CN103965338A - Polypeptide, detector containing polypeptide and detection kit - Google Patents
Polypeptide, detector containing polypeptide and detection kit Download PDFInfo
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- CN103965338A CN103965338A CN201310037395.8A CN201310037395A CN103965338A CN 103965338 A CN103965338 A CN 103965338A CN 201310037395 A CN201310037395 A CN 201310037395A CN 103965338 A CN103965338 A CN 103965338A
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- polypeptide
- malaria
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- detection means
- detection
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Classifications
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- 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
Abstract
The invention relates to a polypeptide, a detector containing the polypeptide and a detection kit. The polypeptide comprises an amino acid sequence shown in the formula of SEQ ID NO: 1. The polypeptide, the detector containing the polypeptide and the detection kit can be used in malaria diagnosis.
Description
Technical field
Detection means and detection kit that the present invention relates generally to polypeptide, comprises this polypeptide, belong to biological technical field.
Background technology
Malaria is a kind of acute infectious disease, is in the world the mankind to be endangered to the most serious mosquito matchmaker Protozool diseases at present, and pathogenic agent is plasmodium, and symptom is the heating of periodically feeling cold.Due to plasmodial life cycle complexity, antigen has the features such as phasic specificity, the control of malaria one of the difficult problem that becomes international.Wherein, pernicious malaria infects 3-5 hundred million people every year, captures millions of people's life.In November, 2011, World Health Organization's recent statistics has 2.16 hundred million case survey of malarias to occur, and mortality ratio is up to 655,000 people.Plasmodium is mainly distributed in the earth torrid zone, subtropics various countries, and the area such as Africa, South East Asia, South America, approximately has 2,000,000,000 populations still to live in epidemic-stricken area, dies from every year the children approximately 1,000,000 of malaria.China in south China, certain areas, particularly Yunnan and the Hainan Province in Central China is district occurred frequently.Therefore, malaria also seriously hampers the Economic development of developing country.
Due to the singularity of malaria treatment medicine, accurately malaria patients can not only be found and treat to diagnosis in time fast, greatly reduces case fatality rate, also can, by the early stage malaria of getting rid of, save in time the life that has infected other communicable disease patient.With this simultaneously, accurately early diagnosis is fast also the necessary means to the real-time prevailing disease monitoring of malaria and control.At present, the classical way of Malaria Diagnosis is still blood smear microscope inspection method, and technical ability and the survey requirement of the method to proofer is very high, cannot meet most needs of patient from far-off regions of living in the developing countries such as Africa and South America far away.
Summary of the invention
The object of the present invention is to provide for example, purposes, for example, purposes in test kit or the detection means of the disease (malaria) causing for the preparation of detection plasmodium of this polypeptide in a kind of disease (malaria) that antibody, the detection means that comprises this polypeptide, the detection kit that comprises this polypeptide or this detection means and this polypeptide of the useful polypeptide of the diagnosis of malaria, anti-this polypeptide are caused detection plasmodium.
, the present invention comprises following technical proposals:
1. the polypeptide being formed by the aminoacid sequence shown in SEQ ID NO:1.
2. a detection means, it comprises:
Solid carrier, and
Be connected in item on this solid carrier polypeptide described in 1.
3. according to the detection means described in item 2, wherein, described solid carrier is SJ modified silica-gel.
4. a detection kit, it comprises the detection means described in polypeptide or the item 2 or 3 described in item 1.
5. the antibody of the polypeptide described in anti-1.
6. the polypeptide described in 1 is in the purposes for the preparation of detecting in test kit or the detection means of the disease that plasmodium causes.
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 the polypeptide described in coding item 1.
10. comprise the expression vector of the nucleic acid described in 9.
11. have imported the host cell of the expression vector described in item 10.
Polypeptide of the present invention is applied to the diagnosis of the disease (for example malaria) that plasmodium causes, can obtains gratifying effect.
Brief description of the drawings
Fig. 1 is fixed on polypeptide the schematic diagram on SJ modified silica-gel surface by chemical covalency.
The HPLC that Fig. 2 confirms the polypeptide of the present invention of chemosynthesis characterizes collection of illustrative plates.
The MS that Fig. 3 confirms the polypeptide of the present invention of chemosynthesis characterizes collection of illustrative plates.
The schematic diagram that Fig. 4 describes the making processes of SJ modified silica-gel (iPDMS film).
The figure that Fig. 5 describes polypeptide microarray chemistry fixation procedure.
Fig. 6 illustrates the schematic diagram of polypeptide microarray spot sample mode.
Fig. 7 is the photo that shows the result that healthy normal people or non-malaria disease human serum are detected.
Fig. 8 is the photo that shows the result that malaria disease human serum is detected.
Embodiment
polypeptide of the present invention
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, that is: LLDYYSPMVHSESHCQEKMQVLFITLRDSK.As shown in the Examples, its serum to malaria patient is positive, and to the reaction that is negative of healthy normal people or non-malaria disease human serum.The diagnostic tool of the disease (for example malaria) that therefore, this 30 peptide for example, causes as plasmodium (subtertian malaria) is useful.
Polypeptide of the present invention, having commercially available in the situation that, can use commercially available product, in addition, can also suitable employing (1) chemical synthesis process or the known method such as (2) enzyme reaction synthetic method obtain, wherein chemosynthesis is more easy.In chemosynthesis polypeptide situation of the present invention, undertaken by or semi-synthetic this polypeptide synthetic with peptide synthesizer.As chemical synthesis process, can list such as peptide solid-phase synthesis etc.Synthetic like this peptide can adopt conventional means such as ion-exchange chromatography, reversed phase high efficiency liquid color spectrum, 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,, in the situation that producing polypeptide of the present invention by enzyme reaction, can adopt the method described in No. WO2004/011653, International Publication brochure for example.; can produce like this: by esterified the C-terminal of a side amino acid or dipeptides or amidation and the amino acid obtaining or dipeptides, amino acid (amino acid of for example carboxy protective) with amino acid in unbound state under the existence of peptide synthetase, react, the dipeptides of generation or tripeptides.As peptide synthetase, can list: bacterial disposing thing or this microbe-derived peptide synthetase with culture, the microbial cells being separated by this culture or this microorganism of the microorganism of the ability that generates peptide.
And except above-mentioned enzyme method, chemical synthesis process, in some cases, polypeptide of the present invention may be also natural existence (but not being separated).In naturally occurring situation, can also be separated.
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), the expression vector (expression vector of the present invention) that comprises this nucleic acid of this polypeptide of coding, the host cell (host cell of the present invention) that has imported this expression vector, they preferably can be used for producing polypeptide of the present invention.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.
detection means of the present invention
The invention still further relates to a kind of detection means (detection means of the present invention), it comprises solid carrier and is connected in the polypeptide of the present invention on this solid carrier.
In the present invention, solid carrier is not particularly limited, as long as for example, carrier as solid or insoluble material (being the material that can separate from reaction mixture by filtration, precipitation, magnetic resolution etc.).
The material that forms 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.Pit, groove, filter membrane bottom etc. can be set on the solid carrier that just as known in the art, film or plate etc. are smooth.
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 ~ approximately 10 μ m scope.The size of magnetic bead can be selected according to specific purposes.
In the present invention, the pearl of being made up of the contour crosslinked spherical agarose of Sepharose has the diameter of approximately 24 μ m ~ approximately 165 μ m scope.Preferably, high crosslinked spherical sepharose 4B has the diameter of approximately 24 μ m ~ approximately 44 μ m scope.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 be from Polysciences, Warrington, PA or Spherotech, Liberville, the polystyrene latex beads such as the goods that IL buys.
Silicon-dioxide (SiO
2)-process or silicon-dioxide (SiO
2) example of solid carrier of base comprises can be from Polysciences, Warrington, the extraordinary magnetic silica pearl that PA buys etc., it can for example, for catching nucleic acid (DNA).Or, can also use the M-280 etc. that can buy from Dynal Biotech.
The magnetic bead with hydrophilic surface can be used for catching bacterial cell, nucleic acid and other composition of proliferation period.As the example of this magnetic bead, can list Polysciences, Warrington, pearl (title: Biomag (registered trademark) carboxyl) or Bangs Laboratory that PA sells, Inc., Fishers, the name of IN is called the pearl of MC02N/2928.Or, can use the M-270 of Dynal Biotech sale etc.
In a preferred embodiment of the present invention, described solid carrier is SJ modified silica-gel.The microarray solid support material (iPDMS film, referring to Chinese patent CN101265329A) of a kind of silicon rubber material of Suzhou Siju Biomaterials Co., Ltd.'s exploitation.This material is taking the conventional PDMS of biological study as basis, add therein specific initiator composition (making this material realize surface-functionalized modification by surface initiated polymerization (SIP)), pass through again polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethylene glycol) methacrylate), pOEGMA) finishing obtain.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 " absolute 0 " level (being near or below the limit of detection of instrument) that approaches, the trouble that not only can exempt sealing and repeatedly clean, can also be by improving the susceptibility of protein microarray by stronger amplification of signal means.And the essence of its silicon rubber has been given mechanical property that this material is stronger and good operability.Suzhou YvonneJu company has successfully been applied to SJ modified silica-gel the combination assay microarray ELISA test kit of 11 tumor markers compositions, realize high-throughput and high-sensitive detection, proved that 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 within the specific limits its surface topography by the controlled modification reaction times.
Polypeptide of the present invention can adopt the method for attachment that well known to a person skilled in the art polypeptide and solid carrier to carry out with being connected of solid carrier.For example, for being connected of protein/polypeptide and modified silica-gel surface, can pass through 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide [1-ethyl-3-(3-dimethyl ami-nopropyl) carbodiimide, EDC] and N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) reaction changes carboxyl (COOH) group on the macromolecular chain of modified silica-gel surface into activating group, this activating group can with on protein/polypeptide with amino (NH2) thus react realize protein/polypeptide is fixed on to solid carrier surface (referring to Fig. 1).
In the sampling liquid using during for point sample, the concentration of polypeptide of the present invention is not particularly limited, and those skilled in the art can select according to conventional, are preferably 1 μ g ~ 1000 μ g/mL, more preferably 10 μ g ~ 500 μ g/mL.In addition, the density distributing on solid carrier for polypeptide of the present invention is not particularly limited, and those skilled in the art can select according to conventional, are preferably 1 ~ 100 points/10mm
2, more preferably 5 ~ 50 points/10mm
2.
The disease (for example malaria) that detection means of the present invention can cause for detection of plasmodium or for example, for the preparation of the test kit that detects the disease (malaria) that causes of plasmodium.
detection kit of the present invention
The invention still further relates to a kind of detection kit (detection kit of the present invention), it comprises polypeptide of the present invention or detection means.This detection kit is preferred for detecting the disease (for example malaria) that plasmodium causes.
Detection means of the present invention or polypeptide of the present invention are the important documents of detection kit of the present invention.Detection kit of the present invention can also comprise:
1. the serum dilution preparing or serum dilution component solution: serum dilution, for example, have sample diluent (production code member 070021-S2), the application of sample variable color sample diluent (production code member bwj010103) of Bo Weijia bio tech ltd, Zhengzhou etc. of Sai Chi bio tech ltd, Beijing.This serum dilution is used for dilute serum, and the serum that test kit detects will dilute suitable multiple, and for example 2 ~ 200 times, preferably 10 ~ 100 times.
Detection kit of the present invention can also comprise:
2. concentrated washing lotion: solid carrier surface is hatched after serum and ELIAS secondary antibody, need wash the unconjugated antibody of solid carrier surface and ELIAS secondary antibody by washing lotion.Concentrated washing lotion is for example 1% the polysorbas20 aqueous solution, need dilute 2 ~ 40 times, preferably 5 ~ 20 times when use.
Detection kit of the present invention can also comprise:
3. ELIAS secondary antibody solution: for example, plasmodium autoantibody in disease (malaria) patients serum that plasmodium causes can be combined by for example, polypeptide of the present invention on solid carrier (SJ modified silica-gel), two anti-can with antibodies, and two markers that resist can react with luminous substrate, thereby send detectable light.ELIAS secondary antibody can be the goat anti-human igg of for example horseradish peroxidase-labeled.As ELIAS secondary antibody solution, can list the mountain goat anti-human igg (H+L) of the horseradish peroxidase-labeled of Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge production, production code member ZB-2304.Concentration to ELIAS secondary antibody in ELIAS secondary antibody solution is not particularly limited, and can be for example 1ng ~ 1000ng/mL.
Detection kit of the present invention can also comprise:
4. luminescent solution component solution: luminescent solution can react with the horseradish peroxidase of two anti-upper marks, makes reaction send the chemical light that instrument can detect.Luminescent solution is mixed by two kinds of solution, is respectively A liquid-superoxol, and B liquid-luminol solution.Luminol (luminol,3-aminophthalic acid cyclic hydrazide) only has that cross just by oxidizer treatment can be luminous.Conventionally 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 G&W:
2 H
2O
2→ O
2+ 2 H
2O
Luminol,3-aminophthalic acid cyclic hydrazide has generated a pairs of anion while reaction with oxyhydroxide, the dioxygen oxidation that it can be decomposited by hydrogen peroxide, and product is an organo-peroxide.This superoxide is very unstable, decomposites immediately nitrogen, 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 part of visible ray.The SuperSignal ELISA Femto Maximum Sensitivity Substrate of for example Thermo Seientific company of the example of luminescent solution component solution, article No. 37074.
Detection kit of the present invention can also comprise:
5. one or more reaction cavity (for example Chinese patent Granted publication CN202054829U).
Detection kit of the present invention can also comprise:
6. the detection molecules of other diseases that cause for detection of plasmodium (such as malaria) (such as polypeptide, protein, nucleic acid etc.).
Detection kit of the present invention can also comprise:
7. working instructions.
Embodiment
Below, by embodiment, the present invention is carried out to more specific description, 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 the present invention to modify/change, and these are included in technical scope of the present invention.
the preparation of 1.30 peptides and confirmation
30 peptides that use in embodiment are made up of the aminoacid sequence shown in SEQ ID NO:1, and it is synthetic by the biochemical (Shanghai) Co., Ltd. of gill, and the sign collection of illustrative plates of this polypeptide is shown in Fig. 2 and Fig. 3, can confirm to have synthesized described polypeptide.
2. the preparation of detection means
Detection chip is taking SJ modified silica-gel (iPDMS film) as solid support material, is prepared from thereon by point sample immobilized polypeptide solution.Modified silica-gel is in traditional polydimethylsiloxane material, to add with initiator olefin-terminal, surface initiated polymerization, and be fixed in the three-dimensional structure of polydimethylsiloxane by heat cross-linking (si-h bond bonding), obtaining a kind of new material is SJ modified silica-gel.Its making processes as shown in Figure 4.
A wherein and B are two components of polydimethylsiloxane, polydimethylsiloxane (Poly (dimethylsiloxane), Sylgard 184) buy from Dow corning (DowCorning) company, comprising liquid composition A(composition is metal platinum catalyzer and the diformazan siloxanes polymer precursor mixture with vinyl) and two kinds of compositions of crosslinking agent B (composition is the dimethyl siloxane precursor with vinyl and Si-H group).C is the initiator of end band vinyl, is purchased from Hangzhou Dong Wei company.Polymer on finally modifying is that oligomeric ethylene glycol methacrylate monomer (Oligo (ethylene glycol) methacrylate, hereinafter to be referred 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 with vinyl end.Make transparent elastic silicone rubber by curing reaction, then carry out finishing by SIP technology and can obtain SJ modified silica-gel.Experiment shows, the surface of SJ modified silica-gel have enough highdensity, by the fixing initiator of covalent linkage, 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 polyoxyethylene glycol (Polyethylene Glycol, PEG) is modified, realize the ability of stronger anti-albumen non-specific adsorption.
The SJ modified silica-gel film making need be kept in 4 DEG C of refrigerators.
Adopt brilliant core
?16 people's point sample instruments of PersonalArrayerTM are prepared polypeptide microarray 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, after 30min, take out with deionized water drip washing 3 times, dry up with nitrogen, at once for point sample.
2) point sample
Sampling liquid is diluted and gets well and transfer in the corresponding micropore of 384 orifice plate, will be placed on point sample instrument base station with 384 orifice plates of sample, pretreated modified silica-gel thin slice is 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 is set as 50%.On the polypeptide microarray of making, the point sample amount of each point is about 0.6nL, and sampling point radius is 200 μ m.
3) chemistry is fixing
The polypeptide microarray just having made will be placed on fixing at least 6h in climatic chamber (26 DEG C, 60% humidity).Chemistry fixation procedure as shown in Figure 5.
First will include the damping fluid point of catching peptide molecule on modified silica-gel film by point sample instrument, then damping fluid start vaporizer, catch peptide molecule and the surface intimate contact of SJ modified silica-gel and interact, by Chemical bond, the high molecular end-COOH of the ploy on modified silica-gel surface (OEGMA) and peptide molecule-NH
2form and stablize covalent linkage, and then will have chemically active peptide molecule to be fixed on SJ modified silica-gel surface.
5) assembling
The polypeptide microarray 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.
6) preserve
The polypeptide microarray assembling, need 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, add purified water or distilled water to dilute in the ratio of 1:10 concentrated cleaning solutions, diluted rear direct use.Use liquid-transfering gun that 2mL scavenging solution is added to chip surface, soak chip 3 minutes, ensure that chip surface is by complete wetting.
2, test serum sample is mixed according to 1:40 dilution with sample diluent.
3, discard the scavenging solution that soaks chip, under the completely moistening state of chip surface, the serum that each serum sample is drawn after 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, 150 revs/min of frequencies, incubated at room 30 minutes.
5, discard the serum sample in chip reactor, use 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
6, after having cleaned, each chip reactor adds respectively 200 μ L enzyme labelled antibody solution, and chip reactor is put into chip permanent seat, is put on shaking table, opens shaking table, 150 revs/min of frequencies, incubated at room 30 minutes.
7, discard the enzyme labelled antibody solution in chip reactor, use 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
8, after having cleaned, take off reaction cavity, each chip surface adds respectively 15 μ L luminous substrate liquid, makes luminescent solution can be laid on uniformly chip surface.
9, the chip that has added luminescent solution is placed in to gel imaging instrument chemoluminescence imaging, and sentence read result.
The malaria patient's that subtertian malaria causes serum and other diseases patients serum sample are provided by chain hospital.Serum is transported or is handed to soon laboratory with parcels such as ice cube/dry ice by related personnel.
Negative control has the contrast of PBS damping fluid (hatch without test serum in the 3rd step, and hatch with PBS solution, all the other steps are identical), the contrast of serum dilution, and the contrast of patients with negative (referring to Healthy People and non-malaria patient) serum.
The spot sample mode of polypeptide microarray as shown in Figure 6.Wherein, the sample of leg-of-mutton 17 points is human IgG, as the locating point of experiment; The sample of foursquare 3 points is PB sampling liquid, as the blank of experiment; The sample polypeptide of star point is polypeptide SEQ ID NO:1 of the present invention, and it is malaria autoantigen protein polypeptide, can produce response to malaria patients' serum; Circular point sample be other malaria autoantigen protein polypeptide, as the detection index (these polypeptide have response explanation to detect in serum and have malaria autoantibody) of experiment.
Experimental result is as shown in Fig. 7 ~ 8.Wherein, Fig. 7 has shown the detected result of negative control, only has the sample of the point shown in trilateral to have response.Fig. 8 has shown the detected result of malaria disease human serum, and trilateral, star point and circular sample have response.It should be noted that, instrument records signal value from low to high, and corresponding signaling point color is by black-white gradual change.
<110> Suzhou Siju Biomaterials Co., Ltd.
<120> polypeptide, the detection means that comprises this polypeptide and detection kit
<130> S07-60
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> malaria antigen polypeptide
<400> 1
LLDYYSPMVHSESHCQEKMQVLFITLRDSK 30
Claims (9)
1. the polypeptide being formed by the aminoacid sequence shown in SEQ ID NO:1.
2. a detection means, it comprises:
Solid carrier, and
Be connected in the polypeptide claimed in claim 1 on this solid carrier.
3. detection means according to claim 2, wherein, described solid carrier is SJ modified silica-gel.
4. a detection kit, it comprises the detection means described in polypeptide claimed in claim 1 or claim 2 or 3.
5. the antibody of anti-polypeptide claimed in claim 1.
6. the purposes of polypeptide claimed in claim 1 in test kit or the detection means of the disease causing for the preparation of detection plasmodium.
7. purposes claimed in claim 6, wherein, described disease is malaria.
8. the purposes described in claim 6 or 7, wherein, described plasmodium is subtertian malaria.
Nucleic acid, the expression vector that comprises described nucleic acid of coding polypeptide claimed in claim 1, imported the host cell of described expression vector.
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|---|---|---|---|
| CN201310037395.8A CN103965338B (en) | 2013-01-31 | 2013-01-31 | Polypeptide, the detection means comprising this polypeptide and detection kit |
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|---|---|---|---|
| CN201310037395.8A CN103965338B (en) | 2013-01-31 | 2013-01-31 | Polypeptide, the detection means comprising this polypeptide and detection kit |
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| CN103965338B CN103965338B (en) | 2016-04-13 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6610836B1 (en) * | 1999-01-29 | 2003-08-26 | Genome Therapeutics Corporation | Nucleic acid amino acid sequences relating to Klebsiella pneumoniae for diagnostics and therapeutics |
-
2013
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6610836B1 (en) * | 1999-01-29 | 2003-08-26 | Genome Therapeutics Corporation | Nucleic acid amino acid sequences relating to Klebsiella pneumoniae for diagnostics and therapeutics |
Non-Patent Citations (1)
| Title |
|---|
| IRIKO,H: "RhopH2 [Plasmodium falciparum]", 《GBANK》 * |
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| CN103965338B (en) | 2016-04-13 |
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