mouse, the expression of 264 probes of BALB.D1-1 mouse lower and 472 The expression of individual probe raise (Fig. 1,2).In the probe of 472 rises, 344 is known.By PGMapper Search engine it was found that in 344 knowns, 61 is related to arthritis.Darc in 472 probes QTL regions (supplementary material table S1) is located at two genes of Fcgr4.In 264 downward probes, Kmo, Fcrla and Ephx1 tri- Individual gene is located in QTL regions.189 in 264 probes are knowns.Wherein 26 knowns are in PGMapper It is classified as arthritis-related genes.(61 up-regulated genes, 26 down-regulated genes and Il1a are analyzed by GeneNetwork, Il1b, Il1r1, Il1r2, Il1rap, Il1rapl1, Il1rn gene, Jing UTHSC Affy MoGene 1.0ST Spleen (http://www.genenetwork.org/webqtl/main.pyFormID=sharinginfo＆GN_AccessionId =283) confirm as 162 probes.Remove and repeat after probe, 138 probes are for GeneNetwork structures.GeneNetwork Show these genes not with II1rn and other il-1s family gene direct correlation (Fig. 3).

BALB.D1-1 and DBA^-/-Gene expression profile

With DBA^-/-The expression of mouse is compared, the expression of 241 probes of BALB.D1-1 mouse lower and 310 The expression of individual probe raise (Fig. 4,5).In the probe of 310 rises, 235 is known.On October 2nd, 2013 We have found that 43 is arthritis-related genes in known by PGMapper search engines.In the spy of 241 downwards In pin, 212 is known.On October 2nd, 2013 we found by PGMapper search engines, 29 in known Individual is arthritis-related genes.Jing UTHSC Affy MoGene 1.0ST Spleen confirm there are 43 rises in 138 probes Gene, 29 down-regulated genes and seven II1rn family genes.Remove and repeat after probe, 101 probes are used to analyze.It is wild with DBA Raw type mouse expression is similar, and the data analysis of GeneNetwork shows, these genes and II1rn family genes expression without It is closely connected (Fig. 6).

BALB.D1-1 and BALB/c gene expression profiles

Compared with the expression of BALB/c mouse, the expression of 31 probes of BALB.D1-1 mouse lower and 70 The expression of individual probe raise (Fig. 7,8).70 rise probes are knowns.And ifi202b genes are located in QTL areas In domain.In 31 downward probes, 30 is known.Compare the list of genes in QTL regions, ifi203 genes are located at the area In domain.70 up-regulated genes and 30 down-regulated gene Jing PGMapper analyses find that Siglec 1 is arthritis-related genes.128 Individual probe represents respectively 70 up-regulated genes, 30 down-regulated genes and II1rn, removes and repeats after probe, and 92 probes are used for GeneNetwork builds.GeneNetwork shows, these genes not with Il1rap or other interleukin 1 family genes Directly link (Fig. 9).

BALB.D1-1 and BALB/c^-/-Gene expression profile

With BALB/c^-/-The expression of mouse is compared, and the expression of 12 probes of BALB.D1-1 mouse is lowered and nothing Probe expression significantly raises (Figure 10).In 12 downward probes, 9 is known or sequence, LOC100040462 (Mndal), LOC639001 (the C5 precursors in T-cell receptors β chain V areas), ifi203, LOC665425 (T-cell receptors β chain V areas LB2 precursors), Lefty1, TRBV6, LOC638301, ifi204, ifi202b.The search-engine results of PGMapper show, this Without arthritis-related genes in a little knowns.Interferon activating gene (ifi202b, ifi203, ifi204, Mndal) and Lefty1 genes are respectively positioned in QTL regions.

6 (Rnpep, ifi203, ifi204, ifi205, Lefty1, ifi202b) in 11 probes are in UTHSC In Affy MoGene 1.0ST spleen databases, this 6 probes and Il1a, Il1b, Il1r1, Il1r2, Il1rap, Il1rapl1, Il1rn carry out GeneNetwork analyses.By the full-length genome transcripting spectrum of spleen, we construct gene net Network (Figure 11), as a result shows the transcriptional level of IL1rn families and interferon activated protein (IFi) family member without significantly correlated Property.We by HZI helper T lymphocyte Affymetrix M430v2 (http://www.genenetwork.org/webqtl/ main.pyFormID=sharinginfo＆GN_AccessionId=319) and HZI regulatory T cells (CD4+CD25+) (http://www.genenetwork.org/webqtl/main.pyFormID=sharinginfo＆GN_AccessionId =122) full genome expression map build GeneNetwork.The GeneNetwork numbers of helper T lymphocyte and regulatory T cells According to display, IL1rn families and ifi family members expression are without significant correlation (Figure 12 and 13).Even if further reducing threshold It is worth to 0.35, IL1rn and IFi kinsfolk still without directly related property.

Interferon activator protein and the potential participation opposing SAD of φt cell receptor β chain genes

Next we are compared the expression of all 12 genes between congeric strains and four parent plants.As schemed Shown in 6, with BALB^-/-Mouse compares, 11 in 12 genes downwards in congeric strains, and with BALB/c wild-type mices ratio More also lower.With DBA^-/-Compare with DBA/1 wild-type mices, without down regulation of gene expression.As shown by data, these genes are in BALB/ C and BALB^-/-Expression in mouse is very high, and in DBA^-/-It is very low with expression in DBA/1 wild-type mices.Consider DBA^-/-Mouse has resistance to SAD and BALB^-/-The fact that mouse is to the sensitiveness of SAD, these gene expressions in congeric strains The reduction of level may be the reason for exactly congenic line mice resistance strengthens.

We are multiple to compare confirmation, and it is to lead to decline in the gene expression dose of congeric strains φt cell receptor β chains and ifi clusters Congenic line mice arthritic is caused to resist compared with BALB/c^-/-It is the high main change of mouse.The expression of these genes is similar to DBA systems mouse and DBA in opposing SAD^-/-It is mouse.The expression of these genes is less than SAD susceptible BALB/c and BALB/c^-/- It is mouse.There are a considerable amount of genes, because different genes group background and Il1r are mutated, in congeric strains and other three parent lines DBA, DBA-/- and BALB/c mouse between differential expression.However, in congeric strains and BALB/c^-/-It is only one of which area between mouse Not.This difference is exactly that the QTL genome areas on No. 1 chromosome of congenic line mice are replaced by the fragment from DBA, and BALB/c^-/-The remainder of genome all is from BALB/c systems mouse.Our analysis suggests that this region candidate gene number Amount is few, and the Disease-causing gene for eventually finding congenic line mice for us provides possibility.

Primary data shows that the cluster of ifi genes 200 resists SAD optimal candidate genes comprising congenic line mice.In congeric strains And BALB/c^-/-Be mouse differential expression gene in, tetra- genes of ifi202b, ifi203, ifi204 and ifi205 be located at ifi On the cluster of gene 200 and congeric strains transgenic fragment.These genes are located at No. 1 chromosome transfer QTL genetic fragment of mouse Between D1Mit110 (167758517-167758517) and D1Mit209 (191493187-191493284).Ifi202 is located at this Individual region is simultaneously considered as one and can activate interferon lupus susceptible gene (Figure 14).Closing tolerance CD8+Ti cells Ifi202b genes can eliminate the rejection ability of these cells.Ifi204 is had begun working at present in reply bacterium infection side The effect in face^[6].It is estimated to can determine ifi family genes in terms of infection and immune innate immune response in the near future Wider function^[7].Therefore, it is very important that future carries out research to their qualifications for being elected.

φt cell receptor β chains are well-known to the adjustment effect of arthritis neurological susceptibility^[8-10].Current information shows that T cell is received Body β chain genes are not in the metastatic gene fragment in congeric strains QTL region.However, research φt cell receptor β chains family and ifi family It is very necessary that whether the gene between race lowers congenic line mice SAD by interacting.

Except φt cell receptor β chains and ifi genes, we also have detected the difference positioned at QTL regions Lefty1 expression.It is early The research of phase shows that the left side of Lefty1, the Lefty2 and Nodal mice embryonic in development is expressed, and relevant with the measure of L-R Connection.At present, due to Lefty have make cell to different differentiation factors such as Nodal, or need Egfcfc as co-receptor its The unique ability of the change cell fate of his signal without response, Lefty has become and has been considered one of cell dryness or differentiation certainly Determine factor.Its effect in arthritis, inflammation or infection is without relevant report.At present, without sufficient information interpretation its same Effect in class system.

Present study sets three repetitions, select every time each be not in female mice.Sex hormone has been demonstrated that difference is adjusted The expression of control ifi202^[11].However, each system is not all measured in same age bracket.Therefore, this species diversity at least reflects Congeric strains and other be other female mice genome change.We use spleen as extraction RNA and full-length genome gene The organ of spectrum^[3-4].Although spleen is important immune organ, the phenotype of SAD is based on joint in these mouse.Therefore, We be not excluded for data may not reflect completely send out genicular institute it is busy.But, it is believed that from this research The molecular mechanism for detecting is critically important for congeric strains causal gene is illustrated.By directly contacting out for candidate gene and disease The further research of exhibition is very necessary.Because expression of the ifi family genes between spleen and haemocyte is with stronger Correlation, so these detections also can be tested using blood cell.

Embodiment 3

The embodiment explanation rheumatoid arthritis neurological susceptibility and ifi202b, ifi203, ifi204 and ifi205 gene The correlation of expression.

Be presented herein below the other mouse of ifi202b, ifi203, ifi204 and ifi205 gene low expression level system and ifi202b, The evidence of the other mouse intercurrent disease difference of ifi203, ifi204 and ifi205 gene high expression level system.ifi202b、ifi203、 Two of ifi204 and ifi205 gene low expression levels are the low incidence of disease or not fall ill in both cases, but Two of ifi202b, ifi203, ifi204 and ifi205 gene high expression are that other mouse has the higher incidence of disease and higher sends out Sick average.

BALB.D1-1^-/-Congeric strains phenotype

Mouse BALB.D1-1-/- and BALB/c-/- germline be under the conditions of identical animal facility raise.Raise Foster condition is the normal condition without the need for any medicine or specially treated.When mouse four months is big, the generation and analysis of disease is observed The process of disease.Every mouse at least passes through three-times-weekly the arthritic occurring degree of external inspection.The score of each limbs All it is 0-4 point.Using the order of severity and the incidence of disease with our previously described identical statistical method analysis of joint inflammation ((0-without erythema and swelling, 1-joint and ankle-joint mild redness, 2-clear and definite swelling, 3-all limbs serious swellings, 4- Limbs damage deformation).Four limbs are calculated clear and definite fraction.The maximum of each mouse score is 16^[12,4].One is The order of severity do not fallen ill is that the total amount by all mouse total scores divided by mouse is calculated.The incidence of disease is little by illness The amount of mouse is calculated divided by mouse total amount.The accurate check analysis incidence of disease of expense She Er and with the variance analysis order of severity Difference.

BALB.D1-1^-/-Phenotype research use 18 BALB D1-1^-/-It is mouse and 18 BALB/c^-/-It is mouse. As shown in figure 16, congeric strains have substantially reduced and have delayed the incidence of disease (the P values respectively 3.4287E-07 of idiopathic arthritis And 3.4287E-06).During by 100 days, although congeric strains morbidity mouse is less than BALB/c^-/-It is mouse, but homology and BALB/ C-/- incidence of disease for being is still almost identical, reaches 100%.

Balb.D1-1^-/-The phenotypic result of homology may refer to Figure 16 and Figure 17.

Similarly, DBA.B-1^-/-The phenotypic result of homology may refer to Figure 18.

BALB.D1-1^-/-And BALB.D1-2^-/-Congeric strains, and DBA/1^-/-Comprising BALB/c^-/-No. 1 chromosome on QTL regions are from DBA.D-1^-/-System.Compared with two other congeric strains, the QTL regions of the BALB/c under DBA/1 backgrounds A piece of genomic DNA increased the risk of disease.DBA.B-1^-/-Phenotype research use 14 mouse.Compare DBA/1^-/-14 Mouse, homology significantly increase the order of severity and idiopathic arthritis incidence (P values be respectively 5.33637E-10 and 9.68472E-12)。

Arthritic level (average (serious journey of the DBA.B-1 congenic line mices relative to DBA/1 systems mouse Degree), the incidence of disease) result may refer to Figure 18 and Figure 19.

Embodiment 4

The embodiment is used to illustrate that ifi202b, ifi203, ifi204 and ifi205 gene transcription level and rheumatoid are closed The relation of the scorching incidence probability of section.

Part I：Determine the technology of gene expression dose

The extraction of total serum IgE and purifying

Take mouse spleen and organize extraction for total serum IgE.Total serum IgE is extracted using Trizol kits (Lifetechnology,CA,USA).Experimental procedure is modified and optimized as follows：

The extraction of RNA：

(1) it is homogenized：The sample tissue less than 50mg is ground in the TRIZOL reagents of 1mL using PRO200 homogenizers (Proscientific, CT, USA).The 5% of volume no more than TRIZOL reagent volumes during homogenised tissue sample.Using being less than 50mg tissues are conducive to improving the quality and yield of total serum IgE.

(2) it is separated：0.2ml chloroforms are added per 1mLTRIZOL reagents.Sample tube is covered tightly.Firmly abundant vortex oscillation 15 Second, it is incubated at room temperature 2-3 minutes.12,000x g, 2-8 DEG C is centrifuged 15 minutes.Carefully upper strata aqueous phase is transferred to into pipe 1, is careful not to Encounter two-phase interface.Remaining upper strata aqueous phase and two-phase interface are transferred to into pipe 2.Pipe 2 is centrifuged 15 minutes in 12,000x g, Upper strata aqueous phase is transferred in pipe 1.The volume of liquid (uses TRIZOL reagent volumes in measurement pipe 1 when water phase volume is homogenate 65%).

Second water is mutually centrifuged and will to greatest extent obtain total serum IgE.

(3) RNA precipitate：The isopropanol for adding 650 μ L cold, 4 DEG C of incubation sample 1h, then 2-4 DEG C, 12,000x g centrifugations 10 minutes.

(4) RNA washings：Discard supernatant.At least the 80% of 1ml cold ethanol is added, washing RNA is once.Vortex oscillation is mixed Sample is closed, 2-8 DEG C, 7,500x g are centrifuged 5 minutes, repetition washing procedure above once, removes the ethanol of all residuals.

(5) RNA purifying：

The nuclease free water for adding 100 μ L dissolves RNA.10 μ L 3M sodium acetates are added, is well mixed；Add 300 μ L100% ethanol；Fully mix -20 DEG C of overnight incubations (16 hours).

We have found that overnight incubation can at utmost regain total serum IgE precipitation.

By 4 DEG C of liquid after overnight, 12,000g centrifugations 30 minutes, supernatant is carefully removed and discarded.Add 500 μ L80% Cold ethanol purge precipitation.4 DEG C, 8,000g centrifugation 5min are carefully removed and are discarded 80% ethanol.Repeat 80% ethanol to wash twice. Ethanol to remove residual, rapid rotated sample pipe suctions out residual liquid with pipettor.It is air-dried precipitation.Use free nucleic acid The resuspended RNA precipitate of enzyme water, makes final concentration of 100ng/ μ L or so.

(6) RNA concentration is detected using NanoDrop 2000.

(7) quality and integrality of the detection of Agilent photometer 2100 RNA.

2. the test of genetic chip and the analysis of expression

(1). test process：

UseTotalPrep^TMRNA detection kits (Ambion, CA, USA).Using 200ng, RIN High-quality total serum IgE of (RNA integrity coefficients) value more than 8, first complementary dna chain (cDNA) of synthesis, the synthesis of cDNA complementary strands, The purifying of cDNA, in-vitro transcription synthesis cRNA, the amplification of cRNA and purifying.2000 points of NanoDrop of the solution concentration of cRNA Light photometer detects the absorptivity at 260nm/280nm, and biotinylated cRNAs (1600ng/ samples) is in 58 DEG C and chip Hybridization 19 hours.The chip of these direct crosses is usedReader scannings (Illumina, San Diego, CA, USA)。

(2) gene information of micro-array chip

Ifi202b, ifi203, ifi204 and ifi205 gene：The annotation of the mouse of service is supported in Illumina data Ifi202b, ifi203, ifi204 and ifi205 (table 1) are searched in file 6_V2_0_R3_11278593_A.

IFI gene informations on the Illumina BeadChip mouse full-length genome chip of expression spectrum of table 1

(3) analysis of gene expression dose

It is proprietary using IlluminaSoftware to the error of initial data and quality testing at Reason.Gene expression data is normalized place using Partek Genomics Suite softwares 6.6 editions (Partec, MI, USA) Reason and summary.Non-paired t test filters out P≤0.05 significant difference and mean difference multiple >=1.5 probes.

Four groups are compared.The same BALB/c of gene expression fragment of D1.BALB-1^-/-, BALB/c wild types, DBA/1^-/-With DBA/1 wild types are compared.The Diff values that quantile method is obtained be to provide this based on reference to group and comparative group average signal it Between the P values of difference be converted, while there is provided the directionality of P values.When P values are that 0.05, Diff values are ± 13；When P values It is ± 22 for 0.01, Diff values；When p value is that 0.001, Diff values are ± 33.Diff values are established as ± 13 by initial analysis, with Guarantee that potential candidate gene will not be missed.

3.realtime PCR (i.e. real-time fluorescence quantitative PCR) technology for detection gene expression dose

(1) realtime PCR test process：

A. the separation of total serum IgE

The separating step and kit of total serum IgE equally adopts above-described Trozol reagents.Our laboratories are unique Optimization Steps：Collect the amount of total serum IgE again to greatest extent and eliminate contaminating genomic DNA.

B. target gene specific design of primers

The use of self-defined TaqMan analysis design tools is ifi202b, ifi203, ifi204, ifi205 and mndal gene Design specific primer (table 2).Design of primers follows following point and is optimized, in order to preferably evaluate table between different sample groups Up to horizontal difference：

Primer pair youngster binds plural extron；Each genetic fragment is between 80-110bps；In having target sequence Without SNP (SNP) between DBA1 and Balb/c；Target sequence is preferably covered, and is overlapped or close micro-array chip Probe sequence.

The primer sequence information of table 2.

C. reverse transcription generates cDNA

The step of TaqMan reverse transcription reagents (Cat#N808-0234, ABI, CA, USA) carry out cDNA and generate, but this experiment Room is modified to it.With 1200ng pure total serum IgE as template.50 DEG C of reactant mixture is incubated 10 minutes, then 37 DEG C 60 minutes, 95 DEG C 15 minutes.For the amount of accurate cDNA, we are in accordance with Qiagen MinElute Reaction Cleanup Kit The amount of the quantitative cDNA of (Cat#28204, Qiagen, CA, USA) kit production handbook.We are quantified with NanoDrop 200 The cDNA for having transcribed.All cDNA samples are dense using the 5ng/ μ L that deionized water is diluted to realtime PCR reaction settings Degree.

D.PCR reacts

TaqMan Gene Expression Master Mix (Cat#4369016, ABI, CA, USA) and self-defined primer The PCR that design is carried out is reacted according to manufacturer's agreement, and this laboratory is equally modified.In order to template more accurately takes 20ng CDNA (4 μ L dilute cDNA) adopts 20 μ L reaction systems.House-keeping gene, beta-actin and GAPDH genes are reference gene.I Using multiple house-keeping genes be in order to explained using each gene expression experiment to single house-keeping gene expression experiment bar Any impact that part is produced.For accuracy, each Setup Experiments multiple holes is added to 96 ultra-fine orifice plates.The PCR reactions of standard set It is set to：50 DEG C 2 minutes, 95 DEG C 10 minutes, 95 DEG C 15 seconds 40 circulation, 60 DEG C 1 minute, using ABI Prism 7900 Detection System (Applied Biosystems, CA, USA).

(2) individuality between gene expression dose difference analysis

Detect period (CT values) and calculate associated retroviral level.Data are painted according to the associated expression for calculating System, cycle threshold be the logarithmic amplification fixed with probe as starting, Δ Ct be genes of interest CT values and house-keeping gene CT values it Between difference.It is the negative exponent value of Δ Ct that data are computed for 2 (probe signals that each circulation increases), with control group and TNF- The associated change of α stimulation groups and draw.According to many house-keeping genes of Δ Ct, we determined that each target gene is optimal one. The parent control group of DBA1 and Balb/c is set respectively as two congeric strains BALB.D1-1^-/-And BALB.D1-1^-/-Control Group.

Part II：Rheumatismal probability is predicted according to gene expression dose

1. the expression in DBA as 0, using the expression in BALB/C as 100.With DBA, BALC and similar The expression of system forms the predictor formula of a prediction morbidity risk rate drawing a straight line, so.

The expression of 2.ifi family genes is directly proportional to rheumathritis.

(1) mouse background introduction

BALB/c systems mouse lacks IL-1 receptor antagonist proteins (Il-1ra) and causes idiopathic arthritis (SAD) and have this The DBA/1IL1rn of defect^-/-It is that mouse does not fall ill^[13].Before, we depict No. 1 chromosome quantitative character site of SAD (QTL) figure, then develops BALB.D1-1^-/-It is mouse, this mouse includes and BALB/c^-/-DBA/1 in background^-/-Opposing is relevant QTL genetic fragments.

(2) result

In congeric strains and BALB/c, DBA/1 and DBA/1^-/-There is fairly large number of gene difference table between three parent lines Reach.But by congeric strains and BALB/c^-/-System compares only minority difference expression gene and is determined.The difference table of these genes Up to essentially from φt cell receptor β chains (Tcrb) and interferon activator protein (ifi) gene.These genes are in congeric strains and BALB/ Differential expression between c, however they co-isogenic expression with DBA/1 and DBA/1-/- be similar.Tcrb-j genes Expression clearly with the cluster of ifi genes 200 in two gene-correlations.

Brief summary：

The transcriptional level that measure ifi202b, ifi203, ifi204 and ifi205 is passed through by above method and step explanation The probability that rheumatism is fallen ill can be predicted.Any other biotechnology is used for determining the methods of genetic transcription and may serve to Determine the gene transcription level in IFI genomes.Therefore, it is any to be predicted with the gene transcription level in IFI genomes is determined The methods of rheumatism incidence probability all should be within the scope of the present application.

Embodiment 5

The embodiment is used to illustrate that ifi202b, ifi203, ifi204 and ifi205 gene is thin in splenocyte and human hematopoietic The correlation of the expression between born of the same parents.

Tested using method substantially the same manner as Example 3.

Test procedure includes：It is determined that the expression of the ifi203 and ifi204 genes in spleen and blood cell sample, And their expression is compared.

The expression of ifi203 and ifi204 genes in identification spleen sample, operating procedure is as follows：1) open GeneNetwork network addresshttp://www.genenetwork.org/webqtl/main.py；2) select from " Type " classification Spleen；3) UTK spleen ILM6.1 are selected in " Data Set " classification；4) in " Get any " classification be input into ifi203 or Ifi204, then searches for the probe of the two genes.

The expression of ifi203 and ifi204 genes in identification hematopoietic cell sample, operating procedure is as follows：1) open GeneNetwork network addresshttp://www.genenetwork.org/webqtl/main.py；2) select from " Type " classification Hematopoietic cell mRNA；3) UMCG stem cell ILM6v1 are selected in " Data Set " classification；4) it is input in " Get any " classification Ifi203 or ifi204, then searches for the probe of the two genes.

After determining the probe sample of each gene, use " addition " key by sample collection to data collection list in.Counting According to acquisition tables, two probes are have selected for comparing, using order " matrix " comparator matrix is built.In a matrix, click The symbol of rho values obtains the correlation graph of two probes.

Comparison of the ifi203 genes in spleen sample and hematopoietic cell has been carried out first, has then compared ifi204 genes in spleen The situation of dirty sample and hematopoietic cell.Analyze data be from above-mentioned GeneNetwork splenocyte and hematopoietic cell the two The ILM6.1 chips (Illumina companies) of cell type.Result of the test is shown in Figure 16, Tu20Zhong.This two groups of as shown by data, this The expression of two kinds of cell is height correlation.Wherein, identical probe is adopted between blood cell and splenocyte It is compared.For ifi203, probe is ILM6420102 (SEQ ID No.5).For ifi204, probe is ILM6520601 (SEQ ID No.6)。

Can be learnt by Figure 16 and Figure 20, expression of the ifi204 genes in blood and splenocyte is also in notable positive Close (Figure 16), expression of the ifi203 genes in blood and splenocyte is in notable positive correlation (Figure 20).

Bibliography

1.Jiao Y,Jin X,Yan J,Zhang C,Jiao F,Li X,Roe BA,Mount DB,Gu W.A deletion mutation in Slc12a6 is associated with neuromuscular disease in gaxp mice.Genomics.2008 May；91(5):407-14.doi:10.1016/j.ygeno.2007.12.010.Epub 2008 Mar 14.PMID:18343091.

2.Jiao Y,Chen H,Yan J,Wang L,Huang Y,Liu X,Williams RW,Lu L,Wang Y,Gu W.Genome-wide gene expression profiles in antioxidant pathways and their potential sex differences and connections to vitamin C in mice.Int J Mol Sci.2013 May 10；14(5):10042-62.PMID:23665904

3.Yanhong Cao,Xiaoyun Liu,Nan Deng,Yan Jiao,Yonghui Ma,Karen A.Hasty, John M.Stuart,Weikuan Gu.Congenic mice provide evidence for a genetic locus that modulates spontaneous arthritis caused by deficiency of IL-1RA.2013,PLoS One.2013 Jun 28；8(6):e68158.doi:10.1371/journal.pone.0068158.Print 2013.PMID: 23840826.

4.Deng N,Jiao Y,Cao Y,Liu X,Ma Y,Hasty KA,Brand DD,Stuart JM,Gu W.Genomic locus on chromosome 1 regulates susceptibility to spontaneous arthritis in mice deficiency of IL-1RA.BMC Immunol.2014 Dec 9；15:57.doi: 10.1186/s12865-014-0057-9.PMID:25488730.

5.Xiong Q,Qiu Y,Gu W.PGMapper:a web-based tool linking phenotype to genes.Bioinformatics.2008 Apr 1；24(7):1011-3.PMID:18204061.

6.Storek KM,Gertsvolf NA,Ohlson MB,Monack DM.cGAS and Ifi204 cooperate to produce type I IFNs in response to Francisella infection.J Immunol.2015 Apr 1；194(7):3236-45.

7.Storek KM,Monack DM.Bacterial recognition pathways that lead to inflammasome activation.Immunol Rev.2015 May；265(1):112-29.doi:10.1111/ imr.12289.PMID:25879288.

8.Trapani JA,Browne KA,Dawson MJ,Ramsay RG,Eddy RL,Show TB,White PC, Dupont B.A novel gene constitutively expressed in human lymphoid cells is inducible with interferon-gamma in myeloid cells.Immunogenetics.1992；36(6): 369-76.PMID:1526658.

9.Yin G,Li X,Li J,Cui H,Hou R,Zhang J,Dong F,Liang H,Zhang K.Screening of differentially expressed genes and predominant expression ofβ variable region of T cell receptor in peripheral T cells of psoriatic patients.Eur J Dermatol.2011 Nov-Dec；21(6):938-44.PMID:21951576.

10.De Andrea M,Zannetti C,Noris E,Gariglio M,Azzimonti B,Landolfo S.The mouse interferon-inducible gene Ifi204 product interacts with the Tpr protein,a component of the nuclear pore complex.J Interferon Cytokine Res.2002 Nov；22(11):1113-21.PMID:12513910.

11.Vogel H,Scherneck S,Kanzleiter T,Benz V,Kluge R,Stadion M,Kryvych S,Blüher M,N,Joost HG,Schürmann A.Loss of function of Ifi202b by a microdeletion on chromosome 1of C57BL/6J mice suppresses 11β-hydroxysteroid dehydrogenase type 1expression and development of obesity.Hum Mol Genet.2012Sep 1；21(17):3845-57.doi:10.1093/hmg/dds213.Epub 2012Jun 12.PMID: 22692684.

12.Griffiths MM,Remmers EF.Genetic analysis of collagen-induced arthritis in rats:a polygenic model for rheumatoid arthritis predicts a common framework of cross-species inflammatory/autoimmune disease loci.Immunol Rev.2001 Dec；184:172-83.Review.PMID:12086311.

13.Zhou,F.,X.He,Y.Iwakura,R.Horai,and J.M.Stuart.Arthritis in mice that are deficient in interleukin-1receptor antagonist is dependent on genetic background.Arthritis and rheumatism 2005.52:3731-3738.PMID:16320323.

The above, is only several embodiments of the application, any type of restriction is not done to the application, although this Shen Please disclosed as above with preferred embodiment, but and be not used to limit the application, any those skilled in the art are not taking off In the range of technical scheme, make a little variation using the technology contents of the disclosure above or modification is equal to Effect case study on implementation, belongs in the range of technical scheme.

Claims (10)

1. whether a kind of nucleic acid is being prepared for diagnosing main body with the presence or absence of the risk with rheumatoid arthritis and/or suffering from Application in the kit of rheumatoid arthritis, it is characterised in that the sequence of the nucleic acid is included as shown in SEQ ID No.1 Sequence in any one section of sequence, any one section of sequence in the sequence as shown in SEQ ID No.2, such as SEQ ID No.3 Any one section of sequence and such as SEQ ID in any one section of sequence in shown sequence, the sequence as shown in SEQ ID No.4 At least one in any one section of sequence in sequence shown in No.23；Or

The sequence of the nucleic acid includes from people, and with the sequence as shown in SEQ ID No.1 in any one section of sequence, such as Any one section of sequence in any one section of sequence in sequence shown in SEQ ID No.2, the sequence as shown in SEQ ID No.3 It is any in any one section of sequence and the sequence as shown in SEQ ID No.23 in row, the sequence as shown in SEQ ID No.4 One section of sequence homology and/or with sequence identity, and at least one in function identical sequence；

It is preferred that the sequence of the nucleic acid includes any one section of sequence in the sequence as shown in SEQ ID No.28.

2. application according to claim 1, it is characterised in that the single chain lengths of the sequence of the nucleic acid are 40-300nt； When by way of biochip to be detected, the single chain lengths of preferred nucleic acid sequence are 50-100nt；When by reversion When the mode of record quantitative fluorescent PCR is to be detected, the single chain lengths of the sequence of the nucleic acid are 80-200nt.

3. application according to claim 1 and 2, it is characterised in that the sequence of the nucleic acid such as SEQ ID Nos.5 and/or Sequence shown in 6.

4. application according to claim 3, it is characterised in that the sequence of the nucleic acid is cDNA sequence and/or RNA sequences Row.

5. application according to claim 1 and 2, it is characterised in that the sequence of the nucleic acid such as SEQ ID Nos.7-10 and At least one in sequence shown in 24.

6. application according to claim 5, it is characterised in that amplification upstream and downstream of sequence as shown in SEQ ID No.7 is drawn As shown in SEQ ID No.11 and SEQ ID No.12, the sequence of the probe of sequence is such as shown in SEQ ID No.7 for the sequence of thing Shown in SEQ ID No.13；Amplification as shown in SEQ ID No.8 the sequence such as SEQ ID No.14 of the upstream and downstream primer of sequence with Shown in SEQ ID No.15, the sequence of the probe of sequence is as shown in SEQ ID No.16 as shown in SEQ ID No.8；Amplification is such as The sequence of the upstream and downstream primer of sequence shown in SEQ ID No.9 as shown in SEQ ID No.17 and SEQ ID No.18, such as SEQ The sequence of the probe of sequence shown in ID No.9 is as shown in SEQ ID No.19；Amplification as shown in SEQ ID No.10 sequence it is upper The sequence of downstream primer as shown in SEQ ID No.20 and SEQ ID No.21, the probe of sequence as shown in SEQ ID No.10 Sequence is as shown in SEQ ID No.22；The sequence such as SEQ ID of amplification upstream and downstream primer of sequence as shown in SEQ ID No.24 Shown in No.25 and SEQ ID No.26, the sequence of the probe of sequence is as shown in SEQ ID No.27 as shown in SEQ ID No.24.

7. application according to claim 6, it is characterised in that primer therein is double-stranded DNA and/or single stranded DNA；Wherein Probe be single stranded DNA.

8. it is a kind of to whether there is the risk with rheumatoid arthritis and/or whether with rheumatoid pass for diagnosing main body The scorching kit of section, it is characterised in that in the application that the kit is included as described in any one in claim 1-7 Nucleotide sequence.

9. it is a kind of to whether there is the risk with rheumatoid arthritis and/or whether with rheumatoid pass for diagnosing main body The scorching kit of section, it is characterised in that in the application that the kit is included as described in any one in claim 1-4 Nucleotide sequence, also including the chip that can fix the nucleotide sequence, preferably the material of the chip is selected from nylon membrane, glass One kind in piece, plastics, silica gel chip and miniature magnetic bead；And also optionally include from from the master in the kit The reagent and/or chip hybridization reagent used by RNA is extracted in the detected sample of body；It is preferred that the detected sample is group living Knit at least one in detection sample, blood, blood plasma, serum and lymph liquid；More preferably described living tissue detection sample be from The living tissue detection sample of spleen, or the sample is hematopoietic cell.

10. it is a kind of to whether there is the risk with rheumatoid arthritis and/or whether with rheumatoid for diagnosing main body Arthritic kit, it is characterised in that include as described in any one in claim 1,2 and 5-7 in the kit Application in nucleotide sequence, also including the reagent carried out used by quantitative fluorescent PCR, and in the kit also optionally Including the reagent and/or reverse transcription quantitative fluorescent PCR examination extracted from the detected sample from the main body used by RNA Agent；It is preferred that the detected sample is at least one in living tissue detection sample, blood, blood plasma, serum and lymph liquid；It is more excellent The living tissue detection sample is selected to be the living tissue detection sample from spleen, or the sample is hematopoietic cell.