CN104945482A - Polypeptide, detection device comprising polypeptide, and detection kit comprising device - Google Patents
Polypeptide, detection device comprising polypeptide, and detection kit comprising device Download PDFInfo
- Publication number
- CN104945482A CN104945482A CN201410852500.8A CN201410852500A CN104945482A CN 104945482 A CN104945482 A CN 104945482A CN 201410852500 A CN201410852500 A CN 201410852500A CN 104945482 A CN104945482 A CN 104945482A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- detection
- present
- detection kit
- detection means
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a polypeptide, a detection device comprising the polypeptide, and a diagnostic kit comprising the detection device. Satisfactory effects can be achieved by applying the polypeptide, the detection device and the diagnostic kit to early screening and clinical auxiliary diagnosis of an EV71 (human enterovirus 71).
Description
Technical field
The invention belongs to field of biological detection, particularly can be used for the polypeptide of the early screening of enterovirns type 71 and clinical assistant diagnosis, comprise the detection kit of the detection means of this polypeptide.
Background technology
Enterovirns type 71 IgM belongs to enterovirus genus A type, and enterovirus, generally with numerical designation, puts in order and represents its precedence found.In order, this virus is named as enterovirns type 71 (Human enterovirus 71), and being called for short EV71, is cause Children (hand-foot and mouth disease; HFMD) one of main pathogens.Mankind's enteric virus71 type to suffer from from California first in 1969 and separates the infant faeces sample of central nervous system disease.
EV71 mainly causes hand foot mouth disease, also can cause the multiple nervous system disorderss such as the paralysis of aseptic meningitis, BBE and poliomyelitis sample.The two large common symptoms that hand foot mouth disease and central nervous system infection are EV71 infection and cause.Virus invades from pharyngeal or enteron aisle, breeds, and by locally discharging, now can cause local symptom in local mucous membrane or Lymphoid tissue.Then virus invades regional nodes again, and enter thus blood circulation cause first time viremia.Virus invades place's amount reproductions such as reticuloendothelium, deep layer lymphoglandula, liver, spleen, marrow through circulation of blood and enters blood circulation thus, causes second time viremia.Virus can enter each organ of whole body with blood flow, as places such as central nervous system, skin mucosa, hearts, breeds and causes pathology further.After susceptible person infects EV71, there is blood vessel transformation reactions and tissue inflammation pathology.When virus involves central nervous system, tissue inflammation comparatively neurotoxic effect is more strong, and central nervous system thin vessels endothelium is vulnerable to infringement most.Cytogamy, vasculitic become, thrombosis can cause ischemic and infarct.In the local organization of funiculi of spinal cord, brain stem, diencephalon, brain and cerebellum, except Neural invasion effect, also there are around blood vessel and parenchyma inflammation road virus 71 type IgM widely very harmful, being badly in need of simple and efficient instrument accurately is at present realize the rapid detection to it.
Market is detected enterovirns type 71 (IgM) and adopt enzyme-linked immunologic detecting kit more, but although test kit detects detection limit lower (0.15mg/L), but detection time is long, be not suitable in situ measurement, Chinese Patent Application No. is the application for a patent for invention of 201010171862.2, a kind of enterovirns type 71 antibody (IgM) diagnostic kit (euzymelinked immunosorbent assay (ELISA)) is proposed, it is front that this test kit detects enterovirns type 71 antibody (IgM), after all need to do necessary instrument cleaning and maintenance work, detection time is long, and require professional and technical personnel in the field to operate.
Summary of the invention
The object of the present invention is to provide a peptide species, comprise the detection means of this polypeptide and comprise the detection kit of this detection means, they to the early screening of enterovirns type 71 and clinical assistant diagnosis useful.The object of the invention is to provide this polypeptide to can be used for the purposes in the detection kit of enterovirns type 71 diagnosis in preparation.
That is, the present invention comprises following technical proposals:
1. a peptide species, its aminoacid sequence as shown in SEQ ID NO:1, that is: FTEMAAPLKSPSAEACGYSD.
2. a detection means, it comprises:
Solid carrier, and
Be connected to the polypeptide according to claim 1 on this solid carrier.
3. detection means according to claim 2, wherein, described solid carrier is SJ modified silica-gel.
4. detection means according to claim 2, its early screening for enterovirns type 71 and clinical assistant diagnosis.
5. a detection kit, it comprises the detection means according to any one of claim 2 ~ 4.
6. detection kit according to claim 5, its early screening for enterovirns type 71 and clinical assistant diagnosis.
7. the purposes of polypeptide according to claim 1 in preparation detection kit.
8. purposes according to claim 7, wherein, described detection kit is used for early screening and the clinical assistant diagnosis of enterovirns type 71.
9. purposes according to claim 7, wherein, described detection kit comprises the detection means according to any one of claim 2 ~ 4.
Apply the present invention to early screening and the clinical assistant diagnosis of enterovirns type 71, gratifying effect can be obtained.
Accompanying drawing explanation
The schematic diagram that the making processes of Fig. 1 to SJ modified silica-gel (iPDMS film) is described.
Fig. 2 illustrates the schematic diagram of polypeptide microarrays spot sample mode.
Fig. 3 is the photo of display to the result that healthy normal people or non-bowel virus 71 type infected patient serum detect.
Fig. 4 is the photo of display to the result that enterovirns type 71 patient infection serum detects.
The embodiment of invention
Polypeptide of the present invention
In one aspect of the method, the invention provides to the early screening of enterovirns type 71 and the useful polypeptide of clinical assistant diagnosis, be made up of the aminoacid sequence shown in SEQ ID NO:1, that is: FTEMAAPLKSPSAEACGYSD.As shown in the Examples, the serum of polypeptide of the present invention to enterovirns type 71 infected patient is positive, and to be negative reaction to healthy normal people or non-bowel virus 71 type infected patient serum.Therefore, polypeptide of the present invention is useful as the early screening of enterovirns type 71 and clinical assistant diagnosis instrument.Polypeptide of the present invention may be used for manufacturing detection means (example detection means of the present invention described as follows) or the detection kit (example detection kit of the present invention described as follows) of early screening and the clinical assistant diagnosis that can be used for enterovirns type 71.
Polypeptide of the present invention can be suitable for adopting the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method to obtain, but is not limited thereto, and wherein chemosynthesis is more easy.In chemosynthesis polypeptide situation of the present invention, undertaken by using peptide synthesizer synthesis or this polypeptide semi-synthetic.As chemical synthesis process, such as peptide solid-phase synthesis etc. can be listed.The peptide of such synthesis can adopt conventional means such as ion-exchange chromatography, reverse phase high performance liquid chromatogram, affinity chromatography etc. to carry out purifying.Such peptide solid phase synthesis process with and subsequent peptide purification be all well-known in the art.
In addition, when producing polypeptide of the present invention by enzyme reaction, the method such as described in No. WO2004/011653, International Publication brochure can be adopted.Namely; can produce like this: by the amino acid of a side or the C-terminal of dipeptides is esterified or amidation and the amino acid that obtains or dipeptides, the amino acid (amino acid of such as carboxy protective) that is in unbound state with amino acid react under the existence of peptide synthetase, the dipeptides of generation or tripeptides.As peptide synthetase, can list: there is the culture of microorganism of the ability generating peptide, the bacterial disposing thing of the microbial cells be separated by this culture or this microorganism or this microbe-derived peptide synthetase.
And, except above-mentioned enzyme method, chemical synthesis process, such as gene engineering method can also be adopted to produce polypeptide of the present invention.
The aminoacid sequence of polypeptide of the present invention can adopt conventional method to carry out analyzing and confirming, such as, can utilize the method such as mass spectrum, chromatogram to carry out and analyze and confirm.
Detection means of the present invention
In another aspect, the present invention also provides a kind of detection means (detection means of the present invention), and it comprises solid carrier and the protein of the present invention that is connected on this solid carrier or polypeptide of the present invention.Described detection means is useful for the early screening of enterovirns type 71 and clinical assistant diagnosis.
In the present invention, solid carrier is not particularly limited, as long as the carrier of solid or insoluble material (be such as can pass through filtration, material that precipitation, magnetic resolution etc. are separated from reaction mixture).
The material forming solid carrier includes but not limited to: silica gel (polydimethylsiloxane, PDMS), Mierocrystalline cellulose, teflon TM, Nitrocellulose, agarose, dextran, chitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polymeric amide, polypropylene, nylon, poly(vinylidene fluoride), latex, silicon-dioxide, glass, glass fibre, gold, platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer, polyethylene, polymine, poly(lactic acid), resin, polyose, albumen (albumin etc.), carbon or their combination etc.
The shape of solid carrier comprises but is not limited to: pearl, magnetic bead, film, microcapillary, filter membrane, plate, micro plate, carbon nanotube, sensor chip etc.Just as known in the art, the solid carrier that film or plate etc. are smooth can be arranged bottom pit, groove, filter membrane etc.
In the present invention, magnetic bead can have the sphere diameter of about 25nm ~ about 1mm scope.In a preferred embodiment, magnetic bead has the diameter of about 50nm ~ about 10 μm scope.The size of magnetic bead can be selected according to specific purposes.
In the present invention, the pearl be made up of the contour crosslinked spherical agarose of Sepharose has the diameter of about 24 μm ~ about 165 μm of scopes.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm ~ about 44 μm of scopes.The size of high crosslinked spherical sepharose 4B can be selected according to specific purposes.
The example with the solid carrier of water repellent surface comprises can from Polysciences, Warrington, PA or Spherotech, the polystyrene latex beads such as the goods that Liberville, IL buy.
The example of the solid carrier of silicon-dioxide (SiO2)-process or silicon-dioxide (SiO2) base comprises can from Polysciences, Warrington, the extraordinary magnetic silica pearl etc. that PA buys, it may be used for catching nucleic acid (such as DNA).Or, the M-280 etc. that can buy from Dynal Biotech can also be used.
The magnetic bead with hydrophilic surface can be used for the bacterial cell of seizure proliferation period, nucleic acid and other composition.As the example of this magnetic bead, Polysciences can be listed, Warrington, the pearl (title: Biomag (registered trademark) carboxyl) that PA sells or Bangs Laboratory, Inc., the name of Fishers, IN is called the pearl of MC02N/2928.Or, the M-270 etc. that Dynal Biotech sells can be used.
In a preferred embodiment of the present invention, described solid carrier is SJ modified silica-gel, it is the microarray solid support material (iPDMS film, see the open CN101265329A of Chinese patent) of a kind of silicon rubber material of Suzhou Siju Biomaterials Co., Ltd.'s exploitation.This material is based on the conventional PDMS of biological study, add specific initiator composition (making this material realize surface-functionalized modification by surface initiated polymerization (SIP)) wherein, obtain through polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethylene glycol) methacrylate), pOEGMA) finishing again.SJ modified silica-gel has outstanding anti-protein non-specific adsorption (Nonspecific protein adsorption, NPA) ability, non-specific protein absorption in complicated protein immunization can being detected controls to close to " absolute 0 " level (being near or below the limit of detection of instrument), not only can exempt the trouble closed and repeatedly clean, can also by the susceptibility using stronger amplification of signal means to improve protein microarray.And the essence of its silicon rubber imparts the stronger mechanical property of this material and good operability.Suzhou Yvonne gathers the combination assay microarray ELISA kit successfully SJ modified silica-gel being applied to 11 tumor markers compositions, achieve high-throughput and high-sensitive detection, demonstrating this material is a kind of outstanding protein microarray solid support material.Meanwhile, this material also has the adjustable characteristic of surface properties, can adjust its surface topography within the specific limits by the controlled modification reaction times.
The connection of polypeptide of the present invention and solid carrier can adopt the method for attachment that well known to a person skilled in the art polypeptide and solid carrier to carry out.Such as, for the connection on protein/polypeptide and modified silica-gel surface, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide [1-ethyl-3-(3-dimethyl ami-nopropyl) carbodiimide can be passed through, EDC] and N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) reaction changes the carboxyl (-COOH) group on the macromolecular chain of modified silica-gel surface into activating group, this activating group can with protein/polypeptide on amino (-NH2) react thus realize protein/polypeptide being fixed on solid carrier surface (see such as Hongwei Ma, Yuanzi Wu, Xiaoli Yang, Xing Liu, Jian ' an He, Long Fu, Jie Wang, Hongke Xu, Yi Shi and Renqian Zhong, Integrated poly (dimethysiloxane) with an intrinsic nonfouling property approaching " Absolute " zero background in immunoassays, Anal.Chem., 82, 6338 – 6342, 2010).
Concentration for polypeptide of the present invention in the sampling liquid used during point sample is not particularly limited, and those skilled in the art can conventionally select, and is preferably 1 μ g ~ 1000 μ g/mL, more preferably 10 μ g ~ 500 μ g/mL.In addition, the density distributed on a solid support for polypeptide of the present invention is not particularly limited, and those skilled in the art can conventionally select, and is preferably 1 ~ 100 points/10mm2, more preferably 5 ~ 50 points/10mm2.
Detection means of the present invention is useful for the early screening of enterovirns type 71 and clinical assistant diagnosis, or it may be used for manufacturing and can be used for the early screening of enterovirns type 71 and the detection kit of clinical assistant diagnosis.
detection kit of the present invention
In one aspect of the method, the present invention also provides a kind of detection kit (detection kit of the present invention), and it comprises detection means of the present invention.This detection kit can be used for early screening and the clinical assistant diagnosis of enterovirns type 71.
Detection kit of the present invention must comprise detection means of the present invention.Detection kit of the present invention can also comprise:
1. the serum sample diluent prepared or serum sample diluent component solution: Sample dilution, such as, have the Sample dilution (production code member 070021-S2) of Sai Chi bio tech ltd, Beijing, the application of sample variable color Sample dilution (production code member bwj010103) etc. of Bo Weijia bio tech ltd, Zhengzhou.Such Sample dilution is the PBS solution containing the composition such as BSA, sucrose, and containing sanitas, clarified liq, can directly use.This Sample dilution is used for dilute serum, and the serum that test kit detects will dilute suitable multiple, such as 2 ~ 200 times, preferably 10 ~ 100 times.
Detection kit of the present invention can also comprise:
2. concentrated washing lotion and washing lotion: after solid carrier surface hatches serum and ELIAS secondary antibody, the unconjugated antibody of solid carrier surface and ELIAS secondary antibody need be washed by washing lotion.Concentrated washing lotion is such as the polysorbas20 aqueous solution of 1%, need dilute 2 ~ 40 times, preferably 5 ~ 20 times during use.Can dilute by 1:9 by concentrating washing lotion (10 ×) purified water or distilled water, configure washing lotion.Such as, add 50mL in 450mL purified water or distilled water and concentrate washing lotion (10 ×), fully mix.The washing lotion do not used is placed on 2 ~ 8 DEG C of preservations, can preserve 3 months.
Detection kit of the present invention can also comprise:
3. ELIAS secondary antibody solution: the IgM in human enterovirus 71 infected person anteserum can be combined by the polypeptide of the present invention on solid carrier (such as SJ modified silica-gel), ELIAS secondary antibody can with antibodies, and the marker in ELIAS secondary antibody can react with luminous substrate mixing solutions, thus send detectable light.ELIAS secondary antibody can be the goat-anti people IgM of such as horseradish peroxidase-labeled.Being not particularly limited the concentration of ELIAS secondary antibody in ELIAS secondary antibody solution, can be such as 1ng ~ 1000ng/mL.
Detection kit of the present invention can also comprise:
4. luminous substrate mixing solutions: luminous substrate mixing solutions can react with the horseradish peroxidase that marks in ELIAS secondary antibody, make to react the chemical light sending instrument and can detect, luminous substrate mixing solutions comprises luminous substrate liquid A-superoxol, and luminous substrate liquid B-luminol (luminol,3-aminophthalic acid cyclic hydrazide) solution.Adopt two kinds of luminous substrate liquid to mix with equal-volume during use in advance, obtain luminous substrate mixing solutions (using in 45 minutes).Luminol only has crosses just meeting luminescence by oxidizer treatment.The mixed aqueous solution of usual use hydrogen peroxide and a kind of hydroxide bases is as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen G&W:
2H
2O
2→O
2+2H
2O
Luminol,3-aminophthalic acid cyclic hydrazide and oxyhydroxide generate a pairs of anion when reacting, the dioxygen oxidation that it can be gone out by peroxide decomposition, and product is an organo-peroxide.This superoxide is very unstable, decomposites nitrogen immediately, generates the 3-aminophthalic acid of excited state.During excited state to ground state transforms, the energy of release exists with the form of photon, and wavelength is positioned at the blue light components of visible ray.The example such as Thermo Seientific company of luminous substrate mixing solutions
eLISA Femto Maximum Sensitivi-ty Substrate, article No. 37074.
Detection kit of the present invention can also comprise:
5. one or more reaction cavity, can adopt such as two-sided biological incubation reactor, Chinese patent ZL 201120177686.3 or ZL201110142518.5 according to demand; Or the biological incubation reactor ZL201220430886.x of one side.
Detection kit of the present invention can also comprise:
6. other are for detecting the detection molecules (such as polypeptide, protein, nucleic acid etc.) of enterovirns type 71.
Detection kit of the present invention can also comprise:
7. working instructions, wherein describe early screening and clinical assistant diagnosis that this detection kit may be used for enterovirns type 71.
Reaction member and reaction kit can such as adopt gel imaging instrument to carry out chemiluminescence imaging.As gel imaging instrument, such as GE LAS4000mini type or sky energy 5500 type Microarray image instrument can be adopted.
Embodiment
Below, by embodiment, more specific description is carried out to the present invention, but be not the restriction to the technology of the present invention scope.By the record of this specification sheets, those skilled in the art can be easy to modify the present invention/change, and these are included in technical scope of the present invention.Scope of the present invention is determined by claims.
1. polypeptide preparation and confirmation
The polypeptide used in embodiment has the aminoacid sequence shown in SEQ ID NO:1, and by Shanghai gill, biochemical company limited synthesizes, and the sign of this polypeptide confirms to have synthesized described polypeptide by hydrogen spectrum and mass spectrum.Liquid phase chromatography detects purity: 95.27% (area normalization method): mass spectroscopy detects (ESI-MS): calculate molecular weight: 2074.31, test value 2074.22.
2. the preparation of detection means
Detection chip be with SJ modified silica-gel (iPDMS film) for solid support material, be prepared from by point sample immobilized polypeptide solution thereon.Modified silica-gel be add in traditional polydimethyl siloxane material band olefin-terminal, the initiator of surface initiated polymerization, and be fixed in the three-dimensional structure of polydimethylsiloxane by heat cross-linking (si-h bond bonding), obtain SJ modified silica-gel, its making processes as shown in Figure 1.
A and B is wherein two components of polydimethylsiloxane, polydimethylsiloxane (Poly (dimethylsiloxane), Sylgard 184) buy from Dow corning (DowCorning) company, comprise liquid composition A (composition is metallic platinum catalyst and the diformazan siloxanes polymer precursor mixture being with vinyl) and crosslinking agent B (composition is the dimethyl siloxane precursor with vinyl and Si-H group) two kinds of compositions.C is the initiator of end strips vinyl, is purchased from Hangzhou Dong Wei company.Polymer on finally modifying is that oligomeric ethylene glycol methacrylate monomer (Oligo (ethylene glycol) methacrylate, hereinafter referred to as OEGMA, molecular weight Mw=526) is bought in Aldrich.Polydimethylsiloxane precursor A and crosslinking agent B are fully mixed with A:B:C=10:1:0.5 ratio with the initiator C of band vinyl end.Make transparent elastic silicone rubber by curing reaction, then carry out finishing by sip technique and can obtain SJ modified silica-gel.Experiment shows, the surface of SJ modified silica-gel have enough highdensity, by the initiator of covalently immobolization, it can pass through surface initiated polymerization (SIP), and to realize surface macromolecule modified.The surface that reaction acquisition polyoxyethylene glycol (Polyethylene Glycol, PEG) is modified is carried out in use poly (OEGMA) (polyethylene glycol methacrylate-styrene polymer), realizes the ability of stronger anti-albumen non-specific adsorption.
The SJ modified silica-gel film made need be kept in 4 DEG C of refrigerators.
Adopt brilliant core
16 people's point sample instruments prepare polypeptide microarrays on modified silica-gel, and process is:
1) pre-treatment
By SJ modified silica-gel thin slice (15 × 15mm
2) be immersed in activation solution, take out with deionized water drip washing 3 times after 30min, dry up with nitrogen, be used for point sample at once.
2) point sample
Sampling liquid is diluted and gets well and transfer in the corresponding micropore of 384 orifice plate, 384 orifice plates of band sample are placed on point sample instrument base station, pretreated modified silica-gel thin slice are placed on the base station of point sample instrument simultaneously, carry out point sample at once.Point sample envrionment conditions is room temperature (25 DEG C), and humidity set is 50%.On the polypeptide microarrays made, the point sample amount of each point is about 0.6nL, and sampling point radius is 200 μm.
3) chemistry is fixing
The polypeptide microarrays just made will be placed on fixing at least 6h in climatic chamber (26 DEG C, 60% humidity).Chemistry fixation procedure is as follows:
First by point sample instrument by include catch peptide molecule damping fluid point on modified silica-gel film, then damping fluid starts evaporation, catch peptide molecule and the surface intimate contact interacting of SJ modified silica-gel, by Chemical bond, the high molecular end-COOH of ploy (OEGMA) on modified silica-gel surface and peptide molecule--NH
2formed and stablize covalent linkage, and then chemically active peptide molecule will be had to be fixed on SJ modified silica-gel surface.
4) assemble
The polypeptide microarrays of fixing 6h must assemble in two days.First by gum, SJ modified silica-gel thin slice is attached on special reaction column, covers reaction cavity.A reactor is made up of two reaction columns and a reaction cavity.
5) preserve
The polypeptide microarrays assembled, needs to vacuumize sealing, is kept in the refrigerator of 4 DEG C, for subsequent use.
3. detect by detection means
Checking procedure
1, before starting to detect, concentrated washing lotion is added purified water in the ratio of 1:10 or distilled water dilutes, must washing lotion after dilute, direct use, use liquid-transfering gun that 2mL washing lotion is added to chip surface, soak chip 3 minutes, ensure that chip surface is fully wet out.
2, test serum sample Sample dilution is mixed according to 1:40 dilution.
3, discard the washing lotion of soaking chip, under the state that chip surface is completely moistening, the serum after each serum sample draws 200 μ L dilutions joins in chip reactor.
4, chip reactor is put into chip permanent seat, be put on shaking table, open shaking table, frequency 150 revs/min, incubated at room 30 minutes.
5, the serum sample in chip reactor is discarded, with 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
6, after having cleaned, each chip reactor adds 200 μ L ELIAS secondary antibody solution respectively, chip reactor is put into chip permanent seat, is put on shaking table, opens shaking table, frequency 150 revs/min, incubated at room 30 minutes.
7, the ELIAS secondary antibody solution in chip reactor is discarded, with 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
8, in advance luminous substrate liquid A and luminous substrate liquid B can be mixed with equal-volume, obtain luminous substrate mixing solutions (using in 45 minutes).To be cleaned complete after, take off reaction cavity, each chip surface adds 15 μ L luminous substrate mixing solutionss respectively, makes luminous substrate mixing solutions can be laid on chip surface uniformly.
9, the chip adding luminescent solution is placed in gel imaging instrument chemiluminescence imaging, and sentence read result.
Healthy normal human serum, enterovirns type 71 infected patient serum and other disease patient's serum samples are provided by chain hospital, and disease criterion all through Clinical Laboratory confirmation, all obtains the Informed Consent Form of supplier.
Negative control has PBS damping fluid (namely to hatch without test serum in the 3rd step, and hatch by PBS solution, all the other steps are identical) contrast, the contrast of serum dilution, and negative serum (refer to Healthy People and non-bowel virus 71 type infected patients serum) contrast.
The spot sample mode of polypeptide microarrays is as shown in Figure 2: wherein, the sample behaviour IgM of 16 points of black circle, as locating point; The sample of foursquare 4 points is PB sampling liquid, as the blank of experiment; The sample of white circular form point is other enterovirns type 71 autoantigen protein polypeptide, as Testing index (these polypeptide have response to illustrate in detected serum have enterovirns type 71 autoantibody); The sample polypeptides of star point is polypeptide of the present invention, and it is enterovirns type 71 autoantigen protein polypeptide, can produce response to enterovirns type 71 infected patient serum.
This polypeptide microarrays is used to detect enterovirns type 71 infected patient serum and negative control according to above-mentioned detecting step, response modes is as shown in figs. 34: wherein, Fig. 3 shows the detected result of negative control, only has the sample of the point shown in black circle to have response.Fig. 4 shows the detected result of enterovirns type 71 infected patient serum, and black is circular, white is circular and sample that is star point has response.It should be noted that, instrument records signal value from low to high, and corresponding signaling point color is by black-red-white gradual change.
Sequence table
<110> Suzhou Siju Biomaterials Co., Ltd.
<120> polypeptide, comprise the detection means of this polypeptide and comprise the detection kit of this device
<130> E02-007
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> PRT
<213> artificial sequence
<220>
<221> misc_feature
<223> Enterovirus 71 antigen polypeptide
<400> 1
Phe Thr Glu Met Ala Ala Pro Leu Lys Ser Pro Ser Ala Glu Ala Cys
1 5 10 15
Gly Tyr Ser Asp
20
Claims (9)
1. a peptide species, its aminoacid sequence as shown in SEQ ID NO:1, that is: FTEMAAPLKSPSAEACGYSD.
2. a detection means, it comprises:
Solid carrier, and
Be connected to the polypeptide according to claim 1 on this solid carrier.
3. detection means according to claim 2, wherein, described solid carrier is SJ modified silica-gel.
4. detection means according to claim 2, its early screening for enterovirns type 71 and clinical assistant diagnosis.
5. a detection kit, it comprises the detection means according to any one of claim 2 ~ 4.
6. detection kit according to claim 5, its early screening for enterovirns type 71 and clinical assistant diagnosis.
7. the purposes of polypeptide according to claim 1 in preparation detection kit.
8. purposes according to claim 7, wherein, described detection kit is used for early screening and the clinical assistant diagnosis of enterovirns type 71.
9. purposes according to claim 7, wherein, described detection kit comprises the detection means according to any one of claim 2 ~ 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410852500.8A CN104945482A (en) | 2014-12-31 | 2014-12-31 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410852500.8A CN104945482A (en) | 2014-12-31 | 2014-12-31 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104945482A true CN104945482A (en) | 2015-09-30 |
Family
ID=54160597
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410852500.8A Pending CN104945482A (en) | 2014-12-31 | 2014-12-31 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104945482A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104650195A (en) * | 2015-03-09 | 2015-05-27 | 盖中涛 | EV71 virus VP1 recombinant antigens as well as monoclonal antibody and application of eV71 virus VP1 recombinant antigens |
| CN105085626A (en) * | 2014-05-22 | 2015-11-25 | 厦门大学 | Broad-spectrum affinity epitope polypeptide and antibody for human enteroviruses and use thereof |
-
2014
- 2014-12-31 CN CN201410852500.8A patent/CN104945482A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085626A (en) * | 2014-05-22 | 2015-11-25 | 厦门大学 | Broad-spectrum affinity epitope polypeptide and antibody for human enteroviruses and use thereof |
| CN104650195A (en) * | 2015-03-09 | 2015-05-27 | 盖中涛 | EV71 virus VP1 recombinant antigens as well as monoclonal antibody and application of eV71 virus VP1 recombinant antigens |
Non-Patent Citations (5)
| Title |
|---|
| BROWN. B. A ET AL: "POLG_HE71B，Accession No:Q66478", 《UNIPROT数据库》 * |
| CHIA-CHYI LIU ET AL.: "Identification and characterization of a cross-neutralization epitope of Enterovirus 71", 《VACCINE》 * |
| 李晨阳等: "EV71结构蛋白VP0的表达及抗体的制备", 《现代生物医学进展》 * |
| 高帆等: "肠道病毒71型灭活疫苗诱导小鼠细胞免疫抗原表位的筛选", 《中国生物制品学杂志》 * |
| 高文超等: "EV71 抗原表位的研究方法及研究进展", 《中国生物制品学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105732775A (en) | Polypeptide, detection device containing polypeptide and detection kit containing device | |
| CN105732772A (en) | Polypeptide, detection device containing polypeptide and detection kit containing device | |
| CN104945481A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN105732776A (en) | Polypeptide, detection device containing polypeptide and detection kit containing device | |
| CN105732771A (en) | Polypeptide, detection device containing polypeptide and detection kit containing device | |
| CN105367626A (en) | Polypeptide, detection device and detection kit for detecting cervical cancer | |
| CN104945472A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104945474A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104945477A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104945476A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN105732774A (en) | Polypeptide, detection device containing polypeptide and detection kit containing device | |
| CN105732769A (en) | Polypeptides, detection device comprising the polypeptides, and detection kit comprising the detection device | |
| CN105732773A (en) | Polypeptide, detection device containing polypeptide and detection kit containing device | |
| CN105732770A (en) | Polypeptides, a detection device comprising the polypeptides, and a detection kit comprising the detection device | |
| CN104945482A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104945479A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104945483A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104945478A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN103880923A (en) | Polypeptide, detector containing the polypeptide, and detection kit containing the polypeptide | |
| CN105384796A (en) | Cervical cancer detection polypeptide and detection device and detection kit | |
| CN104945475A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104945480A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104945473A (en) | Polypeptide, detection device comprising polypeptide, and detection kit comprising device | |
| CN104447959A (en) | Polypeptide, detection device containing polypeptide and detection reagent kit provided with device | |
| CN104725479A (en) | Polypeptide, detection device comprising polypeptide, and detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150930 |