CN106544448A - The cytokine related to clinical prognosis and application in EBOV infected patient blood - Google Patents
The cytokine related to clinical prognosis and application in EBOV infected patient blood Download PDFInfo
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Abstract
The invention discloses cytokine related to clinical prognosis in EBOV infected patient blood and application.In EBOV infected patients blood disclosed by the invention, the cytokine related to clinical prognosis is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and FGF20, and raise relative to expression in surviving populations in this dead colony of 8 cytokines in EBOV the infected, show that this 8 cytokines can be identified as biomolecule, for the prognosis of EBOV the infected.
Description
Technical field
The present invention relates in biomedical sector, in EBOV infected patient blood the cytokine related to clinical prognosis with
Using.
Background technology
Ebola disease viral disease is by the Ebola virus (Ebola virus, EBOV) of Filoviridae (filoviridae)
A kind of caused acute hemorrhage sexually transmitted disease.Case fatality rate is very high, up to 50%～90%.
Ebola virus genome is sub-thread strand RNA, and about long 19kb can encoding nuclear proteins (NP) and envelope protein
7 structural protein such as (VP35, VP40, VP30, VP24), glycoprotein (GP) and RNA polymerase, wherein GP gene pairss EBOV are replicated
There are the coding and functional transcription of uniqueness.Virus is replicated in the kytoplasm of infection cell, assembling, is discharged in bud life mode.Ebola
It is viral mainly to include Zaire's type (EBOV-Z), the Sudan's type (EBOV-S), Cote d'lvoire's type (EBOV-C) and Reston type
(EBOV-R) etc., the characteristic of different subtype is different, and wherein Zaire's type virulence is most strong, and the Sudan's type takes second place, and both are to the mankind and non-
The fatality rate of people primatess is very high.Reston type and Cote d'lvoire's type are relatively low to the virulence of people, show as subclinical infection, but right
Non-human primates have mortality.
EBOV is a kind of Zoonosis virus, and its route of transmission is still not clear, generally by with mankind's close contact to infecting
The blood of animal, secretions, organ or other body fluid cause infection, and body fluid of directly contact the infected etc. can cause interpersonal
Propagate.Various non-human primate and the mankind are generally susceptible.Therefore, family member of HIV-infected people, medical worker, reviewer, angstrom
The rich staff for drawing viral prevalence scene, infection animal contactee etc. are high-risk group.
EBOV is a kind of virus of pantropic, can invade each system organ, is attached most importance to liver, spleen infringement especially.Ebola virus
The generation of disease is relevant with the immunne response level of body.Especially phagocyte is to be attacked by virus first to mononuclear phagocyte system
The target cell for hitting, subsequent fibroblast and endotheliocyte it is infected, vascular permeability increases, and fibrin is calm.Infection
2 days virus is detected first in lung afterwards, is detected after 4 days in the tissue such as liver, spleen, and 6 Tian Hou body tissues can detect.
Many researchs with regard to pathogenicity at present are all carried out in animal model and experiment in vitro, and with regard to people
The correlational study of precursor virus infection is few.Each stage of virus infection body, from cell entry cell, partial copy, propagation (disease
Toxemia), in the target organ breeding, Rehabilitation (but still band poison) or dead, there is the mutual restriction of two kinds of factors：Virus
Infection and host body response and defence.Research pathogenesis and Immune escaping mechanism of the EBOV to the mankind, and then according to these
Committed step, determines modulation site, researches and develops medicine, for the Synthetical prevention of ebola hemorrhagic fever has important meaning
Justice.Meanwhile, the response feature of human infection EBOV is studied, the molecular marker of infection and prognosis characterizations is found, it is fast for setting up
Fast diagnostic method, set up timely and effective intervention stratege theoretical foundation can be provided.
The content of the invention
How the technical problem to be solved is to EBOV the infected's prognosis.
To solve above-mentioned technical problem, present invention firstly provides following applications 1) or 2)：
1) detect application of the system of G1 expressions in EBOV the infected's prognosis or auxiliary prognosis product is prepared；It is described
G1 be THPO genes, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes, GDF10 genes and/or
FGF20 genes；
2) detect application of the system of G1 expressions in EBOV the infected's prognosis or auxiliary prognosis；The G1 is THPO
Gene, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes, GDF10 genes and/or FGF20 bases
Cause.
In above-mentioned application, the system of the detection G1 expressions can be to detect the G1 using method of the prior art
Reagent and/or instrument needed for expression, reagent as described in being detected using high-flux sequence needed for G1 expressions and/or
Instrument, or using the reagent and/or instrument needed for G1 expressions described in quantitative PCR detection, or hybridize skill using northern
Art detects the reagent and/or instrument needed for the G1 expressions, or using needed for G1 expressions described in genechip detection
Reagent and/or instrument.
In above-mentioned application, the high-flux sequence can be carried out using Illumina microarray datasets.
In above-mentioned application, the system of the detection G1 expressions may also include data handling system, the data processing
System is used for analyzing the result that method of the prior art is directly obtained, and determines the expression of the G1.The data processing
System can be software and/or module.
To solve above-mentioned technical problem, present invention also offers following M1) or application M2)：
M1) detect application of the system of P1 contents in EBOV the infected's prognosis or auxiliary prognosis product is prepared；
M2) detect application of the system of the P1 contents in EBOV the infected's prognosis or auxiliary prognosis；
The P1 is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and/or FGF20 protein.
In above-mentioned application, the system of the detection P1 contents can be to detect the P1 contents using method of the prior art
Required reagent and/or instrument, the reagent and/or instrument needed for using mass spectrum or its P1 content as described in correlation technique detection,
Or using the reagent and/or instrument needed for P1 contents described in immuning hybridization technology for detection, or the P1 is detected using elisa technique
Reagent and/or instrument needed for content, or using the reagent and/or instrument needed for P1 contents described in protein chip or detection paper
Device.
For solve above-mentioned technical problem, present invention also offers following N1)-N4) in arbitrary application：
Used by N1) using G1 as in EBOV the infected's prognosis of EBOV the infected's prognostic marker or auxiliary method of prognosis
System is in following a1) or a2) in application：
A1 EBOV the infected's prognosis or auxiliary prognosis product) are prepared；
A2) EBOV the infected's prognosis or auxiliary prognosis；
The G1 be THPO genes, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes,
GDF10 genes and/or FGF20 genes；
Used by N2) using P1 as in EBOV the infected's prognosis of EBOV the infected's prognostic marker or auxiliary method of prognosis
System is in above-mentioned a1) or a2) in application；
The P1 is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and/or FGF20 protein；
N3) using the G1 as EBOV the infected's prognostic marker in above-mentioned a1) or a2) in application；
N4) using the P1 as EBOV the infected's prognostic marker in above-mentioned a1) or a2) in application.
It is used in EBOV the infected's prognosis using G1 as EBOV the infected's prognostic marker or auxiliary prognosis to be
System can be the system of detection G1 expressions mentioned above.
To solve above-mentioned technical problem, present invention also offers following X1) or product X2)：
X1) EBOV the infected's prognosis or auxiliary prognosis product, are the system of the detection G1 expressions；
The G1 be THPO genes, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes,
GDF10 genes and/or FGF20 genes；
X2) EBOV the infected's prognosis or auxiliary prognosis product, are the system for detecting P1 contents；
The P1 is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and/or FGF20 protein.
To solve above-mentioned technical problem, present invention also offers EBOV the infected's prognosis related molecule of non-diagnostic purpose is special
Levy the construction method of spectrum.
The construction method of EBOV the infected's prognosis related molecule characteristic spectrum of non-diagnostic purpose provided by the present invention, bag
Include：Quantitative analyses are carried out to the RNA in EBOV the infected's survival group and the dead group blood of EBOV the infected respectively, according to analysis knot
Fruit obtains EBOV the infected's prognosis related molecule characteristic spectrum；EBOV the infected's prognosis related molecule characteristic spectrum is G1 or P1；
The G1 be THPO genes, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes,
GDF10 genes and/or FGF20 genes；
The P1 is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and/or FGF20 protein.
In said method, it is fixed that the RNA in EBOV the infected's survival group and the dead group blood of EBOV the infected is carried out
Amount analysis specifically may include：RNA or protein in purification EBOV the infected's survival group and the dead group blood of EBOV the infected, profit
The content of RNA is detected with Illumina microarray datasets.
RNA or protein in purification EBOV the infected survival group and the dead group blood of EBOV the infected can utilize band
The magnetic bead for having Oligo (dT) is carried out.
The content of the utilization Illumina microarray datasets detection RNA may include：By RNA fragmentations after purification, and with
This is template reverse transcription synthetic double chain cDNA：End is carried out to double-strand cDNA and repairs polishing so as to 5 ' end phosphorylations, 3 ' ends
Plus " A "；Double-strand cDNA joint with 3 ' dTMP ends is connected in sequence measuring joints, is expanded by PCR, using AMPure XP magnetic beads
Enriching and purifying joint product, obtains library；The library for building is carried out into double end sequencings, detection with Illumina microarray datasets
The content of RNA in sample to be tested.
In said method, EBOV the infected's survival group may include at least one EBOV infection survivors.The EBOV
The infected's death group may include at least one EBOV infection dieds.
In the present invention, the G1 expressions can be the expression of G1 described in blood；The P1 contents can be blood
Described in P1 content.
In the present invention, EBOV the infected's prognosis or auxiliary prognosis product can be EBOV the infected's prognosis or auxiliary prognosis
Medicine and/or health equipment, such as reagent, reagent paper, material or instrument.
In actual applications, can by analyze THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and
FGF20 this expressions of 8 cytokines in object blood to be measured comes to object prognosis to be measured.Specific criterion can
For：When with EBOV the infected as object to be measured, the such as THPO of object to be measured, STC2, IL17D, SEMA3C, SEMA3F, EDN2,
In GDF10 and FGF20 this 8 cytokines, the expression of at least one is corresponding higher than the survival object in EBOV the infected
The 150% of Cytokine Expression Level, the object to be measured is or candidate is high mortality risk EBOV the infected, as described to be measured
The expression of THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and FGF20 of object this 8 cytokines
The 150% of the corresponding Cytokine Expression Level of survival object being not higher than in EBOV the infected, the object to be measured is or waits
Select not high mortality risk EBOV the infected.That is, THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and
In FGF20 the mortality risk of the high EBOV the infected of at least one expression higher than THPO, STC2, IL17D, SEMA3C,
The low EBOV the infected of the expression of SEMA3F, EDN2, GDF10 and FGF20.
The expression height can specifically be presented as：A kind of expression in 8 cytokines is felt for EBOV
More than 1.5 times of the corresponding cytokine of survival object in dye person.
The present invention passes through the difference expression gene for analyzing EBOV the infected and non-EBOV the infected, further by dependency
Analysis, finds the differential expression (p related to virus infection prognosis of 8 cytokines<0.01), this 8 cytokines are respectively：
THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and FGF20, and this 8 cytokines are in EBOV the infected
In dead colony in raise relative to expression in surviving populations, show that this 8 cytokines can be used as biomolecule mark
Know, for the prognosis of EBOV the infected.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
The molecular marker related to virus load in embodiment 1, EBOV the infected's blood
Total serum IgE is extracted from EBOV the infected with non-EBOV the infected's whole blood, it is pure with the absorption of the magnetic bead with Oligo (dT)
Change the mRNA in total serum IgE, in a heated condition by mRNA fragmentations, and as template reverse transcription synthetic double chain cDNA.To double
Chain cDNA carries out end and repairs polishing so as to 5 ' end phosphorylations, and 3 ' ends add " A ".Double-strand cDNA joint with 3 ' dTMP ends is connected
To in sequence measuring joints, expanded by PCR, using AMPure XP enrichment with magnetic bead purification joint products, by PCR reaction identification texts
Storehouse.The library for building is carried out into double end sequencings with Illumina microarray datasets.Filter what sequencing was produced using perl script
Joint sequence, two ends low quality sequence (Q in initial data<=20 base number accounts for more than the 50% of whole piece reads sequence
Row), Sequences of Low Complexity.
Valid data are compared into people with reference on genome using Tophat softwares, Cutfflinks software combinations compare knot
Really, differential expression of the EBOV the infected (n=22) compared with non-EBOV the infected (n=10) is drawn by Cutffdiff softwares
Gene, further by correlation analysiss, finds the differential expression related to EBOV virus loads in EBOV the infected's blood point
Son.As a result show, the differential expression (r related to EBOV virus loads of 89 genes2>0.64,p<0.01), wherein, before coming
10 molecule is：DPAGT1(dolichyl-phosphate N-acetylglucosaminephosphotransferase
1),GPR125(G protein-coupled receptor125),CYP4B1(cytochrome P450family
4subfamily B member 1),PTPRK(protein tyrosine phosphatase,receptor type K),
NEK10(NIMA related kinase 10),NCAPD3(non-SMC condensin II complex subunit
D3),ABCA13(ATP binding cassette subfamily A member 13),DNAH11(dynein,
axonemal,heavy chain 11),KDELC1(KDEL motif containing 1),MTA3(metastasis
Associated 1family member 3), and this 10 molecules in EBOV the infected relative in non-EBOV the infected
Expression increases.Concrete outcome as shown in table 1, shows that this 10 molecules can be identified as biomolecule, infects for EBOV
The clinical diagnosises of person.
The related molecular marker of table 1, EBOV infection
| Title | Ensembl Gene ID | Differential expression multiple | Dependency (r2) | P values |
| DPAGT1 | ENSG00000172269 | 7.27 | 0.79 | <0.0001 |
| GPR125 | ENSG00000152990 | 15.17 | 0.77 | <0.0001 |
| CYP4B1 | ENSG00000142973 | 52.44 | 0.77 | <0.0001 |
| PTPRK | ENSG00000152894 | 9.96 | 0.76 | <0.0001 |
| NEK10 | ENSG00000163491 | 31.53 | 0.76 | <0.0001 |
| NCAPD3 | ENSG00000151503 | 3.81 | 0.75 | <0.0001 |
| ABCA13 | ENSG00000179869 | 16.72 | 0.74 | <0.0001 |
| DNAH11 | ENSG00000105877 | 40.70 | 0.74 | <0.0001 |
| KDELC1 | ENSG00000134901 | 34.19 | 0.73 | <0.0001 |
| MTA3 | ENSG00000057935 | 8.17 | 0.73 | <0.0001 |
Note：Difference of 10 molecules relative to expression in non-EBOV the infected in EBOV the infected is listed in table 1
Different multiple and 10 molecules and EBOV virus load correlation analysiss results.
In actual applications, can by compare DPAGT1, GPR125, CYP4B1, PTPRK, NEK10, NCAPD3,
ABCA13, DNAH11, KDELC1 and MTA3 are to be measured to judge with the expression in non-EBOV the infected's blood in object to be measured
Whether object is EBOV the infected.Specifically, the criterion for being obtained according to the above results is as follows：Such as object to be measured
DPAGT1, GPR125, CYP4B1, PTPRK, NEK10, NCAPD3, ABCA13, DNAH11, KDELC1 and MTA3 this 10 genes
In the expression of at least one higher than 3 times of non-EBOV the infected's corresponding gene, the doubtful EBOV the infected of the object to be measured,
As object to be measured DPAGT1, GPR125, CYP4B1, PTPRK, NEK10, NCAPD3, ABCA13, DNAH11, KDELC1 and
The expression of MTA3 this 10 genes is not higher than 3 times of non-EBOV the infected's corresponding gene, and the object to be measured is non-EBOV
The infected.
Tiny RNA s (sRNAs) related to virus load in embodiment 2, EBOV the infected's blood
Total serum IgE is extracted from EBOV the infected with non-EBOV the infected's whole blood, using 15% denaturing polyacrylamide gel
Electrophoretic techniquess (PAGE) separate small fragment (15-30nt) RNA from total serum IgE, after purification to detached small fragment RNA connection 3 ' and
5 ' end connectors, then the RNA for being connected with joint is inverted with SuperScriptII reverse transcription (Invitrogen products)
Record, synthesizes cDNA.Then, performing PCR amplification, amplification program are entered：98 DEG C of denaturations 30s, 98 DEG C of degeneration 10s, 60 DEG C of annealing 30s,
Thus 72 DEG C of extension 15s, 14 circulations, 72 DEG C of extension 8min build library.Finally, product utilization Illumina sequencings are extended
Platform carries out high-flux sequence.Joint sequence, the two ends low quality being sequenced in the initial data for producing is filtered using perl script
Sequence (sQ<=5 base number accounts for the sequence of more than the 50% of whole piece reads sequence, the ratio of N more than 10%, polyA/T/
G/C sequences).
SRNA valid data after screened by length compare people with reference on genome, analyze
Distribution and expression of the sRNAs in reference gene group, the sequence in comparison is compared with miRBase sequences, obtains
MiRNA information, while removing rRNA, tRNA, snRNA, snoRNA as far as possible.EBOV the infected (n is drawn with reference to sample information
=differential expression miRNA 12) compared with non-EBOV the infected (n=3), further by correlation analysiss, has found and EBOV
The related miRNA of virus infection.As a result show, 11 miRNAs and their target gene are related to EBOV virus loads (VL)
(p<0.01, differential expression multiple>3), this 11 miRNAs are respectively：hsa-miR-151a-5p,hsa-miR-744-5p,
hsa-miR-423-5p,hsa-miR-331-3p,hsa-miR-1306-5p,hsa-miR-1285-3p,hsa-miR-185-5p,
hsa-miR-550a-5p,hsa-miR-3940-3p,hsa-miR-574-5p,hsa-miR-941.MiRNAs is in high level (VL>
107Copy/mL whole bloods) and middle level (105Copy/mL whole bloods<VL<107Copy/mL whole bloods) virus load EBOV infection
Expression value in sample sees table 2 below, and this 11 miRNA in EBOV the infected relative in non-EBOV the infected
Expression declines.Concrete outcome as shown in table 2, shows that this 11 miRNA can be identified as biomolecule, infects for EBOV
The clinical diagnosises of person.
The related miRNA of table 2, EBOV infection
Note：List in table 2 11 miRNA expression (median TPM values) and with EBOV virus load dependencys
Analysis result.
In actual applications, can be by comparing tables of this 11 miRNA in object to be measured with non-EBOV the infected's blood
Judge up to level whether object to be measured is EBOV the infected.Specifically, the criterion for being obtained according to the above results is as follows：Such as
Hsa-miR-1285-3p, hsa-miR-1306-5p, hsa-miR-151a-5p, hsa-miR-185-5p, hsa- of object to be measured
miR-331-3p、hsa-miR-3940-3p、hsa-miR-423-5p、hsa-miR-550a-5p、hsa-miR-574-5p、hsa-
There is at least one expression corresponding less than non-EBOV the infected in miR-744-5p and hsa-miR-941 this 11 miRNA
The 1/3 of miRNA expressions, the such as doubtful EBOV the infected of the object to be measured, hsa-miR-1285-3p, hsa- of object to be measured
miR-1306-5p、hsa-miR-151a-5p、hsa-miR-185-5p、hsa-miR-331-3p、hsa-miR-3940-3p、
Hsa-miR-423-5p, hsa-miR-550a-5p, hsa-miR-574-5p, hsa-miR-744-5p and hsa-miR-941 this 11
The expression of individual miRNA is not less than the 1/3 of the corresponding miRNA expressions of non-EBOV the infected, and the object to be measured is non-
EBOV the infected.
The molecular marker related to clinical prognosis in embodiment 3, EBOV the infected's blood
Total serum IgE is extracted from EBOV the infected with non-EBOV the infected's whole blood, it is pure with the absorption of the magnetic bead with Oligo (dT)
Change the mRNA in total serum IgE, in a heated condition by mRNA fragmentations, and as template reverse transcription synthetic double chain cDNA.To double
Chain cDNA carries out end and repairs polishing so as to 5 ' end phosphorylations, and 3 ' ends add " A ".Double-strand cDNA joint with 3 ' dTMP ends is connected
To in sequence measuring joints, expanded by PCR, using AMPure XP enrichment with magnetic bead purification joint products, by PCR reaction identification texts
Storehouse.The library for building is carried out into double end sequencings with Illumina microarray datasets.Filter what sequencing was produced using perl script
Joint sequence, two ends low quality sequence (Q in initial data<=20 base number accounts for more than the 50% of whole piece reads sequence
Row), Sequences of Low Complexity.
Valid data are compared into people with reference on genome using Tophat softwares, Cutfflinks software combinations compare knot
Really, differential expression of the EBOV the infected (n=22) compared with non-EBOV the infected (n=10) is drawn by Cutffdiff softwares
Gene, further by correlation analysiss, finds the molecular marker related to EBOV virus the infected's prognosis.As a result show, 22
The differential expression of individual molecule (p related to EBOV virus infection prognosis<0.01, differential expression multiple>2), wherein, 15 genes are compiled
Code protein, its coding protein it is entitled：MS4A2(membrane spanning 4-domains A2),SYCN
(syncollin),LCE3E(late cornified envelope 3E),FJX1(four jointed box 1),ARMS2
(age-related maculopathy susceptibility 2),RMI2(RecQ mediated genome
instability 2),PHLDA2(pleckstrin homology like domain family A member 2),
OPRM1(opioid receptor mu 1),TGFBR3L(transforming growth factor beta receptor
3like),BEND5(BEN domain containing 5),TP53AIP1(tumor protein p53regulated
apoptosis inducing protein 1),SBK2(SH3domain binding kinase family member 2),
GJC2(gap junction protein gamma 2),LDHD(lactate dehydrogenase D),CHST1
(carbohydrate sulfotransferase 1)；7 is lincRNA, and this 7 lincRNA are specially：RP11-
552E20.4,RP4-758J18.10,RP11-142G7.2,RP5-1065P14.2,RP11-255A11.21,LINC00639,
LINC00092.And raise relative to expression in surviving populations in this dead colony of 22 molecules in EBOV the infected.
Concrete outcome as shown in table 3, shows that this 22 molecules can be identified as biomolecule, for the prognosis of EBOV the infected.
Table 3, the molecular marker of prognosis EBOV virus infection
Note：List in table 3 23 molecules expression (median FKPM values) and its with EBOV the infected's prognosis
Analysis result.
In actual applications, can be by analyzing this expression of 22 molecules in object blood to be measured come to object to be measured
Prognosis.Specifically, the criterion for being obtained according to the above results is as follows：It is when with EBOV the infected as object to be measured, such as to be measured
The MS4A2 of object, SYCN, LCE3E, FJX1, ARMS2, RMI2, PHLDA2, OPRM1, TGFBR3L, BEND5, TP53AIP1,
SBK2、GJC2、LDHD、CHST1、RP11-552E20.4、RP4-758J18.10、RP11-142G7.2、RP5-1065P14.2、
The expression for having at least one in RP11-255A11.21, LINC00639 and LINC00092 this 22 molecules is felt higher than EBOV
2 times of corresponding developed by molecule level in survival object in dye person, the object to be measured are doubtful for high mortality risk EBOV the infected,
As the MS4A2 of object to be measured, SYCN, LCE3E, FJX1, ARMS2, RMI2, PHLDA2, OPRM1, TGFBR3L, BEND5,
TP53AIP1、SBK2、GJC2、LDHD、CHST1、RP11-552E20.4、RP4-758J18.10、RP11-142G7.2、RP5-
The expression of 1065P14.2, RP11-255A11.21, LINC00639 and LINC00092 this 22 molecules is not higher than EBOV
2 times of corresponding developed by molecule level in survival object in the infected, the object to be measured is not high mortality risk EBOV the infected,
That is MS4A2, SYCN, LCE3E, FJX1, ARMS2, RMI2, PHLDA2, OPRM1, TGFBR3L, BEND5, TP53AIP1, SBK2,
GJC2、LDHD、CHST1、RP11-552E20.4、RP4-758J18.10、RP11-142G7.2、RP5-1065P14.2、RP11-
In 255A11.21, LINC00639 and LINC00092 this 22 molecules, the high EBOV the infected's of at least one expression is dead
Risk of dying higher than MS4A2, SYCN, LCE3E, FJX1, ARMS2, RMI2, PHLDA2, OPRM1, TGFBR3L, BEND5,
TP53AIP1、SBK2、GJC2、LDHD、CHST1、RP11-552E20.4、RP4-758J18.10、RP11-142G7.2、RP5-
The low EBOV senses of the expression of 1065P14.2, RP11-255A11.21, LINC00639 and LINC00092 this 22 molecules
Dye person.
The cytokine related to clinical prognosis in embodiment 4, EBOV the infected's blood
Total serum IgE is extracted from EBOV the infected with non-EBOV the infected's whole blood, it is pure with the absorption of the magnetic bead with Oligo (dT)
Change the mRNA in total serum IgE, in a heated condition by mRNA fragmentations, and as template reverse transcription synthetic double chain cDNA.To double
Chain cDNA carries out end and repairs polishing so as to 5 ' end phosphorylations, and 3 ' ends add " A ".Double-strand cDNA joint with 3 ' dTMP ends is connected
To in sequence measuring joints, expanded by PCR, using AMPure XP enrichment with magnetic bead purification joint products, by PCR reaction identification texts
Storehouse.The library for building is carried out into double end sequencings with Illumina microarray datasets.Filter what sequencing was produced using perl script
Joint sequence, two ends low quality sequence (Q in initial data<=20 base number accounts for more than the 50% of whole piece reads sequence
Row), Sequences of Low Complexity.
Valid data are compared into people with reference on genome using Tophat softwares, Cutfflinks software combinations compare knot
Really, differential expression of the EBOV the infected (n=22) compared with non-EBOV the infected (n=10) is drawn by Cutffdiff softwares
Gene, further by correlation analysiss, finds the cytokine related to virus infection prognosis.As a result show, 8 cells because
The differential expression of son (p related to virus infection prognosis<0.01, differential expression multiple>1.5), this 8 cytokines are respectively：
THPO(thrombopoietin),STC2(stanniocalcin 2),IL17D(interleukin 17D),SEMA3C
(semaphorin 3C),SEMA3F(semaphorin 3F),EDN2(endothelin 2),GDF10(growth
Differentiation factor 10), FGF20 (fibroblast growth factor 20), and this 8 cells because
Son is raised relative to expression in surviving populations in the dead colony in EBOV the infected.Concrete outcome as shown in table 4, shows
This 8 cytokines can be identified as biomolecule, for the prognosis of EBOV the infected.
Table 4, the cytokine of prognosis EBOV virus infection
Note：List in table 48 cytokines expression (median FKPM values) and its with EBOV the infected's prognosis
Analysis result.
In actual applications, can be by analyzing this expression of 8 cytokines in object blood to be measured come to be measured
Object prognosis.Specifically, the criterion for being obtained according to the above results is as follows：When with EBOV the infected as object to be measured, such as
In THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and FGF20 of object to be measured this 8 cytokines at least
There is the expression of higher than 150% of the corresponding Cytokine Expression Level of survival object in EBOV the infected, this is to be measured
Object is doubtful for high mortality risk EBOV the infected, the such as THPO of object to be measured, STC2, IL17D, SEMA3C, SEMA3F,
The survival object that the expression of EDN2, GDF10 and FGF20 this 8 cytokines is not higher than in EBOV the infected is accordingly thin
The 150% of intracellular cytokine expression, the object to be measured be not high mortality risk EBOV the infected, i.e. THPO, STC2, IL17D,
In SEMA3C, SEMA3F, EDN2, GDF10 and FGF20, the mortality risk of the high EBOV the infected of at least one expression is higher than
The low EBOV the infected of the expression of THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and FGF20.
The present invention also have chosen the blood of non-EBOV the infected, survival EBOV the infected and dead EBOV the infected in addition
Sample, the THPO genes and STC2 genes that have detected using quantitative RT-PCR technology in this 8 cytokines are right in these researchs
As the expression in blood.Wherein, the primer sequence of THPO genes is 5'-TCACTGCCTCAGCCAGAACTAC-3'(sequences
1) non-EBOV the infected 15, survival EBOV the infected 31 with 5'-GGTTCAGCAGACCAGGAATCTT-3'(sequences 2), are tested
Example, the blood sample of dead EBOV the infected 18；The primer sequence of STC2 genes is 5'-CACAGGTTCGGCTGCATAAG-
3) with 5'-AGTTCACGAGGTCCACGTAGG-3'(sequences 4) 3'(sequences, test non-EBOV the infected 15, survival EBOV senses
Dye person 26, the blood sample of dead EBOV the infected 14.
As a result find, expressions of the THPO and STC2 in survival EBOV the infected's blood is significantly lower than death EBOV
In the infected's blood, corresponding Cytokine Expression Level (table 5), shows, THPO and STC2 can be EBOV to be felt as mark
Dye person carries out prognosis.
The RT-PCR experimental results of STC2 and THPO in table 5, object of study blood
<110>Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL
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<170> PatentIn version 3.5
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<213>Artificial sequence
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<213>Artificial sequence
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Claims (9)
1. application of the system of G1 expressions in EBOV the infected's prognosis or auxiliary prognosis product is prepared is detected；The G1 is
THPO genes, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes, GDF10 genes and/or
FGF20 genes.
2. application of the system of G1 expressions in EBOV the infected's prognosis or auxiliary prognosis is detected；The G1 is THPO bases
Cause, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes, GDF10 genes and/or FGF20 genes.
3. application according to claim 1 and 2, it is characterised in that：The system of the detection G1 expressions is detection institute
State the reagent and/or instrument needed for G1 expressions.
4. following M1) or application M2)：
M1) detect application of the system of P1 contents in EBOV the infected's prognosis or auxiliary prognosis product is prepared；
M2) detect application of the system of the P1 contents in EBOV the infected's prognosis or auxiliary prognosis；
The P1 is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and/or FGF20 protein.
5. application according to claim 4, it is characterised in that：Detect the system of the P1 contents to detect the P1 contents
Required reagent and/or instrument.
6. following N1)-N4) in arbitrary application：
N1) using G1 as system used in EBOV the infected's prognosis of EBOV the infected's prognostic marker or auxiliary method of prognosis
In following a1) or a2) in application：
A1 EBOV the infected's prognosis or auxiliary prognosis product) are prepared；
A2) EBOV the infected's prognosis or auxiliary prognosis；
The G1 is THPO genes, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes, GDF10
Gene and/or FGF20 genes；
N2) using P1 as system used in EBOV the infected's prognosis of EBOV the infected's prognostic marker or auxiliary method of prognosis
In above-mentioned a1) or a2) in application；
The P1 is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and/or FGF20 protein；
N3) using the G1 as EBOV the infected's prognostic marker in above-mentioned a1) or a2) in application；
N4) using the P1 as EBOV the infected's prognostic marker in above-mentioned a1) or a2) in application.
7. following X1) or product X2)：
X1) EBOV the infected's prognosis or auxiliary prognosis product, are the system for detecting G1 expressions；
The G1 is THPO genes, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes, GDF10
Gene and/or FGF20 genes；
X2) EBOV the infected's prognosis or auxiliary prognosis product, are the system for detecting P1 contents；
The P1 is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and/or FGF20 protein.
8. according to arbitrary application or product described in claim 7 in claim 1-6, it is characterised in that：The G1 expression
Level is the expression of G1 described in blood；The P1 contents are the content of P1 described in blood.
The construction method of 9.EBOV the infected's prognosis related molecule characteristic spectrum, including：Respectively to EBOV the infected's survival group and
RNA in the dead group blood of EBOV the infected carries out quantitative analyses, obtains related point of EBOV the infected's prognosis according to analysis result
Subcharacter is composed；EBOV the infected's prognosis related molecule characteristic spectrum is G1 or P1；
The G1 is THPO genes, STC2 genes, IL17D genes, SEMA3C genes, SEMA3F genes, EDN2 genes, GDF10
Gene and/or FGF20 genes；
The P1 is THPO, STC2, IL17D, SEMA3C, SEMA3F, EDN2, GDF10 and/or FGF20 protein.
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