CN108866246B

CN108866246B - Diagnose the biomarker of childrens respiratory tract virus infection

Info

Publication number: CN108866246B
Application number: CN201811049148.9A
Authority: CN
Inventors: 李然然
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-09-10
Filing date: 2018-09-10
Publication date: 2019-06-04
Anticipated expiration: 2038-09-10
Also published as: CN108866246A

Abstract

The invention discloses the biomarkers of diagnosis childrens respiratory tract syncytial virus infection, and the biomarker can be with auxiliary diagnosis childrens respiratory tract syncytial virus infection.The present invention discloses the combination of at least two biomarkers relevant to childrens respiratory tract syncytial virus, the childrens respiratory tract syncytial virus can be used in combination with bioinformatics method, and the invention also discloses products relevant to biomarker.

Description

Diagnose the biomarker of childrens respiratory tract virus infection

Technical field

The invention belongs to biomedicine field, it is related to diagnosing the biomarker of childrens respiratory tract virus infection.

Background technique

Respiratory Syncytial Virus(RSV) (Respiratory syncytial virus, RSV) is clinically infant's respiration in recent years One of the important pathogen of road infection, which infects in the world, and has apparent season fashion trend, and winter-spring season is high Hair.RSV can cause upper respiratory tract infection symptoms (such as catching a cold), can also cause lower respiratory tract infection symptom (such as ramuscule gas Guan Yan and pneumonia etc.).It is shown according to the data of the Center for Disease Control (CDC), the children more than half infected before 1 years old RSV, by 2 years old, almost all of children had rsv infection at least once.25%~40% children have capillary bronchitis and pneumonia Symptom, 0.5%~2% children are because rsv infection needs hospitalization, and serious rsv infection person can lead to death.RSV Infection can adjust host gene expression, influence host anti-virus response and virus replication, but still lack effective base at present Because come the pathogenesis of analyzing childrens respiratory tract syncytial virus infection.

The fast development and application of high throughput sequencing technologies are the pathogenetic research of childrens respiratory tract syncytial virus infection It provides more comprehensively with quick analysis means, is also provided for the further therapeutic scheme of childrens respiratory tract syncytial virus infection Brand-new thinking.Rsv infection can cause systemic immune response.During rsv infection, immunocyte is existed by the transport of blood It is developed in circulation, into center and peripheral lymph node organ, finally moves to focus of infection.Peripheral blood is as storage immunocyte Place provides the material of whole reflection immunity-associated cell and signal path.Further investigate childrens respiratory tract syncytial virus The regulatory mechanism of infected related gene facilitates the genetic molecule mechanism for understanding childrens respiratory tract syncytial virus infection, for breathing The diagnosis and treatment of road syncytial virus infection provide theoretical foundation and clinical means.

Summary of the invention

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide can be used for diagnosing childrens respiratory tract virus sense The biomarker of dye, to realize the early diagnosis of childrens respiratory tract virus infection.

To achieve the goals above, the present invention adopts the following technical scheme:

The reagent that the first aspect of the present invention provides detection gene expression dose closes born of the same parents in preparation diagnosis childrens respiratory tract Application in virus infection product, the gene include the one or two of GYG1, LMNB1.

The reagent that the second aspect of the present invention provides detection gene expression dose closes born of the same parents in preparation diagnosis childrens respiratory tract Application in virus infection product, the gene include two or more of GYG1, LMNB1, GPR84 or IFI27.

Further, the reagent for detecting gene expression dose is selected from:

The probe of gene described in specific recognition；Or

The primer of gene described in specific amplification；Or

Specifically bind the specific binding agent of the albumen of the gene coding；

The third aspect of the present invention provides a kind of product, and the product of stating includes the reagent for detecting gene expression dose, The gene includes the one or two of GYG1, LMNB1.

The fourth aspect of the present invention provides a kind of product, and the product of stating includes the reagent for detecting gene expression dose, The gene includes two or more in GYG1, LMNB1, GPR84 or IFI27.

Further, the product includes chip, kit, nucleic acid film item.

Further, the reagent is selected from:

The probe of gene described in specific recognition；Or

The primer of gene described in specific amplification；Or

Specifically bind the specific binding agent of the albumen of the gene coding.

The fifth aspect of the present invention provides product described in third aspect present invention or fourth aspect present invention and is preparing Diagnose the application in the tool of childrens respiratory tract syncytial virus infection.

The sixth aspect of the present invention provides gene marker in the meter of building diagnosis childrens respiratory tract syncytial virus infection The application in model is calculated, the gene marker includes GYG1 or LMNB1.

The seventh aspect of the present invention provides gene marker in the meter of building diagnosis childrens respiratory tract syncytial virus infection The application in model is calculated, the gene marker includes two or more in GYG1, LMNB1, GPR84 or IFI27.

The advantages of the present invention:

Present invention firstly discovers that GYG1 and LMNB1 is to childrens respiratory tract syncytial virus infection related, it is tested by detecting The expression of said gene in person's peripheral blood, may be implemented the Accurate Diagnosis of childrens respiratory tract virus infection.

Present invention firstly discovers that several carry out Combining diagnosis in GYG1, LMNB1, GPR84, IFI27, with higher Specificity and sensibility and accuracy.

Detailed description of the invention

Fig. 1 is expression figure of the biomarker in respiratory syncytial virus infection children, wherein figure A is GYG1, Scheming B is LMNB1, and figure C is GPR84, and figure D is IFI27.

Specific embodiment

The present invention after extensive and in-depth study, by largely screening, has found close born of the same parents' disease in childrens respiratory tract for the first time The gene that differential expression is presented in malicious infected patient is that the early detection of childrens respiratory tract syncytial virus infection finds preferably way Diameter and method.

Gene marker

" gene marker " is also referred to as " molecular marker ", " biomarker ", is the expression water in tissue or cell The flat any gene or albumen to change compared with the expression of normal or healthy cell or tissue.

Herein comprising the methods availalbe any in the prior art for detection molecules marker expression.Molecular marker of the present invention The expression of object can be detected (e.g., RNA transcript) or protein level in nucleic acid level.It is intended to determine by " detection expression " The quantity or presence of the expression product of RNA transcript or its molecular marker gene.Therefore, " detection expression " includes a molecule mark Will object is determined to be expressed, cannot being detected expression, and expression is in low-level, expression in normal level or the reality of overexpression Example.It is overexpressed to determine, the detected body sample can be compared with the body sample accordingly from Healthy People.That That is " normal " level of the expression is the expression of molecular marker.This sample can be in normalized form It is existing.In some embodiments, the determination that molecular marker is overexpressed need to compare body sample and accordingly from the body of Healthy People Sample.

It would be recognized by those skilled in the art that practicability of the invention is not limited to marker gene of the invention The gene expression of any specific variants is quantified.

As unrestricted example, a kind of nucleotides sequence of representative people GYG1, LMNB1, GPR84, IFI27 gene Column such as NC_000003.12 (148991408..149027669), NC_ in current international public nucleic acid database GeneBank 000005.10(126776623..126837020)、NC_000012.12(54362445..54365224,complement)、 Shown in NC_000014.9 (94110733..94116699).

Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.

Polypeptide of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.Of the invention is more Peptide may also include or not include the methionine residues of starting.

The polynucleotides for encoding the mature polypeptide of albumen of the present invention include: the coded sequence of an encoding mature polypeptide； The coded sequence of mature polypeptide and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide And non-coding sequence.The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, can also be with It is the polynucleotides for further including additional code and/or non-coding sequence.

The invention further relates to the variant of above-mentioned polynucleotides, coding has the more of identical amino acid sequence with the present invention The segment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant naturally occurred or The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insertion variant.Such as this Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution, Missing or insertion, but not from substantially change its encode polypeptide function.

Detection method

The present invention can be used multiple nucleic acids and protein techniques known to persons of ordinary skill in the art and detect, this A little technologies include but is not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, protein immunization technology.

Nucleic acid amplification technologies of the present invention are selected from polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and be based on nucleic acid sequence The amplification (NASBA) of column.Wherein, PCR needs directly expand RNA reverse transcription at DNA (RT-PCR), TMA and NASBA before amplification Increase RNA.

In general, PCR uses the annealing of denaturation, primer pair and opposite strand and multiple circulations of primer extend, with index side The copy number of formula increase target nucleic acid sequence；Reverse transcriptase (RT) is then used for the DNA (cDNA) complementary from mRNA preparation by RT-PCR, Then cDNA is generated to multiple copies of DNA by PCR amplification；TMA is in substantially constant temperature, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, wherein multiple RNA copy of target sequence is autocatalytically given birth to At other copy, TMA is optionally included using blocking, part, terminate part and other modified parts, to improve TMA process Sensitivity and accuracy；LCR uses the two groups of complementary DNA oligonucleotides hybridized with the adjacent area of target nucleic acid.

DNA oligonucleotides is covalently attached in thermal denaturation, hybridization and the multiple circulations of the repetition of connection by DNA ligase, To generate detectable double-strand connection oligonucleotide product；SDA uses multiple circulations of following steps: primer sequence pair and target sequence The opposite strand of column is annealed, and carries out primer extend under there are dNTP α S to generate half thiophosphorylation of double-strand (hemiphosphorothioated) primer extension product, in the nucleic acid that semi-modified restriction enzyme enzyme recognition site carries out The nicking that enzyme cutting mediates, and the polymerase-mediated object drawn carried out from the end notch 3' extend to replace existing chain and generate under confession One takes turns the chain of primer annealing, nicking and strand displacement, expands so as to cause the geometry of product.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or Northern trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH is for measuring with position tissue slice or entirely MRNA and other transcripts (for example, ncRNA) in organization embedding.Usually sample cell and tissue are handled in situ solid Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes off extra probe.Point Not Shi Yong autoradiograph, fluorescence microscopy or immunohistochemistry, in tissue with radiation, fluorescence or antigenic mark base The probe of label is positioned and is quantified.ISH can also be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, to detect two or more transcripts simultaneously.

Southern and Northern trace is respectively used to detection specific DNA or RNA sequence.Make to extract from sample DNA or RNA fracture, it is separated by electrophoresis on matrix gel, be then transferred on molecular filter.Make filter combine DNA or RNA with and the complementary label probe of sequence of interest hybridize.Detection is integrated to the hybridization probe of filter.A kind of change of the program Change form is reverse northern trace, wherein the substrate nucleic acid for being fixed to film is the set of isolated DNA fragmentation, and probe is From tissue extraction and the RNA that is marked.

Protein immunization technology includes sandwich immunoassay, such as sandwich ELISA, wherein using identifying on biomarker not The detection of the biomarker is carried out with two kinds of antibody of epitope；Radiommunoassay (RIA), direct, indirect or comparison are enzyme-linked Immunosorbent assay (ELISA), enzyme immunoassay (EIA) (EIA), fluorescence immunoassay (FIA), immunoblotting, immuno-precipitation With the immunoassays based on any particle (as used gold particle, Argent grain or latex particle, magnetic-particle or quantum dot).It can example Such as implement immunization in the form of microtiter plate or item.

Immunization according to the present invention can be based on, for example, any one of following methods.

Immuno-precipitation is simplest method of immunity；The amount of this method measurement sediment, reagent antibodies With sample be incubated with and with its present in target antigen react to be formed after insoluble aggregate and form the precipitating.It is immune Precipitation reaction can be qualitative or quantitative.

In particle immunoassays, Multiple Antibodies are connect with the particle, and the particle can be in combination with many antigens Molecule.This has greatly speeded up the speed of visible reaction.This allows the quick and sensitive detection of biomarker.

In immunoturbidimetry (immunonephelometry), the phase interaction of the target antigen on antibody and biomarker With the formation for causing immune complex, the immune complex is too small and cannot precipitate.But these compounds will scatter incidence Light, nephometer can be used to measure in this.The concentration of antigen (i.e. biomarker) can be measured within a few minutes of reaction.

Radiommunoassay (RIA) method comes labelled antigen or antibody using radioactive isotope such as I125.It is used Isotope emits gamma-rays, and the ray is usually measured after removing uncombined (free) radioactive label.With it is other Immunoassays compare, the main advantage of RIA be higher sensitivity, easy signal detection and confirmation, quickly Measurement.Main disadvantage be the health and safety risk as caused by the use of radiation and with maintenance license radiation safety and Processing routine relevant time and expense.For this reason, routine clinical laboratory practice in, RIA largely by Replaced enzyme immunoassay (EIA).

Enzyme immunoassay (EIA) (EIA) development is the substitute of radiommunoassay (RIA).These methods are marked anti-using enzyme Body or target antigen.The sensitivity of EIA close to RIA sensitivity, and be not present the danger as caused by radioactive isotope.For examining Survey most widely used EIA method first is that enzyme linked immunosorbent assay (ELISA) (ELISA).Two kinds of antibody can be used in ELISA method, One is specific for target antigen, and another and enzyme is coupled, and the addition of zymolyte causes chemiluminescence signal or fluorescence to be believed Number generation.

Fluorescence immunoassay (FIA) refers to the immunoassays using fluorescent marker or enzyme label, the fluorescent marker or enzyme mark Be denoted as on substrate to form fluorescence-causing substance.Fluorescence measurement is inherently sensitiveer than colorimetric (spectrophotometry) measurement. Therefore, FIA method has sensitivity for analysis more higher than the EIA method measured using absorption (optical density).

Chemiluminescence immunoassay uses chemiluminescent labeling, and light is generated when it is excited by chemical energy；Use light detection Device measurement transmitting.

Therefore, method known to can be used carries out immunization according to the present invention.In the inspection of biomarker of the invention Any direct (as used sensor chip) or round-about way can be used in survey.

Probe

" probe " refers to the molecule that can be used in measuring the expression of specific gene.Exemplary probe includes PCR primer And gene specific DNA oligonucleotide probe, such as be fixed on microarray substrate micro probe array, quantitative nucleic acid enzyme protect Shield examines probe, the probe connecting with molecular barcode and the probe being fixed on pearl.

Term " probe ", which refers to, in the present invention to divide in conjunction with the particular sequence of another molecule or subsequence or other parts Son.Unless otherwise indicated, " probe " is often referred to match and another polynucleotides (often referred to as " target multicore by complementary base Thuja acid ") combine polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and with the probe lack sufficient sequence it is mutual The target polynucleotide of benefit property combines.Probe can make direct or indirect label, and range includes primer.Crossing system, including, but It is not limited to: solution phase, solid phase, mixed phase or in situ hybridization measuring method.

As probe, fluorescent marker, radio-labeled, biotin labeling etc. can be used, cancer detection is carried out with polynucleotides The label probe of label.Labeling method of polynucleotides itself is well known.Can check by the following method in sample whether There are subject nucleic acids: fixed subject nucleic acid or its amplified matter are hybridized with label probe, are washed, and then measurement with The label of solid phase binding.Alternatively, cancer detection polynucleotides can be also fixed, subject nucleic acid is hybrid with it, then application mark The detections such as note probe are incorporated into the subject nucleic acid in solid phase.In this case, the cancer detection multicore glycosides being incorporated into solid phase Acid is also referred to as probe.It the use of the method for polynucleotide probes measurement subject nucleic acid in this field is also well known.Can as follows into Row this method: connect polynucleotide probes and subject nucleic acid (preferably within ± 4 DEG C) at or near Tm Touching is washed, the label probe or the template nucleic acid in conjunction with solid phase probe for then measuring hybridization for hybridizing.

The polynucleotides used as probe be preferably sized to 18 or more nucleotide, more preferably 20 or The overall length or less of more nucleotide and coding region.As primer in use, the polynucleotides are preferably sized to 18 Or more nucleotide and 50 or more Oligonucleotide.These probes have mutual with the specific base sequence of target gene The base sequence of benefit.Here, so-called " complementation ", as long as hybridization, can not be complete complementary.These polynucleotides are usual Have 80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% relative to the specific base sequence Homology.These probes can be DNA, be also possible to RNA, furthermore it is possible to be logical in part of it or whole nucleotides Cross the polynucleotides that the artificial replacement nucleic acid such as PN, LNA, ENA, GNA, TNA obtains.

Chip, kit, nucleic acid film item

Term " chip " is also referred to as " array ", refers to the solid support of the nucleic acid comprising connection or peptide probes.Array is usual A variety of different nucleic acid or peptide probes comprising being connected to substrate surface according to different known locations.These arrays, also referred to as " microarray " can use mechanical synthesis methods or light guidance synthetic method usually to generate these arrays, and the light guidance is closed The combination of photolithography method and solid phase synthesis process is incorporated at method.Array may include flat surface, or can be pearl Son, gel, polymer surfaces, the fiber of such as optical fiber, glass or nucleic acid or peptide in any other suitable substrate.It can be with Certain mode carrys out array of packages, to allow to carry out the manipulation of diagnosis of global function device or other means.

" microarray " is that hybridised arrays original part is ordered in matrix, and the hybridised arrays original part such as polynucleotide is visited Needle (such as oligonucleotides) or bonding agent (such as antibody).The matrix can be solid matrix, for example, glass or silica Slide, pearl, fibre optics binder or semi-solid matrix, such as nitrocellulose filter.Nucleotide sequence can be DNA, RNA or Any arrangement therein.

Various probe arrays have been described in the literature and can be used in the context of the invention detecting may be with this paper The relevant marker of the phenotype.For example, DNA probe array chip or biggish DNA probe array chip (otherwise, Ke Yitong Cross and interrupt chip and obtain each individual chip) for one embodiment of the invention.DNA probe array chip generally comprises glass Glass chip placed high-density DNA probe (short dna segment) array thereon.These chips can respectively keep for example, about 6000 The DNA of ten thousand longer sample DNA sequences (for example, from individual or group, for example, including marker of interest) for identification Probe.It is carried out with the DNA probe group identification sample DNA on chip glass by DNA hybridization.When DNA sample and DNA probe array When hybridization, sample is incorporated into those of sample DNA sequence complementation probe.It is more steady by evaluation individual sample DNA and those probes Admittedly hybridize, it is possible to determine that known nucleic acid sequence whether there is in sample, thereby determine that the marker found in nucleic acid It whether there is.It can also be using this means by controlling hybridization conditions to allow to distinguish single nucleotide, for example, being used for SNP It identifies with the sample gene parting of one or more SNP and carries out ASH.It is multiple that array provides a kind of (or series winding) simultaneously detection The convenience embodiment of polymorphic marker.

In the present invention, nucleic acid film item includes substrate and the oligonucleotide probe that is fixed in the substrate；The substrate Can be any substrate suitable for immobilized oligonucleotide probe, for example, nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass, Silica gel chip, miniature magnetic bead etc..

In the present invention, kit can be used for detecting gene or albumen (GYG1, LMNB1, GPR84, IFI27) of the present invention Expression, comprising for genetic test and/or quantitative ligand, and/or chip of the invention.It is optional with kit Specification is together.

Kit includes one or more sterile chambers, such container can be box, ampoule, bottle, phial, pipe, Bag, pouch, blister package or other suitable vessel forms known in the art.Such container can by plastics, glass, Laminated paper, metal foil or the other materials suitable for container drug.

In the present invention, term " includes " is for referring to phrase " including but not limited to ", and with phrase " including but not limited to " It may be used interchangeably.

Specific binding agent

In a preferred embodiment of the invention, the concentration of the albumen of gene coding of the present invention is measured.In a reality Apply in scheme, in vitro by using specific binding agent from sample specific assay marker protein concentration.

Specific binding agent is the receptor of such as protein, the agglutinin of conjugated protein, the antibody for protein, needle To peptide antibody (peptidebody), the agent of bispecific dual combination or the bispecific antibody form of protein.Specific binding Agent at least has 10 to its correspondence target molecule⁷The affinity of mol/l.Specific binding agent preferably has its target molecule 10⁸Mol/l or further preferably 10⁹The affinity of mol/l.

The example of specific binding agent is peptide, peptide mimics, aptamer, spiegelmer, darpin, ankyrin repetition Albumen, Kunitz type domain, antibody, single domain antibody and monovalent antibody fragments.

In certain embodiments, specific binding agent is antibody or monovalent antibody fragments, preferably is selected from monoclonal antibody and spreads out Raw monovalent fragment.

Monovalent antibody fragments include but is not limited to Fab, Fab '-SH, single domain antibody, Fv and scFv segment, offer as follows 's.

Term " antibody " clearly covers monoclonal antibody, polyclonal antibody, from extremely herein with broadest use The multi-specificity antibody (such as bispecific antibody) and antibody fragment that few two kinds of complete antibodies are formed, as long as they show the phase The biological activity of prestige.In certain preferred embodiments, specific binding agent is antibody or monovalent antibody fragments, preferably is selected from list Monovalent fragment derived from clonal antibody.

" monoclonal antibody " refers to the antibody that the antibody from a group substantially homogeneity obtains, that is, constitutes each antibody phase of group Together and/or same epitope is combined, during producing monoclonal antibody other than issuable possibility variant, such variant one As with indivisible presence.Such monoclonal antibody is typically include the antibody comprising the polypeptide sequence in conjunction with target, wherein target It in conjunction with polypeptide sequence is obtained by the process including selecting single target combination polypeptide sequence in the more peptide sequence of comforming 's.

Monoclonal antibody further includes " chimeric " antibody, wherein a part of heavy chain and/or light chain be derived from particular species Or the corresponding sequence belonged in the antibody of specific antibodies classification or subclass is identical or homologous, and the remainder of chain with derived from another One species belong to that corresponding sequence in the antibody of another antibody isotype or subclass is identical or homologous and the piece of such antibody Section, as long as they show desired biological activity.

" humanization " form of inhuman (such as mouse) antibody refers to that bottom line includes the sequence derived from non-human immunoglobulin The gomphosis immunoglobulins of column, immunoglobulin chain or its segment such as Fv, Fab, Fab ', F (ab ')²Or antibody is other anti- Original combines subsequence.

" antibody fragment " includes a part of full length antibody, usually its antigen binding domain or variable region.Antibody fragment Example includes Fab, Fab ', F (ab ')²With Fv segment；Double antibody；Linear antibodies；Single-chain antibody molecules；And by antibody fragment shape At multi-specificity antibody.

" Fv " is the minimum antibody fragment comprising intact antigen identification and binding site.The segment is by close, non-covalent knot The dimer composition of the heavy chain variable domain and a light-chain variable domain that close.It is distributed from the foldable structure of the two structural domains Six hypervariable loops (heavy chain and each 3 rings of light chain) out, facilitate the amino acid residue in conjunction with antigen and assign antibody with antigen binding Specificity.However, even single variable domain (or half of Fv only comprising three CDR to antigen-specific) can also have knowledge Ability that is other and combining antigen, only affinity is lower than entire binding site.

" functional fragment " of antibody of the invention refers to that those retain and derive their complete full chain molecule with substantially Identical affinity combination polypeptide and it is active at least one measuring method (such as inhibit TH2 induction asthma approach, such as In mouse model, or in vitro inhibit antibody fragment combine antigen biological activity) segment.

Polyclonal antibody includes that antibody obtained by the protein is immunized to the animal (for example, mouse) for generating human antibody. After being prepared for chimeric antibody or humanized antibody, the amino acid in variable region (for example, FR) and/or constant region can be used Other amino acid substitutions etc..

Marker combination

The present invention relates to the purposes that gene marker carries out assessment childrens respiratory tract syncytial virus infection, such marker packets Include the one or more of GYG1, LMNB1, GPR84, IFI27.

As those of skill in the art can understand, improved there are many mode using the measurement of two or more markers Diagnosis problem in investigation.

Biochemical marker can measure individually, or in one embodiment of the invention, they can be measured simultaneously, Such as using chip or based on the array technique of pearl.Then the independent concentration for interpreting biomarker, such as indicated using every kind Individual retentions of object or their combinations are interpreted.

As those of skill in the art can understand, can be implemented in various ways and realize by marker levels and certain can The step of energy property or risk association get up.Preferably, the mathematically survey of combined protein matter and one or more other markers Determine concentration, and combined value is associated with basic diagnosis problem.Any suitable prior art mathematical method can be passed through It combines the measurement of marker levels.

Preferably, the mathematical algorithm applied in marker combination is a kind of logarithmic function.Preferably, using such mathematics Algorithm or such logarithmic function the result is that single value.According to basic diagnosis problem, can easily by such value with it is for example a Body about childrens respiratory tract syncytial virus infection risk or with help to assess childrens respiratory tract syncytial virus infection patient Other intentional diagnostic uses associate.In an advantageous manner, such logarithmic function obtains as follows: a) by individual point Class enters group, for example, normal person, have childrens respiratory tract syncytial virus infection risk individual, there is the sense of childrens respiratory tract syncytial virus Patient of dye etc., b) marker of the significant difference between these groups, c are identified by univariate analysis) logarithm regression point Analysis is to assess the independent difference values that can be used for assessing these difference groups of marker, and d) to carry out composition independency poor for building logarithmic function It is not worth.In such analysis, marker is no longer independent, but represents a marker combination.

Logarithmic function for marker combination to be got up with disease association is preferably developed using by applied statistical method With the algorithm of acquisition.For example, suitable statistical method is discriminant analysis (DA) (i.e. linear, secondary, regular DA), Kernel method (i.e. SVM), nonparametric technique (i.e. k- nearest neighbor classifiers), PLS (partial least square), method (the i.e. logic based on tree Recurrence, CART, random forest method, boosting/bagging method), generalized linear model (i.e. logarithm regression), the side based on principal component Method (i.e. SIMCA), broad sense Additive Model, the method based on fuzzy logic, the method based on artificial neural network and genetic algorithms.Skillfully Technical staff merges in the suitable statistical method of selection to assess marker group of the invention thus to obtain suitable mathematical algorithm Aspect will not be problematic.In one embodiment, for obtaining number used in assessment childrens respiratory tract syncytial virus infection The statistical method for learning algorithm is selected from DA (i.e. linear, secondary, rule based judgment analysis), Kernel method (i.e. SVM), nonparametric technique (i.e. k- nearest neighbor classifiers), PLS (partial least square), method (i.e. logistic regression, CART, random forest based on tree Method, propelled method) or generalized linear model (i.e. logarithm regression).

Area (=AUC) is the Xiang Zhibiao for diagnosing the performance or accuracy of regulation under receiver operating curve.Diagnosis side The accuracy of method describes best by its recipient's operating characteristics (ROC).ROC figure is derived from the entire data area in observation The line chart of the upper continuous all sensitivity/specificities pair for changing decision threshold.

The clinical performance of laboratory test depends on its diagnosis accuracy, or subject is correctly classified into clinical related The ability of subgroup.The ability of the different situations of correct two kinds for distinguishing investigated subject of diagnosis accuracy measurement test.It is such Situation is such as health and disease or progression of disease to no progression of disease.

In each case, ROC line chart by the entire scope for decision threshold by sensitivity to 1- specificity draw come Describe overlapping between two kinds of distributions.It is that sensitivity or true-positive fraction [are defined as (number of true positives test result in y-axis Mesh)/(number+false negative test results numbers of true positives)].This is also referred to as the existing positive of disease or situation.It is only Only calculated from impacted subgroup.Be in x-axis false-positive fraction or 1- specificity [be defined as (numbers of false positive results)/ (number+false positive results number of true negative)].It is a Xiang Zhibiao of specificity, and completely from impregnable Asia Group calculates.Because true and false fraction positive is by using the completely separable calculating of test result from two different subgroups, institute Popularity degree with ROC line chart independent of disease in sample.Each point on ROC line chart represents one and corresponds to specific decision The sensitivity of threshold/1- specificity is right.One test with perfect differentiation (two kinds of distribution of results do not overlap), which has, passes through a left side The ROC line chart at upper angle, there true-positive fraction are 1.0 or 100% (perfect sensitivity), and false-positive fraction is 0 (perfect special It is anisotropic).The assumption diagram of one test for not distinguishing (distribution of results of two groups is identical) is 45 ° from the lower left corner to the upper right corner Diagonal line.Most of line charts are fallen between these two extremes.If (ROC line chart entirely fall within 45 ° of diagonal lines hereinafter, so this It is easy to correct by the way that the standard of " positive " is inverted to " being less than " or vice versa from " being greater than ".) qualitatively, line chart is closer to upper left The whole accuracy at angle, test is higher.

A convenient target for quantifying the diagnosis accuracy of laboratory test is that its performance is stated by single numerical value. The most common global measurement is area under ROC curve (AUC).Routinely, this area is always >=0.5 (, if it is not, so Decision rule can be overturned to be allowed in this way).Numberical range (test values that perfection separates two groups) and 0.5 (two between 1.0 Without obvious distributional difference between the test value of group) between.Area depends not only on the specific part of line chart such as closest to right Sensitivity at the point of linea angulata or 90% specificity, but also depend on entire line chart.This is the how close perfection person of ROC line chart The one kind of (area=1.0) is quantitative, descriptive statement.

Whole measuring method sensitivity can depend on implementing the specificity that method disclosed herein requires.It is preferably provided with certain In, specificity 75% may be sufficient, and statistical method and gained algorithm can be based on this specific requirements.It is excellent at one Select in embodiment, for assess have childrens respiratory tract syncytial virus infection risk individual method be based on specificity 80%, 85% or further preferably 90% or 95%.

Certain marker combinations can be advantageous in screening childrens respiratory tract syncytial virus infection.

In one embodiment, this invention address that assessing childrens respiratory tract syncytial virus infection by biochemical marker In-vitro method, including one of GYG1, LMNB1, GPR84, IFI27 or a variety of in measurement sample, wherein significant difference in The presence of reference concentration instruction childrens respiratory tract syncytial virus infection.

As preferred embodiment, the gene marker be GYG1, LMNB1, GPR84 one or more with The combination of IFI27；In a preferred embodiment, the gene marker is GYG1 and IFI27；It is preferred at another In embodiment, the gene marker is LMNB1 and IFI27；In yet another embodiment, the gene marker is GPR84 and IFI27；In yet another embodiment, the gene marker is GYG1, LMNB1 and IFI27；In another reality It applies in scheme, the gene marker is GYG1, GPR84 and IFI27；In yet another embodiment, the gene marker For LMNB1, GPR84 and IFI27；In yet another embodiment, the gene marker be GYG1, LMNB1, GPR84 and IFI27。

Below with reference to embodiment, the present invention is described in further detail.

Following embodiment is only illustrative of the invention and is not intended to limit the scope of the invention.

Embodiment 1 screens gene marker relevant to childrens respiratory tract syncytial virus infection

1, sample collection

Each blood sample for collecting 5 normal child's blood and childrens respiratory tract syncytial virus infection patient, writes sample exactly The acquirement of situations such as title, number, Date of Sampling, sample process process, above-mentioned all samples pass through the same of Ethics Committee Meaning.

2, the preparation of RNA sample

RNA sample is extracted using the blood rna extracts kit of Invitrogen, concrete operations are detailed in specification.

3, the quality analysis of RNA sample

The RNA of said extracted is subjected to agarose gel electrophoresis, using Nanodrop2000 to the concentration of mentioned RNA and pure Degree is detected, and agarose gel electrophoresis detects RNA integrality, and Agilent2100 measures RIN value.Single requirement for construction data base RNA is total 5ug is measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.

4, construction cDNA library

The rRNA in total serum IgE is removed using Ribo-Zero kit, utilizes Illumina TruseqTM RNA Sample Prep Kit carries out the building of cDNA library, and concrete operations by specification carries out.

5, upper machine sequencing

CDNA library is sequenced using Illumina x-ten microarray dataset, concrete operations by specification carries out.

6, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out to sequencing result, carries out the positioning of RNA-seq read using TopHat v1.3.1, it will Valid data comparison is examined on genome to ginseng, is analyzed using metaMA packet, and p value merging method therefor is in meta analysis inverse normal method.When FDR<0.05, | Combined.ES |>0.8, it is believed that the expression of gene significant difference

7, result

Sequencing result shows that GYG1, LMNB1, GPR84 and IFI27 present aobvious in childrens respiratory tract patients with viral infections The up-regulation of work property.

The differential expression of embodiment 2QPCR sequence verification gene

1, large sample QPCR verifying is carried out to difference expression gene according to the testing result of high-flux sequence.According to embodiment Sample collection mode in 1 selects blood samples of patients 45 and normal child's blood 18 of respiratory syncytial virus infection.

2, RNA is extracted

3, reverse transcription:

It is operated using the reverse transcription reagent box of TAKARA company.Specific step is as follows:

(1) it takes 2 μ g of total serum IgE to carry out reverse transcription, Oligo (dT) 2 μ l is added, mixes well.70 DEG C of water-baths are after five minutes immediately Ice bath 2-3 minutes.

(2) 25 μ l reaction systems are constructed, including 5 × RT Buffer 5 μ l, dNTP (2.5mM) 5 μ l, RNasin 40U/ μ l, M-MLV 200U/ μ l mends nuclease free water to anticipated volume.

(3) 42 DEG C of water-baths after sixty minutes, 95 DEG C water-bath 5 minutes to inactivate M-MLV.

(4) -20 DEG C store for future use.

4, QPCR is expanded

(1) design of primers

QPCR amplimer is designed according to the coded sequence of gene in Genbank and GAPDH gene, by the raw work biology in Shanghai The synthesis of engineering services Co., Ltd.Specific primer sequence is as follows:

GYG1 gene:

Forward primer is 5 '-ACTGCTGGTCGCTTACAC-3 ' (SEQ ID NO.1)；

Reverse primer is 5 '-TGCTGCTGACAATTCTTCTCT-3 ' (SEQ ID NO.2)；

LMNB1 gene:

Forward primer is 5 '-GAGTAGTAGTGTTAGCATCT-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-TGTTCTTCAAGCGGATAA-3 ' (SEQ ID NO.4)；

GPR84 gene:

Forward primer is 5 '-GAAGGTGACTCGAATGTG-3 ' (SEQ ID NO.5)；

Reverse primer is 5 '-ATGTTGAGCAGCAAGAAG-3 ' (SEQ ID NO.6)；

IFI27 gene:

Forward primer is 5 '-ACTGGGAGCAACTGGACT-3 ' (SEQ ID NO.7)；

Reverse primer is 5 '-CAATGACAGCCGCAATGG-3 ' (SEQ ID NO.8)；

GAPDH gene:

Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.9)；

Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.10).

(2) PCR reaction system: forward primer and each 0.6 μ l, 2 × SuperReal PreMix Plus, 10 μ of reverse primer L, DNA profiling 2 μ l, ddH₂7.4 μ l, 50 × ROX Reference Dye of O^△2 μ l, 4.8 μ l of sterile purified water.

(3) PCR reaction condition: 95 DEG C of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C of 15s, 60 DEG C of 60s, 95 DEG C of 15s.PCR reaction is carried out on 7300 type fluorescence quantitative PCR instrument of ABI, passes through melt curve analysis analysis and electrophoresis Determine that purpose band, Δ Δ CT method carry out relative quantification.

5, statistical method

Experiment is tested using 3 repetitions, and result data is indicated in a manner of mean+SD, is used SPSS13.0 statistical software is next for statistical analysis, and difference between the two is examined using t, it is believed that has system as P < 0.05 Meter learns meaning.

6, result

As a result as shown in Figure 1, compared with normal child, GYG1, LMNB1, GPR84 and IFI27 are in Respiratory Syncytial Virus(RSV) Expression in infected children blood is significantly raised, and difference has statistical significance (P < 0.05), consistent with RNA-sep result.

The ROC of 3 difference expression gene of embodiment is analyzed

4 genes for screening differential expression above are combined, using area under ROC curve method and calculated curve (AUC) to evaluate significant influence factor for the diagnostic value of childrens respiratory tract syncytial virus infection, AUC and its 95% can Letter section is assessed by MedCalc, can be divided into no estimated performance (AUC < 0.5), low prediction according to AUC value difference Performance (0.5≤AUC≤0.7), moderate estimated performance (0.7≤AUC≤0.9) and high estimated performance (0.9≤AUC≤1), P < 0.05 is statistically significant for difference.

The results are shown in Table 1, and gene association checkout and diagnosis childrens respiratory tract syncytial virus infection area under the curve is 0.878 ~0.986；Prompting GYG1, LMNB1, GPR84 and IFI27 to be applied to diagnosis childrens respiratory tract syncytial virus infection has compared with Gao Zhun True property.

The area under the curve of 1 difference expression gene of table

Gene	AUC
		GYG1	0.895
LMNB1	0.878
		GRP84	0.907
IFI27	0.941
		GYG1+LMNB1	0.896
GYG1+GRP84	0.916
		GYG1+IFI27	0.979
LMNB1+GRP84	0.919
		LMNB1+IFI27	0.968
GRP84+IFI27	0.986
		GYG1+LMNB1+GRP84	0.924
GYG1+LMNB1+IFI27	0.975
		GYG1+GRP84+IFI27	0.982
LMNB1+GRP84+IFI27	0.977
		GYG1+LMNB1+GRP84+IFI27	0.974

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

Sequence table

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<213>artificial sequence (Artificial Sequence)

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gagtagtagt gttagcatct 20

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tgttcttcaa gcggataa 18

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gaaggtgact cgaatgtg 18

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atgttgagca gcaagaag 18

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actgggagca actggact 18

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caatgacagc cgcaatgg 18

<210> 9

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tttaactctg gtaaagtgga tat 23

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ggtggaatca tattggaaca 20

Claims

1. application of the reagent of gene expression dose in preparation diagnosis childrens respiratory tract syncytial virus infection product is detected, it is special Sign is that the gene includes the one or two of GYG1, LMNB1.

2. application of the reagent of gene expression dose in preparation diagnosis childrens respiratory tract syncytial virus infection product is detected, it is special Sign is that the gene includes two or more of GYG1, LMNB1, GPR84 or IFI27.

3. application according to claim 1 or 2, which is characterized in that the reagent is selected from:

The probe of gene described in specific recognition；Or

The primer of gene described in specific amplification；Or

Specifically bind the specific binding agent of the albumen of the gene coding.

4. a kind of application of product in the tool of preparation diagnosis childrens respiratory tract syncytial virus infection, which is characterized in that described Product includes the reagent for detecting gene expression dose, and the gene includes the one or two of GYG1, LMNB1.

5. a kind of application of product in the tool of preparation diagnosis childrens respiratory tract syncytial virus infection, which is characterized in that described Product includes the reagent for detecting gene expression dose, the gene include two kinds in GYG1, LMNB1, GPR84 or IFI27 or It is a variety of.

6. application according to claim 4 or 5, which is characterized in that the product includes chip, kit, nucleic acid film item.

7. application according to claim 6, which is characterized in that the reagent is selected from:

The probe of gene described in specific recognition；Or

The primer of gene described in specific amplification；Or

Specifically bind the specific binding agent of the albumen of the gene coding.