CN103965336B - Polypeptide, the detection device comprising this polypeptide and detection kit - Google Patents
Polypeptide, the detection device comprising this polypeptide and detection kit Download PDFInfo
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- CN103965336B CN103965336B CN201310033098.6A CN201310033098A CN103965336B CN 103965336 B CN103965336 B CN 103965336B CN 201310033098 A CN201310033098 A CN 201310033098A CN 103965336 B CN103965336 B CN 103965336B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/445—Plasmodium
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56905—Protozoa
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
- G01N2333/445—Plasmodium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The detection device the present invention relates to polypeptide, comprising this polypeptide and detection kit.The polypeptide of the present invention is made up of the aminoacid sequence shown in SEQ ID NO:1.The polypeptide of the present invention, the detection device comprising this polypeptide and detection kit are useful in the diagnosis of malaria.
Description
Technical field
The detection device the invention mainly relates to polypeptide, comprising this polypeptide and detection kit, belong to biological technical field.
Background technology
Malaria is a kind of acute infectious disease, is mosquito matchmaker's Protozool diseases the most serious to mankind's harm the most in the world,
Pathogen is plasmodium, and symptom is heating of periodically feeling cold.Owing to plasmodial biocycle is complicated, it is special that antigen has the stage
The features such as property, the preventing and treating of malaria becomes international one of difficult problem.Wherein, pernicious malaria infects 3-5 hundred million people every year, captures number
The life of million people.In November, 2011, World Health Organization's recent statistics has 2.16 hundred million case survey of malarias to occur, and mortality rate is up to
655,000 people.Plasmodium is mainly distributed on the areas such as the earth torrid zone, subtropical zone various countries, i.e. Africa, Southeast Asia, South America, there are about 20
Hundred million populations still live in epidemic-stricken area, die from the child about 1,000,000 of malaria every year.China in south China, the certain areas in Central China, particularly
Yunnan and Hainan Province are district occurred frequently.Therefore, malaria the most seriously hampers the economic development of developing country.
Due to the particularity of malaria treatment medicine, the most quickly diagnosis can not only find in time and treat malaria patients,
It is substantially reduced case fatality rate, also can save, by getting rid of malaria in early days, the life having infected other infectious disease patient in time.With
This simultaneously, the most quickly early diagnosis is also the necessary means that epidemic diseases real-time to malaria is monitored and controlled.At present, diagnosis malaria
The classical way of disease is still blood smear microscopy, and the method is the highest to technical ability and the examination requirements of trier, far away cannot
Meet most needs living in remote districts patient in the developing countries such as Africa and South America.
Summary of the invention
It is an object of the invention to provide and a kind of malaria is diagnosed useful polypeptide, the antibody of this polypeptide anti-, comprise this
The detection device of polypeptide, the detection kit comprising this polypeptide maybe this detection device and this polypeptide cause at detection plasmodium
Disease (such as malaria) in purposes, this polypeptide in preparation for detecting the reagent of the disease (such as malaria) that plasmodium causes
Purposes in box or detection device.
That is, the present invention comprises following technical proposals:
1. the polypeptide being made up of the aminoacid sequence shown in SEQ ID NO:1.
2. a detection device, comprising:
Solid carrier, and
The polypeptide described in item 1 being connected on this solid carrier.
3. according to the detection device described in item 2, wherein, described solid carrier is SJ modified silica-gel.
4. a detection kit, it includes the polypeptide described in item 1 or the detection device described in item 2 or 3.
The antibody of the polypeptide described in the most anti-item 1.
6. the polypeptide described in 1 in preparation in the test kit detecting the disease that plasmodium causes or detection device
Purposes.
7. the purposes described in 6, wherein, described disease is malaria.
8. the purposes described in 6 or 7, wherein, described plasmodium is subtertian malaria.
9. the nucleic acid of coding polypeptide described in item 1.
10. comprise the expression vector of nucleic acid described in item 9.
11. host cells having imported the expression vector described in item 10.
The polypeptide of the present invention is applied to the diagnosis of the disease (such as malaria) that plasmodium causes, it is possible to obtain satisfactory
Effect.
Accompanying drawing explanation
Polypeptide is fixed on the schematic diagram on SJ modified silica-gel surface by Fig. 1 by chemical covalent.
The HPLC that the polypeptide of the present invention of chemosynthesis is confirmed by Fig. 2 characterizes collection of illustrative plates.
The MS that the polypeptide of the present invention of chemosynthesis is confirmed by Fig. 3 characterizes collection of illustrative plates.
The schematic diagram that the manufacturing process of SJ modified silica-gel (iPDMS thin film) is illustrated by Fig. 4.
The figure that polypeptide microarrays chemistry fixation procedure is illustrated by Fig. 5.
Fig. 6 illustrates the schematic diagram of polypeptide microarrays spot sample mode.
Fig. 7 is to show the photo to the result that healthy normal person or non-malaria disease human serum detect.
Fig. 8 is to show the photo to the result that malaria disease human serum detects.
Detailed description of the invention
The polypeptide of the present invention
The polypeptide of the present invention is 30 peptides.30 peptides of the present invention are made up of the aminoacid sequence shown in SEQ ID NO:1, it may be assumed that
YCNRLKDENNCISNLQVEDQGNCDTSWIFA.As shown in the Examples, the serum of malaria patient is positive by it, and right
Healthy normal person or non-malaria disease human serum are negative.Therefore, this 30 peptide causes as plasmodium (such as subtertian malaria)
The diagnostic tool of disease (such as malaria) is useful.
The polypeptide of the present invention is in the case of commercially available, it is possible to use commercially available product, further, it is also possible to suitably use (1) to change
The known method such as synthetic method or (2) enzyme reaction synthetic method of learning obtains, and wherein chemosynthesis is the easiest.Close at chemistry
In the case of becoming the polypeptide of the present invention, by using peptide synthesizer synthesis or this polypeptide semi-synthetic to carry out.As chemosynthesis
Method, can list such as peptide solid-phase synthesis etc..So peptide of synthesis can use conventional means such as ion to exchange color
Spectrum, reverse phase high performance liquid chromatograph, affinity chromatography etc. are purified.Such peptide solid phase synthesis process with and subsequent peptide purification all
It is well-known in the art.
Additionally, in the case of the polypeptide being produced the present invention by enzyme reaction, such as International Publication pamphlet can be used
No. WO2004/011653 described method.I.e., it is possible to so produce: by aminoacid or the carboxyl terminal quilt of dipeptides of a side
Esterification or amidatioon and the aminoacid that obtains or dipeptides and aminoacid be in free state aminoacid (such as carboxy protective
Aminoacid) react in the presence of peptide synthetase, the dipeptides of generation or tripeptides.As peptide synthetase, can list: tool
There are the culture of microorganism, this culture microbial cells separated or the thalline of this microorganism of the ability generating peptide
Reason thing or this microbe-derived peptide synthetase.
And, in addition to above-mentioned enzyme method, chemical synthesis process, in some cases, the polypeptide of the present invention is also possible to
It is naturally-occurring (but not being separated).In the case of naturally occurring, it is also possible to be separated.
The nucleic acid of the present invention, expression vector, host cell
The invention still further relates to the nucleic acid (nucleic acid of the present invention) encoding this polypeptide, expression vector (this comprising this nucleic acid
Bright expression vector), imported the host cell (host cell of the present invention) of this expression vector, they may be preferably used for producing
The polypeptide of the present invention.The nucleic acid of the present invention, expression vector, host cell can use the method for well known to a person skilled in the art
Preparation.
The detection device of the present invention
The invention still further relates to a kind of detection device (the detection device of the present invention), it includes solid carrier and is connected to
The polypeptide of the present invention on this solid carrier.
In the present invention, solid carrier is not particularly limited, as long as (e.g. may be used as solid or insoluble material
Material to be separated from reactant mixture by filtration, precipitation, Magnetic Isolation etc.) carrier.
The material constituting solid carrier includes but not limited to: silica gel (polydimethylsiloxane, PDMS), cellulose, Teflon
GrandTM, NC Nitroncellulose, agarose, glucosan, chitosan, polystyrene, polyacrylamide, polyester, Merlon, polyamide,
Polypropylene, nylon, polyvinylidene fluoride, latex, silicon dioxide, glass, glass fibre, gold, platinum, silver, copper, ferrum, rustless steel, ferrum
Oxysome, silicon wafer, polyethylene, polymine, polylactic acid, resin, polysaccharide, albumen (albumin etc.), carbon or their group
Close.
The shape of solid carrier includes but is not limited to: pearl, magnetic bead, thin film, micro cautery, filter membrane, plate, micro plate, carbon
Nanotube, sensor chip etc..The most as known in the art, the solid carrier that thin film or plate etc. are smooth can set
Put 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 μ m.The size of magnetic bead can select according to specific purposes.
In the present invention, the pearl being made up of Sepharose contour crosslinked spherical agarose has about 24 μm ~ about 165 μm
The diameter of scope.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm ~ about 44 μ m.High crosslinked spherical fine jade
The size of lipolysaccharide pearl can select according to specific purposes.
The example of the solid carrier with hydrophobic surface include can from Polysciences, Warrington, PA or
The polystyrene latex beads such as the goods that Spherotech, Liberville, IL buy.
Silicon dioxide (SiO2)-process or silicon dioxide (SiO2) example of solid carrier of base includes can be from
The extraordinary magnetic silica pearl etc. that Polysciences, Warrington, PA buy, it may be used for catching nucleic acid (such as
DNA).Or, it is also possible to use the M-280 etc. that can buy from Dynal Biotech.
The magnetic bead with hydrophilic surface can be used for catching the bacterial cell of proliferation period, nucleic acid and other composition.As
The example of this magnetic bead, can list Polysciences, pearl (title: the Biomag (registration that Warrington, PA sell
Trade mark) carboxyl) or Bangs Laboratory, the pearl of the entitled MC02N/2928 of Inc., Fishers, IN.Or
Person, it is possible to use the M-270 etc. that Dynal Biotech sells.
In a preferred embodiment of the present invention, described solid carrier is SJ modified silica-gel.Suzhou consor thing material
(iPDMS thin film, sees Chinese patent to the microarray solid support material of a kind of silicone rubber material of material company limited exploitation
CN101265329A).This material is based on the PDMS that biological study is commonly used, and adds specific initiator wherein
Part (make this material can pass through surface initiated polymerization (SIP) and realize surface-functionalized modification), then through Polyethylene Glycol methyl
Acquisition is modified on acrylate (poly (oligo (ethylene glycol) methacrylate), pOEGMA) surface.SJ
Modified silica-gel has outstanding anti-protein non-specific adsorption (Nonspecific protein adsorption, NPA) energy
Power, can control the non-specific protein absorption in complicated protein immunization detection to (close or low close to " absolute 0 " level
Detectable limit in instrument), it is possible not only to exempt the trouble closed and be cleaned multiple times, it is also possible to by using higher signal to expand
Increasing means improve the susceptiveness of protein microarray.And the essence of its silicone rubber imparts the mechanical performance that this material is stronger
With good operability.SJ modified silica-gel is the most successfully applied to 11 tumor markers compositions by the poly-company in Suzhou
Combination assay microarray ELISA kit, it is achieved that high flux and high-sensitive detection, it was demonstrated that this material is a kind of excellent
Elegant protein microarray solid support material.Meanwhile, this material also has the adjustable characteristic of surface nature, can be by control
The modification reaction time processed adjusts its surface topography within the specific limits.
The polypeptide of the present invention and the connection of solid carrier can use and well known to a person skilled in the art that polypeptide carries with solid
The method of attachment of body is carried out.Such as, for the protein/polypeptide connection with modified silica-gel surface, 1-second can be passed through
Base-3-(3-dimethyl aminopropyl)-carbodiimides [1-ethyl-3-(3-dimethyl ami-nopropyl)
Carbodiimide, EDC] and the reaction of N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) by modified silica-gel
Carboxyl (-COOH) group on the macromolecular chain of surface changes activated group into, and this activated group can be with the ammonia that carried on protein/polypeptide
Base (-NH2) reacts thus realizes being fixed on protein/polypeptide solid carrier surface (seeing Fig. 1).
The concentration of the polypeptide of the present invention in the sampling liquid of use during point sample is not particularly limited, those skilled in the art
Can select according to routine, preferably 1 μ g ~ 1000 μ g/mL, more preferably 10 μ g ~ 500 μ g/mL.Additionally, for the polypeptide of the present invention
The density being distributed on a solid support is not particularly limited, and those skilled in the art can select according to routine, and preferably 1 ~ 100
Point/10mm2, more preferably 5 ~ 50 points/10mm2。
The detection device of the present invention may be used for detecting the disease (such as malaria) that causes of plasmodium or preparation is used for examining
Survey the test kit of the disease (such as malaria) that plasmodium causes.
The detection kit of the present invention
The invention still further relates to a kind of detection kit (detection kit of the present invention), it polypeptide including the present invention or inspection
Survey device.This detection kit is preferred for detecting the disease (such as malaria) that plasmodium causes.
The detection device of the present invention or the polypeptide of the present invention are the important documents of the detection kit of the present invention.The detection of the present invention
Test kit can also include:
1. the serum dilution prepared or serum dilution component solution: serum dilution, such as, have Beijing to match biology of speeding
The Sample dilution (production code member 070021-S2) of Science and Technology Ltd., the sample-adding of Bo Weijia bio tech ltd, Zhengzhou
Variable color Sample dilution (production code member bwj010103) etc..This serum dilution is used for dilute serum, the serum of test kit detection
Suitable multiple to be diluted, such as 2 ~ 200 times, preferably 10 ~ 100 times.
The detection kit of the present invention can also include:
2. concentrate washing liquid: after solid carrier surface hatches serum and ELIAS secondary antibody, solid carrier table need to be washed by washing liquid
The unconjugated antibody in face and ELIAS secondary antibody.Concentrate washing liquid e.g. 1% polysorbas20 aqueous solution, need to dilute during use 2 ~ 40 times, excellent
Select 5 ~ 20 times.
The detection kit of the present invention can also include:
3. ELIAS secondary antibody solution: the plasmodium autoantibody in disease (such as malaria) patients serum that plasmodium causes can
The polypeptide of the present invention on solid carrier (such as SJ modified silica-gel) is combined, two anti-can with antibodies, and two anti-on marks
Note thing can react with luminous substrate, thus sends detectable light.ELIAS secondary antibody can be such as horseradish peroxidase-labeled
Goat anti-human igg.As ELIAS secondary antibody solution, the Radix Cochleariae officinalis that Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge produces can be listed
The Goat anti human IgG (H+L) of peroxidase labelling, production code member ZB-2304.To ELIAS secondary antibody in ELIAS secondary antibody solution
Concentration is not particularly limited, and can be such as 1ng ~ 1000ng/mL.
The detection kit of the present invention can also include:
4. luminescent solution component solution: luminescent solution can resist the horseradish peroxidase of upper labelling to react with two so that reaction is sent out
Go out the chemical light that instrument can detect that.Luminescent solution is mixed by two kinds of solution, is A liquid hydrogenperoxide steam generator respectively, and B
Liquid luminol solution.Luminol (luminol) is only crossed by oxidizer treatment just can luminescence.Generally use hydrogen peroxide and one
The mixed aqueous solution of hydroxide bases is as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen and water:
2 H2O2 → O2 + 2 H2O
A pairs of anion is generated with hydroxide, the oxygen oxygen that it can be decomposited by hydrogen peroxide when luminol reacts
Changing, product is an organic peroxide.This peroxide is the most unstable, decomposites nitrogen immediately, generates the 3-ammonia of excited state
Base phthalic acid.During excited state converts to ground state, the energy of release is presented in photon, and wavelength is positioned at the indigo plant of visible ray
Light part.The SuperSignal ELISA of the example of luminescent solution component solution such as Thermo Seientific company
Femto Maximum Sensitivity Substrate, article No. 37074.
The detection kit of the present invention can also include:
5. one or more reaction cavity (such as Chinese patent Granted publication CN202054829U).
The detection kit of the present invention can also include:
6. other are for detecting detection molecules (such as polypeptide, protein, the core of the disease (such as malaria) that plasmodium causes
Acid etc.).
The detection kit of the present invention can also include:
7. operation instructions.
Embodiment
Hereinafter, by embodiment, the present invention carried out more specific description, but be not the limit to the technology of the present invention scope
Fixed.By the record of this specification, the present invention readily can be modified/change by those skilled in the art, and these comprise
In the technical scope of the present invention.
The preparation of 1.30 peptides and confirmation
30 peptides used in embodiment are made up of the aminoacid sequence shown in SEQ ID NO:1, its by gill biochemical (on
Sea) company limited's synthesis, the sign collection of illustrative plates of this polypeptide is shown in Fig. 2 and Fig. 3, can confirm that and synthesized described polypeptide.
2. detect the preparation of device
Detection chip is as solid support material with SJ modified silica-gel (iPDMS thin film), fixing many by point sample thereon
Peptide solution is prepared from.Modified silica-gel be add in traditional polydimethyl siloxane material band olefin-terminal, surface draws
Send out the initiator of polyreaction, and be fixed in the three dimensional structure of polydimethylsiloxane by heat cross-linking (si-h bond bonding),
Obtain a kind of new material i.e. SJ modified silica-gel.Its manufacturing process is as shown in Figure 4.
A and B therein is two components of polydimethylsiloxane, polydimethylsiloxane (Poly
(dimethylsiloxane), Sylgard 184) buy from Dow corning (DowCorning) company, comprise liquid composition A
(composition is band for the diformazan siloxanes macromolecule precursor mixture of metallic platinum catalyst and band vinyl (composition be) and crosslinking agent B
Have the dimethyl siloxane precursor of vinyl and Si-H group) two kinds of compositions.C is the initiator of end strips vinyl, is purchased from
Hangzhou Dong Wei company.Macromolecule on finally modifying is oligomeric ethylene glycol methacrylate monomer (Oligo (ethylene
Glycol) methacrylate, hereinafter referred to as OEGMA, molecular weight Mw=526) buy in Aldrich.By poly dimethyl silicon
Oxygen alkane precursor A and crosslinking agent B are sufficiently mixed with A:B:C=10:1:0.5 ratio with the initiator C of band vinyl end.Pass through
Curing reaction makes transparent elastic silicone rubber, then carries out surface modification by SIP technology and i.e. can get SJ modified silica-gel.
Experiment shows, the surface of SJ modified silica-gel have the most highdensity, by the initiator of covalently immobolization, it can pass through surface
It is macromolecule modified that initiated polymerization (SIP) realizes surface.Poly (OEGMA) (polyethylene glycol methacrylate-styrene polymer) is used to carry out
Reaction obtains the surface that Polyethylene Glycol (Polyethylene Glycol, PEG) is modified, it is achieved stronger anti-albumen is non-specific
Property absorption ability.
The SJ modified silica-gel thin film made need to be saved in 4 DEG C of refrigerators.
Use brilliant core®16 people's point sample instruments of PersonalArrayerTM prepare polypeptide microarrays, mistake on modified silica-gel
Cheng Wei:
1) pretreatment
By SJ modified silica-gel thin slice (15 × 15mm2) be immersed in activating solution, take out with deionized water drip washing 3 after 30min
Secondary, dry up with nitrogen, be immediately used to point sample.
2) point sample
Sampling liquid diluted good and transfer in the 384 corresponding micropores of orifice plate, 384 orifice plates of band sample are placed in point sample
On instrument base station, the modified silica-gel thin slice of pretreatment is placed on the base station of point sample instrument simultaneously, carries out point sample at once.Point sample environment bar
Part 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, sampling point half
Footpath is 200 μm.
3) chemistry is fixing
Fixing at least 6h in the polypeptide microarrays climatic chamber to be placed on (26 DEG C, 60% humidity) just made.Chemistry is fixing
Process is as shown in Figure 5.
First pass through point sample instrument by include capture peptide molecule buffer point on modified silica-gel thin film, then buffer
Liquid starts evaporation, and capture peptide molecule and the surface intimate contact of SJ modified silica-gel also interacts, by chemical bond, modified silicon
Ploy (OEGMA) high molecular end-COOH on glue surface and the NH of peptide molecule2Formed and stablize covalent bond, and then will have
Chemically active peptide molecule is fixed on SJ modified silica-gel surface.
5) assembling
The polypeptide microarrays of fixing 6h must assemble in two days.First pass through gum to be attached to by SJ modified silica-gel thin slice
On special reaction column, cover reaction cavity.One reactor is made up of two reaction columns and a reaction cavity.
6) preserve
The polypeptide microarrays assembled, needs evacuation to seal, and is saved in the refrigerator of 4 DEG C, standby.
3. detect with detection device
Testing sequence
1, before starting detection, concentrated cleaning solutions is added purified water in the ratio of 1:10 or distilled water is diluted, dilution
Directly use after completing.Use liquid-transfering gun that 2mL cleanout fluid is added to chip surface, soak chip 3 minutes, it is ensured that chip surface quilt
Complete wetting.
2, by test serum sample Sample dilution according to 1:40 dilution mixing.
3, discarding the cleanout fluid of immersion chip, when chip surface moistens completely, each serum sample draws 200
Serum after μ L dilution joins in chip reactor.
4, chip reactor is put into chip and fix seat, be put on shaking table, open shaking table, frequency 150 revs/min, room temperature
Hatch 30 minutes.
5, discard the serum sample in chip reactor, clean reaction cavity and chip surface 3 times by 15mL washing liquid.
6, after having cleaned, each chip reactor is separately added into 200 μ L enzyme labelled antibody solution, is put into by chip reactor
Chip fixes seat, is put on shaking table, opens shaking table, frequency 150 revs/min, incubated at room 30 minutes.
7, discard the enzyme labelled antibody solution in chip reactor, clean reaction cavity and chip surface 3 times by 15mL washing liquid.
8, after having cleaned, taking off reaction cavity, each chip surface is separately added into 15 μ L luminous substrate liquid, makes luminescent solution
Chip surface can be laid on uniformly.
9, the chip adding luminescent solution is placed in chemiluminescence imaging in gel imaging instrument, and sentence read result.
Serum and other disease patient's serum samples of the malaria patient that subtertian malaria causes are provided by chain hospital.Serum by
Related personnel transports with parcels such as ice cube/dry ice or is handed to laboratory soon.
Negative control have PBS (i.e. hatch without test serum in the 3rd step, and hatch by PBS solution, remaining
Step is identical) comparison, the comparison of serum dilution, and the comparison of patients with negative (referring to Healthy People and non-malaria patient) serum.
The spot sample mode of polypeptide microarrays is as shown in Figure 6.Wherein, the sample of 17 points of triangle is human IgG, as reality
The anchor point tested；The sample of foursquare 3 points is PB sampling liquid, as the blank of experiment；The sample polypeptides of star point
Being the polypeptide SEQ ID NO:1 of the present invention, it is malaria autoantigen protein polypeptide, can produce malaria patients' serum and ring
Should；Circular dot sample be other malaria autoantigen protein polypeptide, as the Testing index of experiment, (these polypeptide have sound
Should illustrate to detect in serum and have malaria autoantibody).
Experimental result is as shown in Fig. 7 ~ 8.Wherein, Fig. 7 shows the testing result of negative control, only shown in triangle
The sample of point has response.Fig. 8 shows that the testing result of malaria disease human serum, the sample of triangle, star point and circle have sound
Should.It should be noted that instrument records signal value from low to high, corresponding signaling point color is by black and white gradual change.
<110>Suzhou consor thing Materials Co., Ltd
<120>polypeptide, the detection device comprising this polypeptide and detection kit
<130> S37-39
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>malaria antigen polypeptide
<400> 1
YCNRLKDENNCISNLQVEDQGNCDTSWIFA 30
Claims (9)
1. the polypeptide being made up of the aminoacid sequence shown in SEQ ID NO:1.
2. a detection device, comprising:
Solid carrier, and
It is connected to the polypeptide described in the claim 1 on this solid carrier.
Detection device the most according to claim 2, wherein, described solid carrier is iPDMS thin film.
4. a detection kit, it includes the polypeptide described in claim 1 or the detector described in Claims 2 or 3
Part.
The antibody of the polypeptide described in the most anti-claim 1.
6. the polypeptide described in claim 1 is used for detecting in the test kit of the disease that plasmodium causes or detection device in preparation
Purposes.
7. the purposes described in claim 6, wherein, described disease is malaria.
8. the purposes described in claim 6 or 7, wherein, described plasmodium is subtertian malaria.
9. encode the nucleic acid of polypeptide, the expression vector comprising described nucleic acid described in claim 1, import described expression vector
Host cell.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1377968A (en) * | 2001-04-04 | 2002-11-06 | 中国医学科学院基础医学研究所 | Malignant malarial parasite new antigen candidate gene pfMAg |
| CN1508249A (en) * | 2002-12-19 | 2004-06-30 | 中国医学科学院基础医学研究所 | Plasmodium falciparum msra-1 gene, its protein product and application |
| CN1649894A (en) * | 2001-01-26 | 2005-08-03 | 英特塞尔股份公司 | Method for identifying, isolating and producing specific pathogenic antigens |
-
2013
- 2013-01-29 CN CN201310033098.6A patent/CN103965336B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1649894A (en) * | 2001-01-26 | 2005-08-03 | 英特塞尔股份公司 | Method for identifying, isolating and producing specific pathogenic antigens |
| CN1377968A (en) * | 2001-04-04 | 2002-11-06 | 中国医学科学院基础医学研究所 | Malignant malarial parasite new antigen candidate gene pfMAg |
| CN1508249A (en) * | 2002-12-19 | 2004-06-30 | 中国医学科学院基础医学研究所 | Plasmodium falciparum msra-1 gene, its protein product and application |
Non-Patent Citations (1)
| Title |
|---|
| parasitophorous vacuole antigen, partial [Plasmodium falciparum];Coppel,R.L.et al;《GenBank》;19930426;第1页 * |
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