CN103965314B - Polypeptide, the detection device comprising this polypeptide and detection kit - Google Patents
Polypeptide, the detection device comprising this polypeptide and detection kit Download PDFInfo
- Publication number
- CN103965314B CN103965314B CN201310031927.7A CN201310031927A CN103965314B CN 103965314 B CN103965314 B CN 103965314B CN 201310031927 A CN201310031927 A CN 201310031927A CN 103965314 B CN103965314 B CN 103965314B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- malaria
- present
- detection device
- detection
- 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.)
- Expired - Fee Related
Links
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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (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)
Abstract
The detection device the present invention relates to polypeptide, comprising this polypeptide and detection kit。The polypeptide of present invention aminoacid sequence shown in SEQ ID NO:1 is constituted。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 in the world that mankind's harm is serious at present, and pathogen is plasmodium, and symptom is heating of periodically feeling cold。Owing to plasmodial biocycle is complicated, antigen has the features such as phase specificity, and the preventing and treating of malaria becomes international one of difficult problem。Wherein, pernicious malaria infects 3-5 hundred million people, the life of claims millions people every year。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 earth torrid zone, subtropical zone various countries, and namely the area such as Africa, Southeast Asia, South America, there are about 2,000,000,000 populations and 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, particularly Yunnan in Central China and Hainan Province be district occurred frequently。Therefore, malaria also seriously hampers the economic development of developing country。
Due to the particularity of malaria treatment medicine, accurately quickly diagnosis can not only find in time and treat malaria patients, is substantially reduced case fatality rate, also can pass through early stage and get rid of malaria, save the life having infected other infectious disease patient in time。With this simultaneously, accurately quickly early diagnosis is also the necessary means to the real-time epidemic diseases monitoring of malaria and control。At present, the classical way of Malaria Diagnosis is still blood smear microscopy, and the method is significantly high to technical ability and the examination requirements of trier, cannot meet far away 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 a kind of polypeptide that the diagnosis of malaria is useful, the antibody of this polypeptide anti-, the detection device comprising this polypeptide, the detection kit comprising this polypeptide maybe this detection device and this polypeptide purposes in the disease (such as malaria) that detection plasmodium causes, this polypeptide purposes in the test kit prepared for detecting the disease (such as malaria) that plasmodium causes or detection device。
That is, the present invention comprises following technical proposals:
1. the polypeptide that the aminoacid sequence shown in SEQIDNO:1 is constituted。
2. a detection device, comprising:
Solid carrier, and
It is connected to the polypeptide described in item 1 on this solid carrier。
3. the detection device according to 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。
5. the antibody of the polypeptide described in anti-item 1。
6. the polypeptide described in 1 in preparation for detecting the purposes in the test kit of the disease that plasmodium causes or detection device。
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. imported the host cell of 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 gratifying 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 shows the photo to the result that healthy normal person or non-malaria disease human serum detect。
Fig. 8 shows 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 present invention aminoacid sequence shown in SEQIDNO:1 is constituted, it may be assumed that VEVEEILPEDKNEKGQHEIVEVEEILPEDD。As shown in the Examples, the serum of malaria patient is positive by it, and reaction that healthy normal person or non-malaria disease human serum are negative。Therefore, the diagnostic tool of the disease (such as malaria) that this 30 peptide causes as plasmodium (such as subtertian malaria) is useful。
The polypeptide of the present invention is when commercially available, it is possible to use commercially available product, adopts the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method to obtain further, it is also possible to suitable, and wherein chemosynthesis is more easy。In the polypeptide situation of the chemosynthesis present invention, by using peptide synthesizer synthesis or this polypeptide semi-synthetic to carry out。As chemical synthesis process, it is possible to list such as peptide solid-phase synthesis etc.。So the peptide of synthesis can adopt conventional means such as ion exchange chromatography, reverse phase high performance liquid chromatograph, affinity chromatography etc. to be purified。Such peptide solid phase synthesis process with and subsequent peptide purification be all well-known in the art。
Additionally, when being produced the polypeptide of the present invention by enzyme reaction, it is possible to adopt such as No. WO2004/011653 described method of International Publication pamphlet。Namely; can so produce: the aminoacid esterified for the carboxyl terminal of the aminoacid of a side or dipeptides or amidatioon obtained or dipeptides and aminoacid are in the aminoacid (aminoacid of such as carboxy protective) of free state and react under the existence of peptide synthetase, the dipeptides of generation or tripeptides。As peptide synthetase, it is possible to list: have the bacterial disposing thing or this microbe-derived peptide synthetase that generate the culture of microorganism of ability of peptide, this culture microbial cells separated or this microorganism。
And, except above-mentioned enzyme method, chemical synthesis process, in some cases, the polypeptide of the present invention is it is also possible that naturally occur (but not being separated)。In naturally occurring situation, 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 coding nucleic acid (nucleic acid of the present invention) of this polypeptide, the expression vector (expression vector of the present invention) comprising this nucleic acid, the host cell (host cell of the present invention) that imported 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 adopt the method for well known to a person skilled in the art to prepare。
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 the polypeptide of solid carrier and the present invention that is connected on this solid carrier。
In the present invention, solid carrier is not particularly limited, as long as the carrier of solid or insoluble material (such as can be by the material that filtration, precipitation, Magnetic Isolation etc. separate from reactant mixture)。
The material constituting solid carrier includes but not limited to: silica gel (polydimethylsiloxane, PDMS), cellulose, polytetrafluoroethyleneTM, 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, ferrite, silicon wafer, polyethylene, polymine, polylactic acid, resin, polysaccharide, albumen (albumin etc.), carbon or their combination etc.。
The shape of solid carrier includes but is not limited to: pearl, magnetic bead, thin film, micro cautery, filter membrane, plate, micro plate, CNT, sensor chip etc.。Just as known in the art, the solid carrier that thin 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 μ m。The size of magnetic bead can select according to specific purposes。
In the present invention, the pearl being made up of the contour crosslinked spherical agarose of Sepharose has the diameter of about 24 μm ~ about 165 μ m。Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm ~ about 44 μ m。The size of high crosslinked spherical sepharose 4B can select according to specific purposes。
The polystyrene latex beads such as having that the example of the solid carrier of hydrophobic surface includes can from Polysciences, Warrington, PA or Spherotech, Liberville, the goods that IL buys。
Silicon dioxide (SiO2)-process or silicon dioxide (SiO2) example of solid carrier of base includes the extraordinary magnetic silica pearl etc. that can buy from Polysciences, Warrington, PA, it may be used for catching nucleic acid (such as DNA)。Or, it is also possible to using can from the DynalBiotech M-280 etc. bought。
The magnetic bead with hydrophilic surface can be used for catching the bacterial cell of proliferation period, nucleic acid and other composition。Example as this magnetic bead, it is possible to list Polysciences, Warrington, the pearl (title: Biomag (registered trade mark) carboxyl) of PA sale or BangsLaboratory, Inc., Fishers, the name of IN is called the pearl of MC02N/2928。Or, it is possible to use the M-270 etc. that DynalBiotech sells。
In a preferred embodiment of the present invention, described solid carrier is SJ modified silica-gel。A kind of microarray solid support material (iPDMS thin film, referring to Chinese patent CN101265329A) of the silicone rubber material of Suzhou consor thing Materials Co., Ltd exploitation。This material is based on the PDMS that biological study is commonly used, add specific initiator composition (make this material can pass through surface initiated polymerization (SIP) and realize surface-functionalized modification) wherein, obtain then through polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethyleneglycol) methacrylate), pOEGMA) finishing。SJ modified silica-gel has outstanding anti-protein non-specific adsorption (Nonspecificproteinadsorption, NPA) ability, non-specific protein absorption in complicated protein immunization detection can be controlled to close to " absolute 0 " level (being near or below the detectable limit of instrument), it is possible not only to exempt the trouble closed and repeatedly clean, it is also possible to by using higher amplification of signal means to improve the susceptiveness of protein microarray。And the essence of its silicone rubber imparts the stronger mechanical performance of this material and good operability。SJ modified silica-gel has successfully been applied to the combination assay microarray ELISA kit of 11 tumor markers compositions by the poly-company in Suzhou, achieve high flux and high-sensitive detection, it was demonstrated that this material is a kind of outstanding protein microarray solid support material。Meanwhile, this material also has the adjustable characteristic of surface nature, it is possible to adjust its surface topography within the specific limits by the controlled modification response time。
The polypeptide of the present invention and the connection of 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)-carbodiimides [1-ethyl-3-(3-dimethylami-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 activated group, this activated group can react thus realizing protein/polypeptide is fixed on solid carrier surface (referring to Fig. 1) with the amino (-NH2) be with on protein/polypeptide。
In the sampling liquid used during for point sample, the concentration of the polypeptide of the present invention is not particularly limited, and those skilled in the art can conventionally select, it is preferred to 1 μ g ~ 1000 μ g/mL, more preferably 10 μ g ~ 500 μ g/mL。Additionally, the density being distributed on a solid support for the polypeptide of the present invention is not particularly limited, those skilled in the art can conventionally select, it is preferred to 1 ~ 100 points/10mm2, more preferably 5 ~ 50 points/10mm2。
The detection device of the present invention may be used for the detection disease (such as malaria) that causes of plasmodium or preparation for detecting 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 includes the polypeptide of the present invention or detects device。This detection kit is preferred for the disease (such as malaria) that detection 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 kit of the present invention can also include:
1. the serum dilution prepared or serum dilution component solution: serum dilution, for instance have the application of sample variable color Sample dilution (production code member bwj010103) etc. of the Sample dilution (production code member 070021-S2) of Sai Chi bio tech ltd, Beijing, Bo Weijia bio tech ltd, Zhengzhou。This serum dilution is used for dilute serum, and the serum of test kit detection to dilute suitable multiple, for instance 2 ~ 200 times, it is preferable that 10 ~ 100 times。
The detection kit of the present invention can also include:
2. concentration washing liquid: after solid carrier surface hatches serum and ELIAS secondary antibody, the unconjugated antibody of solid carrier surface and ELIAS secondary antibody need to be washed by washing liquid。Concentration washing liquid is such as the polysorbas20 aqueous solution of 1%, need to dilute 2 ~ 40 times, preferably 5 ~ 20 times during use。
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) be combined, two anti-can with antibodies, and two labels resisted can react with luminous substrate, thus sending detectable light。ELIAS secondary antibody can be the goat anti-human igg of such as horseradish peroxidase-labeled。As ELIAS secondary antibody solution, it is possible to list Goat anti human IgG (H+L), the production code member ZB-2304 of the horseradish peroxidase-labeled that Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge produces。ELIAS secondary antibody concentration in ELIAS secondary antibody solution is not particularly limited, it is possible to 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 sends 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 the mixed aqueous solution of hydrogen peroxide and a kind of hydroxide bases as exciting agent。Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen and water:
2H2O2→O2+2H2O
Generating a pairs of anion with hydroxide when luminol reacts, the dioxygen oxidation that it can be gone out by peroxide decomposition, product is an organic peroxide。This peroxide is very unstable, decomposites nitrogen immediately, generates the 3-aminophthalic acid of excited state。During excited state converts to ground state, the energy of release exists with the form of photon, and wavelength is positioned at the blue light components of visible ray。The SuperSignal of the example of luminescent solution component solution such as ThermoSeientific company?ELISAFemtoMaximumSensitivitySubstrate, 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 the detection molecules (such as polypeptide, protein, nucleic acid etc.) of the disease (such as malaria) that plasmodium causes。
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 restriction to the technology of the present invention scope。By the record of this specification, those skilled in the art can be easy to the present invention is modified/changes, and these are included in the technical scope of the present invention。
1.30The preparation of peptide and confirmation
30 peptides aminoacid sequence shown in SEQIDNO:1 used in embodiment forms, and it is by the synthesis of gill biochemistry (Shanghai) Co., Ltd., and Fig. 2 and Fig. 3 is shown in by the sign collection of illustrative plates of this polypeptide, it is possible to confirm to have synthesized described polypeptide。
2.The preparation of detection device
Detection chip is with SJ modified silica-gel (iPDMS thin film) for solid support material, is prepared from by point sample immobilized polypeptide solution thereon。Modified silica-gel is to add with olefin-terminal, surface initiated polymerization initiator in traditional polydimethyl siloxane material, and it is fixed in the three dimensional structure of polydimethylsiloxane by heat cross-linking (si-h bond bonding), obtain a kind of new material and 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), Sylgard184) buying from Dow corning (DowCorning) company, comprising liquid composition A(composition is metallic platinum catalyst and the diformazan siloxanes macromolecule precursor mixture with vinyl) and two kinds of compositions of crosslinking agent B (composition is the dimethyl siloxane precursor with vinyl and Si--H)。C is the initiator of end strips vinyl, is purchased from Hangzhou Dong Wei company。Macromolecule on finally modifying is that oligomeric ethylene glycol methacrylate monomer (Oligo (ethyleneglycol) methacrylate, hereinafter referred to as OEGMA, molecular weight Mw=526) is bought in Aldrich。Polydimethylsiloxane precursor A and crosslinking agent B and the initiator C with vinyl end are sufficiently mixed with A:B:C=10:1:0.5 ratio。Make transparent elastic silicone rubber by curing reaction, then pass through sip technique and carry out finishing and can obtain SJ modified silica-gel。Experiments show that, 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。Use poly (OEGMA) (polyethylene glycol methacrylate-styrene polymer) to react and obtain the surface that Polyethylene Glycol (PolyethyleneGlycol, PEG) is modified, it is achieved the ability of stronger anti-albumen non-specific adsorption。
The SJ modified silica-gel thin film made need to be saved in 4 DEG C of refrigerators。
Adopting brilliant PersonalArrayerTM16 people's point sample instrument of core to prepare polypeptide microarrays on modified silica-gel, process is:
1) pretreatment
By SJ modified silica-gel thin slice (15 × 15mm2) be immersed in activating solution, take out with deionized water drip washing 3 times after 30min, 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 with sample being placed on point sample instrument base station, the modified silica-gel thin slice of pretreatment is placed on the base station of point sample instrument simultaneously, carrying out point sample at once。Point sample environmental condition 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 to be placed in climatic chamber (26 DEG C, 60% humidity) and fix at least 6h。Chemistry fixation procedure is as shown in Figure 5。
First pass through point sample instrument and will include the buffer point catching peptide molecule on modified silica-gel thin film, then buffer starts evaporation, catch peptide molecule and the surface intimate contact of SJ modified silica-gel and interact, by chemical bond, the high molecular end-COOH of ploy (OEGMA) on modified silica-gel surface and the NH of peptide molecule2Formed and stablize covalent bond, and then chemically active peptide molecule will be had to be fixed on SJ modified silica-gel surface。
4) assembling
The polypeptide microarrays of fixing 6h must assemble in two days。First pass through gum to be attached on special reaction column by SJ modified silica-gel thin slice, cover reaction cavity。One reactor is made up of two reaction columns and a reaction cavity。
5) preserve
The polypeptide microarrays assembled, it is necessary to evacuation seals, 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, directly use after having diluted。Use liquid-transfering gun that 2mL cleanout fluid is added to chip surface, soak chip 3 minutes, it is ensured that chip surface is fully wet out。
2, by test serum sample Sample dilution according to 1:40 dilution mixing。
3, discarding the cleanout fluid soaking chip, when chip surface moistens completely, each serum sample is drawn the serum after 200 μ L dilute and is joined in chip reactor。
4, chip reactor is put into the fixing seat of chip, is put on shaking table, opens shaking table, frequency 150 revs/min, incubated at room 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, chip reactor is put into the fixing seat of chip, 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 can be laid on chip surface uniformly。
9, the chip adding luminescent solution is placed in chemiluminescence imaging in gel imaging instrument 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 is transported by related personnel with parcels such as ice cube/dry ice or is handed to laboratory soon。
Negative control has the comparison of PBS (namely hatch without test serum in the 3rd step, and hatch by PBS solution, all the other steps are identical), 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 the anchor point of experiment；The sample of foursquare 3 points is PB sampling liquid, as the blank of experiment；The sample polypeptides of star point is the polypeptide SEQIDNO:1 of the present invention, and it is malaria autoantigen protein polypeptide, malaria patients' serum can be produced response；Circular dot sample be other malaria autoantigen protein polypeptide, as the Testing index (these polypeptide have response to illustrate there is malaria autoantibody in detection serum) of experiment。
Experimental result is such as shown in Fig. 7 ~ 8。Wherein, Fig. 7 shows the testing result of negative control, and only the sample of the point shown in triangle has response。Fig. 8 shows the testing result of malaria disease human serum, and the sample of triangle, star point and circle has response。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>S7-68
<160>1
<170>PatentInversion3.1
<210>1
<211>30
<212>PRT
<213>artificial sequence
<220>
<223>malaria antigen polypeptide
<400>1
VEVEEILPEDKNEKGQHEIVEVEEILPEDD30
Claims (9)
1. the polypeptide that the aminoacid sequence shown in SEQIDNO:1 is constituted。
2. a detection device, comprising:
Solid carrier, and
It is connected to the polypeptide described in the claim 1 on this solid carrier。
3. detection device 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 detection device described in Claims 2 or 3。
5. the antibody of the polypeptide described in anti-claim 1。
6. the polypeptide described in claim 1 is used for detecting the purposes in the test kit of the disease that plasmodium causes or detection device in preparation。
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. the coding nucleic acid of polypeptide described in claim 1, the expression vector comprising described nucleic acid, imported the host cell of described expression vector。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310031927.7A CN103965314B (en) | 2013-01-28 | 2013-01-28 | Polypeptide, the detection device comprising this polypeptide and detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310031927.7A CN103965314B (en) | 2013-01-28 | 2013-01-28 | Polypeptide, the detection device comprising this polypeptide and detection kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103965314A CN103965314A (en) | 2014-08-06 |
| CN103965314B true CN103965314B (en) | 2016-06-22 |
Family
ID=51235360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310031927.7A Expired - Fee Related CN103965314B (en) | 2013-01-28 | 2013-01-28 | Polypeptide, the detection device comprising this polypeptide and detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103965314B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628928A (en) * | 2014-11-11 | 2016-06-01 | 深圳国际旅行卫生保健中心 | Kit for assisted diagnosis of malaria |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5231168A (en) * | 1988-09-16 | 1993-07-27 | Statens Seruminstitut | Malaria antigen |
-
2013
- 2013-01-28 CN CN201310031927.7A patent/CN103965314B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5231168A (en) * | 1988-09-16 | 1993-07-27 | Statens Seruminstitut | Malaria antigen |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103965314A (en) | 2014-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103965321B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965314B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965311A (en) | Polypeptide, detection device containing polypeptide, and detection kit containing polypeptide | |
| CN103965333A (en) | Polypeptide, detection device containing polypeptide, and detection kit containing polypeptide | |
| CN103965313B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103965312B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965324B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965323B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965319B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965322B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965338B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103965329B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103965334B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103965328B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965339B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965315B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965318B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965317B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965327B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965332B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965336B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965330B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103965325A (en) | Polypeptide, detective device and detective kit containing the polypeptide | |
| CN105646691A (en) | Polypeptide, detection device containing polypeptide, and detection kit containing polypeptide | |
| CN103965320A (en) | Polypeptide, detection device and detection kit containing the polypeptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| CB02 | Change of applicant information |
Address after: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 BioBAY No. 218 building C20 Room 201 Applicant after: SUZHOU SJ BIOMATERIALS Co.,Ltd. Address before: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 BioBAY No. 218 building A4 room 209 Applicant before: SUZHOU SJ BIOMATERIALS Co.,Ltd. |
|
| COR | Change of bibliographic data | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190606 Address after: 215123 11/F, Building D, 398 Ruoshui Road, Suzhou Industrial Park, Jiangsu Province Patentee after: SUZHOU INDUSTRIAL PARK QIANGDONG PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Address before: 215123 Room 201, C20 Building, 218 Xinghu Street, Suzhou Industrial Park, Jiangsu Province Patentee before: SUZHOU SJ BIOMATERIALS Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160622 Termination date: 20220128 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |