CN104965087A - Method for efficiently detecting toxoplasma acute infection, and target protein thereof - Google Patents
Method for efficiently detecting toxoplasma acute infection, and target protein thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting toxoplasma acute infection without a non-disease diagnosis or treatment purpose, a kit thereof, and an antibody used in the method. The method is characterized in that a sample to be detected reacts with a toxoplasma Coronin protein or the antibody of the toxoplasma Coronin protein. The detection method can be used to rapidly detect the toxoplasma acute infection, and has high specificity and sensitivity.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to method and the target proteins thereof of the acute infection of a kind of efficient detection Infection of Toxoplasma Gondii.
Background technology
Infection of Toxoplasma Gondii is the entozoic protozoon of a kind of world wide, can cause that people and animals miscarry, stillborn foetus and immunosuppressed patient dead, its complicated clinical manifestation, makes a definite diagnosis difficulty, brings high risks to public health and animal husbandry.In the past, immunological detection method is the Main Diagnosis method of toxoplasmosis, but the detection of resisting toxoplasmosis IgG is difficult to distinguish acute infection and previous infection, IgM and the IgA positive can not as the mark of acute infection.Therefore, the early diagnosis marker of Infection of Toxoplasma Gondii acute infection needs to be found further to determine.
Arch insect circulating antigen (Circulating Antigens, CAg) be the excretion of Infection of Toxoplasma Gondii acute infection period polypide self and pyrolysis product, zoogenetic infection Infection of Toxoplasma Gondii can detect CAg in 1-2 days, after 2 weeks, titre significantly reduces, CAg positive prompting toxoplasmosis is acute, Active infection, being the important symbol that pathogen exists, is the reliability index of early infection.Excretory-secretory antigen (Excretory/Secretory Antigens, ESA) be the principal ingredient of CAg, existing research is extract ESA antigen or express certain secretory protein to prepare the CAg resisting to detect in serum mostly, because the content of CAg antigen is less, and complicated component, thus make the specificity of these methods, sensitivity limited.Therefore, the method for a species specificity and all higher detection Infection of Toxoplasma Gondii of sensitivity is still lacked at present.
Summary of the invention
Technical matters to be solved by this invention overcomes the method detecting at present arch insect infection to be difficult to distinguish acute infection and previous infection, to utilize circulating antigen to the method detecting acute infection because of the reason specificitys such as circulating antigen content is low, complicated component and the limited problem of sensitivity, provide a kind of detection method being applicable to high specific that the early stage acute infection of Infection of Toxoplasma Gondii detects, hypersensitivity, Infection of Toxoplasma Gondii acute infection can be detected fast by described detection method, and there is higher specificity and sensitivity.
One of technical solution of the present invention: a kind of method of detection Infection of Toxoplasma Gondii acute infection of non-diseases diagnoses and treatment object, it comprises the steps: the antibody of sample to be detected and Infection of Toxoplasma Gondii Coronin albumen or described Infection of Toxoplasma Gondii Coronin albumen is reacted.
In the present invention, described sample to be detected can be the conventional indication in this area by the sample of arch insect infection, be preferably by the serum sample after arch insect infection.
In the present invention, described Infection of Toxoplasma Gondii Coronin albumen can be the conventional indication in this area, preferably for amino acid sequence is if the GenBank number of including is for shown in the sequence of EPT25607.1, the preparation method of described Infection of Toxoplasma Gondii Coronin albumen can be the method for this area routine, as synthetic method or expression and purification method, it is preferably Prokaryotic expression, purification method.Described Prokaryotic expression, purification method preferably comprises the steps: the prokaryotic expression bacterial strain of the prokaryotic expression carrier cultivated containing described Infection of Toxoplasma Gondii Coronin gene, and smudge cells, extracts described Infection of Toxoplasma Gondii Coronin albumen from lysate.The bacterial strain of described prokaryotic expression can be the expression strain of this area routine, is preferably E. coli expression strains, is more preferably e. coli strain bl21.The carrier of described prokaryotic expression can be the expression vector of this area routine, and being preferably pET series expression vector, is pET28a carrier best.Described extracting method can be that this area is conventional, and being preferably chromatography, is more preferably affinity chromatography, is nickel ion-6 histidine affinity chromatography best.
In the present invention, the antibody of described Infection of Toxoplasma Gondii Coronin albumen can be the antibody of the conventional indication in this area, as monoclonal antibody or polyclonal antibody.The preparation method of the antibody of described Infection of Toxoplasma Gondii Coronin albumen can be the preparation method of this area routine, preferably comprise the steps: described antibody obtained after described Infection of Toxoplasma Gondii Coronin protein immune animal, more preferably comprise: by described Infection of Toxoplasma Gondii Coronin protein immune animal, gather blood after secondary immunity and be separated the serum containing described antibody.The animal that described animal can use for this area routine, being preferably rabbit or mouse, is more preferably mouse.Described inoculation can be the injection system of this area routine, as directly by as described in Infection of Toxoplasma Gondii Coronin protein injection in animal body, preferably for described Infection of Toxoplasma Gondii Coronin albumen is expelled in described animal body together with Freund's adjuvant.The interval time of described secondary immunity is preferably 2 weeks, and described collection blood is preferably the tail venous collection from described animal.
In the present invention, described reaction can be the conventional indication in this area, preferably comprises the steps: that the antibody of described Infection of Toxoplasma Gondii Coronin albumen or described Infection of Toxoplasma Gondii Coronin albumen is coated in matrix by (1); (2) matrix being coated with the antibody of described Infection of Toxoplasma Gondii Coronin albumen or described Infection of Toxoplasma Gondii Coronin albumen in step (1) adds sample to be detected, the antibody of itself and described Infection of Toxoplasma Gondii Coronin albumen or described Infection of Toxoplasma Gondii Coronin albumen is reacted.
In the present invention, preferably can also comprise step (3): the result of detecting step (2) described reaction.Described detection can be the detection method of this area routine, it is preferably the detection method of detectable antigens and antibody response, such as when matrix is wrapped quilt for Coronin albumen time, hatch add the antibody of the anti-igg that horseradish peroxidase (HRP) marks after step (2) after, develop the color and measure values of chemiluminescence; When matrix is wrapped quilt be the antibody of described detection Infection of Toxoplasma Gondii acute infection time, after step (2), add the Coronin antibody with the antibody allos of described detection Infection of Toxoplasma Gondii acute infection, hatch again after adding the antibody of the anti-igg that horseradish peroxidase (HRP) marks after hatching again, develop the color and measure values of chemiluminescence.
Technical solution of the present invention two: a kind of antibody detecting Infection of Toxoplasma Gondii acute infection, it is obtained by the preparation method by comprising the steps: by described antibody obtained after Infection of Toxoplasma Gondii Coronin protein immune animal.
In the present invention, the antibody of described detection Infection of Toxoplasma Gondii acute infection can be the antibody of the conventional indication in this area, as monoclonal antibody or polyclonal antibody.The preparation method of the antibody of described detection Infection of Toxoplasma Gondii acute infection can be the preparation method of this area routine, preferably comprise the steps:, by aforementioned Infection of Toxoplasma Gondii Coronin protein immunization injection animal, to gather blood after secondary immunity and be separated the serum containing described antibody.The animal that described animal can use for this area routine, being preferably rabbit or mouse, is more preferably mouse.Described inoculation can be the injection system of this area routine, as directly by as described in Infection of Toxoplasma Gondii Coronin protein injection in animal body, preferably for described Coronin albumen is expelled in animal body together with Freund's adjuvant.The interval time of described secondary immunity is preferably 2 weeks, and described collection blood is preferably the tail venous collection blood from animal.
Technical solution of the present invention three: a kind of kit detecting Infection of Toxoplasma Gondii acute infection, it comprises the antibody of aforementioned Coronin albumen and/or the acute infection of aforementioned detection Infection of Toxoplasma Gondii.
In the present invention, the kit of described detection Infection of Toxoplasma Gondii acute infection preferably can also comprise the antibody that immune response confining liquid, immune response cleansing solution, immune response stop buffer and coupling have the anti-igg of chromogenic reaction enzyme.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can combination in any, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: a kind of target proteins and antibody thereof detecting Infection of Toxoplasma Gondii acute infection provided by the present invention, and a kind of method of the detection Infection of Toxoplasma Gondii acute infection of described target proteins and antibody thereof that utilizes can detect the early stage acute infection of Infection of Toxoplasma Gondii effectively and quickly,, and there is higher specificity and sensitivity.
Accompanying drawing explanation
The phylogenetic analysis of Fig. 1, different plant species Coronin amino acid sequence shows.
The RT-PCR result of Fig. 2, Infection of Toxoplasma Gondii Coronin gene.
Fig. 3, Infection of Toxoplasma Gondii Coronin albumen HisBind post purification result.1: stream wears liquid, 2: rinsing liquid, 3: eluent.
The tissue location of Fig. 4, Infection of Toxoplasma Gondii Coronin albumen.
Embodiment
Further illustrate the present invention by embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises." room temperature " described in embodiment refers to the temperature of carrying out the operation room tested, and is generally 25 DEG C.
The source of the experiment material in following embodiment, instrument and reagent is as follows:
Animal used as test and microorganism: kunming mice and BALB/c are purchased from Shanghai Slac Experimental Animal Co., Ltd.; Beasle dog is purchased from Xin Gang experimental animal field, Shanghai; RH strain of Toxoplasma gondii is purchased from National Veterinary Microbiological Culture Collection administrative center; E. coli bl21 (DE3) and Escherichia coli TOP 10 competent cell are purchased from Beijing CoWin Bioscience Co., Ltd..
Experimental cell and reagent: Vero cell is biochemical and cell research institute purchased from Chinese Academy of Sciences Shanghai; DMEM, pancreatin are purchased from Gibco company; BSA, Freund's adjuvant available from Sigma; Compound protease inhibitors is purchased from Merck company; Normal Mouse IgG, Protein A agarose microbeads, DAPI are purchased from green skies Bioisystech Co., Ltd; The donkey against murine IgG that Alexa Fluor 488 marks is purchased from Jackson company; HisBand Resin kit is purchased from Merck & Co., Inc.; BCA protein quantification kit and dithiothreitol (DTT) (DTT) are purchased from Bio-Rad company; Total serum IgE Reverse Transcription box is purchased from Thermo Scientific company; PMD19T plasmid is purchased from precious bioengineering (Dalian) company limited; PET28a plasmid is purchased from Novagen company; Ampicillin purchased from kanamycins purchased from Sheng Gong bioengineering incorporated company; 3,3', 5,5'-tetramethyl benzidine, paraformaldehyde, Triton-X 100 are purchased from Chemical Reagent Co., Ltd., Sinopharm Group; Primer is synthesized by Suzhou Jin Weizhi Bioisystech Co., Ltd; DNA sequencing is undertaken by precious bioengineering (Dalian) company limited.
Experimental apparatus: chromatographic column RP-C18 is purchased from Column Technology Inc.; Chromatograph Zorbax300SB-C18peptide traps purchased from Agilent Technologies, Wilmington, DE; QExactive mass spectrometer is purchased from Thermo Fisher Scientific; Laser confocal microscope Nikoneclipse C1-Si is purchased from Nikon (Nikon) company.
Embodiment 1 obtains Infection of Toxoplasma Gondii ESA antigen and preparation resists more
1, the preparation of ESA
By kunming mice abdominal cavity aseptic inoculation RH strain of Toxoplasma gondii, within three days, collect the polypide in ascites afterwards, with the syringe pump twice of No. 20 syringe needles with broken peritoneal macrophage, with 5 μm of frit after polypide dilution, the serum-free DMEM of the polypide after gained purifying to clean after twice with serum-free DMEM according to 1 × 10
8/ ml concn suspension polypide, bring up again suspension 5% CO2gas incubator 37 DEG C and cultivate 2h to make polypide secretion ESA, the centrifugal 10min of 1000rpm removes polypide and collects supernatant, after adding compound protease inhibitors in supernatant, utilize 3KDa protein concentration pipe 20 times to concentrate, by ESA concentration the packing after aseptic PBS dialysis under 4 DEG C of conditions of the albumen after concentrated ,-80 DEG C of preservations.
2, the preparation of polyclonal antibody
After ESA described in 1 being added the emulsification of same volume Freund's adjuvant, according to 100 μ g/ only immune BALB/c mouse, each immunization interval 2 weeks, exempts from tail venous collection blood separation of serum after ten days first and detects for antibody titer ELISA.
Embodiment 2
The different preparation infecting the acute infection Infection of Toxoplasma Gondii dog serum sample of number of days
Select cleaning grade beasle dog, test group and each 3 of control group, test group abdominal cavity aseptic inoculation 1 × 10
9rH strain of Toxoplasma gondii polypide described in the embodiment 1 that aseptic PBS after the purifying of individual fresh collection is resuspended, control group inoculates aseptic PBS, detects Temperature changing and gathers blood every day to infecting latter 7 days, is used as Infection of Toxoplasma Gondii acute infection animal blood serum after separation of serum.
Embodiment 3
The immunoaffinity chromatography of circulating antigen in the serum of Infection of Toxoplasma Gondii acute infection different number of days
1, the removal of dog serum IgG
Dog serum 60 μ l in the strain of toxoplasma gondii infection RH described in embodiment 23 days is added in the aseptic PBS of 540 μ l (pH=7.2), add the Protein A agarose microbeads of 100 μ l 25% (v/v), collected by centrifugation supernatant after 4 DEG C of slow stirring 2h, supernatant is the dilute serum of removing most IgG.
2, non-specific binding is removed
The ProteinG agarose microbeads of 5 μ l Normal Mouse IgG and 100 μ l 25% will be added, collected by centrifugation supernatant after 4 DEG C of slow stirring 2h in the supernatant of above-mentioned collection.
3, immunoprecipitation enrichment destination protein
Spend the night adding the many anti-rear 4 DEG C of stirrings of ESA obtained in 5 μ l embodiments 1 in the supernatant of above-mentioned collection, add the Protein G agarose microbeads of 100 μ l 25%, collected by centrifugation agarose microbeads after 4 DEG C of stirring 3h, add 60 μ l 2 × SDS-PAGE sample-loading buffers after microballoon being cleaned 3 times, 100 DEG C are boiled the centrifugal 5min of 2500rpm after 5min and collect supernatant.
4, the enzymolysis of film auxiliary protein group preparation (FASP) sample
The albumen of the enrichment obtained in 3 being added DTT to final concentration is 100mM, and boiling water bath 5min, is cooled to room temperature.Add 200 μ L urea buffer (8M urea, 150mM TrisHCl pH8.0) mixing, proceed to 10kd ultra-filtration centrifuge tube, the centrifugal 15min of 14000g.Add the centrifugal 15min of 200 μ L urea buffer14000g, abandon filtrate.Add 100 μ L 50Mm iodoacetamides, described iodoacetamide is dissolved in 8M urea, and this 8M urea is by 150mM Tris-HCl, pH 8.0 is mixed with (woulding you please here confirm or amendment), 600rpm vibrates 1min, the centrifugal 10min of lucifuge room temperature 30min, 14000g.Add 100 μ L aforementioned urea damping fluids, the centrifugal 10min of 14000g repeats 2 times.Add 100 μ L and dissolve damping fluid (Dissolution buffer), described dissolving damping fluid is 25mM NH
4hCO
3, the centrifugal 10min of 14000g repeats 2 times.Add 40 μ l Trypsin damping fluids (2 μ g Trypsin join 40 μ L dissolve damping fluids be made into), 600rpm vibrates 1min, 37 DEG C of 16-18h.Renew collection tube, the centrifugal 10min of 14000g, gets filtrate, prepares to carry out liquid chromatography coupling mass spectrophotometry.
5, capillary high performance liquid chromatography
Liquid phase A liquid is 0.1% (v/v) aqueous formic acid, and B liquid is 0.1% (v/v) formic acid acetonitrile solution, and wherein acetonitrile is 84% (v/v).
Chromatographic column 0.15mm*150mm (RP-C18) (Column Technology Inc.) balances with the A liquid of 95% (v/v).Gained sample in 4 is loaded to Zorbax 300SB-C18peptidetraps by automatic sampler, then is separated through chromatographic column, related fluid phase gradient is as follows: 0 minute-50 minutes, and B linear gradient is from 4% to 50%; 50 minutes-54 minutes, B linear gradient was from 50% to 100%; 54 minutes-60 minutes, B liquid maintained 100%.
6, ESI Mass Spectrometric Identification
The product be separated to through capillary high performance liquid chromatography in 5 is carried out mass spectrophotometry through capillary high performance liquid chromatography desalination and after being separated with Q Exactive mass spectrometer again.Detection mode: positive ion.The mass-charge ratio of the fragment of polypeptide and polypeptide gathers according to following method: each full scan (full scan) gathers 10 fragment patterns stored (MS2scan) afterwards.
7, ESI MASS SPECTRAL DATA ANALYSIS
The corresponding database of source document (raw file) use Mascot 2.2 software search.Finally obtain the protein results identified.Correlation parameter is as follows: Enzyme=Trypsin, Missed cleavage=2, Fixedmodification:Carbamidomethyl (C), Variable modification:Oxidation (M) search uses Uniprot database: uniprot_Toxoplasma_gondii_26517_20140612.fasta protein library (accession sequence 26517, under be loaded in 201406013).Peptides tolerance:20ppm, MS/MS tolerance:0.1Da, Mascot result filtration parameter is: Mascot score >=20.Search storehouse result and show that the protein sequence coverage rate of Coronin albumen is 3.70%, unique peptide hop count is 3, and unique peptide hop count >=2 are successful identification.
Embodiment 4 sequential analysis
Coronin gene nucleotide series and albumen peace amino acid sequence are analyzed with NCBI online database and DNAstar software.The phylogenetic analysis of different plant species Coronin amino acid sequence shows, Infection of Toxoplasma Gondii Coronin recombinant protein is except the Hammondia hammondi (Hammondia hammondi) of the non-pathogenic with cat parasitism is except Coronin homology is higher, with Demodiosis canis homology 81%, with other species homologies lower than 50%, the results are shown in Figure 1.
Embodiment 5
The marker protein that prokaryotic expression is relevant to Infection of Toxoplasma Gondii early diagnosis
1, the Design and synthesis of primer
According to the known array (the Gen-Bank number of including: AY713297) of toxoplasma gondii Coronin gene, utilize Primer 5.0 for its open reading frame design primer, the length of expection amplification gene is 1251bp, BamH I and Xho I restriction enzyme site is introduced respectively, upstream primer FP:5'GTC at upstream and downstream primer 5 ' end
gGATCCcACGATGGATCGAAGGCAT 3', downstream primer RP:5'CGC
cTCGAGaGCGGCTTCGTCCTGA 3',
2, the collection of RH strain of Toxoplasma gondii and the extraction of RNA
From liquid nitrogen, take out the RH strain of Toxoplasma gondii recovery of conservation, intraperitoneal inoculation mouse, rinses abdominal cavity with sterile saline after 3d, collect peritoneal fluid, the centrifugal 15min of 1000rpm, abandons supernatant, gained RH strain of Toxoplasma gondii polypide PBS washes 3 times, adopts Trizol method to extract RNA.Total serum IgE be stored in-70 DEG C for subsequent use.
3, the RT-PCR amplification of Coronin gene
By Reverse Transcription box, the total serum IgE of extraction is illustrated that reverse transcription obtains cDNA, and carry out pcr amplification as template.Reaction system (25 μ L): 2 × Taq Mix 12.5 μ L, upstream and downstream primer and each 1 μ L of cDNA template, add dd H
2o mends to 25 μ L.Reaction conditions: 94 DEG C of 4min; 94 DEG C of 45s, 58 DEG C of 50s, 72 DEG C of 50s, 30 circulations; Last 72 DEG C extend 10min.Amplified production reclaims kit with Ago-Gel and reclaims after 1% agarose gel electrophoresis qualification, as shown in Figure 2.The PCR primer reclaiming purifying is connected with pMD19T carrier, connects product conversion to Escherichia coli TOP 10 competent cell, amicillin resistance screening positive clone.After bacterium colony PCR identifies, positive plasmid is sent.Positive plasmid is named as pMD19T-CORONIN.
4, the structure of recombinant expression plasmid and qualification
PMD19T-CORONIN and pET-28a expression vector all uses restriction enzyme BamH I and Xho I double digestion, enzyme is cut rear Ago-Gel and is reclaimed kit recovery, reclaim product to connect and transformation of E. coli BL21 (DE3) competent cell, picking single bacterium colony cultivation extraction plasmid, carries out PCR and enzyme cuts qualification.
5, the abduction delivering of recombinant plasmid in Escherichia coli
The positive recombinant bacterium of screening is inoculated in the LB fluid nutrient medium of 10mL containing kanamycins 70 μ g/mL by the expression of recombinant plasmid and soluble analysis, in 37 DEG C, 200rpm jolting cultivates after 8h, be inoculated in 250ml LB kanamycins nutrient culture media, when the same terms expansion is cultured to OD600nm value for 0.6-0.8, add 0.5mmol/L IPTG, 20 DEG C of abduction delivering 12h.The centrifugal 10min of 8000rpm collects thalline, the abduction delivering thalline of collected by centrifugation is resuspended with 1 × binding buffer liquid of 1/10 original fluid volume, ultrasonication on ice bath, the centrifugal 10min of 12000rpm, collect supernatant and precipitation respectively, analyze the existence form of recombinant protein and purifying by SDS-PAGE.
6, the purifying of recombinant protein c oronin
By the thalline collected after abduction delivering, resuspended with 1 × binding buffer liquid, through ultrasonication, collect supernatant, after 0.22 μm of membrane filtration, collect albumen according to HisBind Resin purification kit step purifying, purification result as shown in Figure 3, main purified product molecular size range is 54kD, meets with destination protein size.Product after the purifying mass concentration of BCA protein quantification kit measurement albumen.
Embodiment 6
The preparation of the antibody of recombinant protein c oronin
After recombinant protein being added the emulsification of same volume Freund's adjuvant, according to 100 μ g/ only immune BALB/c mouse, each immunization interval 2 weeks, two exempt from tail venous collection blood separation of serum after ten days detects for antibody titer ELISA.
Effect example 1
Coronin albumen is located at the tissue of Toxoplasma
1) inoculate Infection of Toxoplasma Gondii after the Vero cell on cell climbing sheet covers with individual layer, after 48h, clean 3 times with the PBS of precooling;
2) 3 times are cleaned with 4% paraformaldehyde 4 DEG C of fixing 20min, PBS;
3) 3 times are cleaned with PBS perforation 30min, PBS containing 0.5%Triton-X 100;
4) PBS37 DEG C closed 30min, the PBS of above-mentioned creep plate containing 1%BSA cleans 3 times;
5) the ELMO mouse polyvalent antibody that dilutes according to 1:4000 of 1%BSA in addition, hatch 1.5h for 37 DEG C, PBS cleans 3 times;
6), after the donkey against murine IgG (Jackson product) that Alexa Fluor 488 marks dilutes according to 1:200 with 1%BSA, hatch 2h for 37 DEG C, PBS cleans 3 times;
7) DAPI redyes rear PBS and cleans 3 times, and laser confocal microscope 100 × oily mirror microscopy also gathers image (Fig. 4).
As seen from Figure 4, Coronin albumen mainly concentrates on the surface of polypide and the top of polypide, and this may participate in the motion of polypide with Coronin albumen and to invade cell relevant, and confirm anti-, Coronin antibody can identify Infection of Toxoplasma Gondii native antigen simultaneously.
Effect example 2
Evaluate the effect that recombinant protein c oronin detects Infection of Toxoplasma Gondii acute infection
Recombinant protein made for embodiment 5 is pressed gradient bag by 96 orifice plates, 4 DEG C of bags are spent the night.1% gelatin is closed, after PBST washes 3 times, using the positive serum of artificial challenge's aforementioned RH strain of Toxoplasma gondii 18d according to 1:10,1:50,1:100,1:200 gradient as primary antibodie, add negative dog serum in contrast simultaneously, 37 DEG C of reaction 1h, add HRP after washing and mark the anti-dog IgG of rabbit, 37 DEG C of reaction 1h, washing; Colour developing: add 100 μ L/ hole 3,3', 5,5'-tetramethyl benzidine (TMB) nitrite ions, hatches colour developing about 15min for 37 DEG C; Stop: add stop buffer 2M H
2sO
450 μ L/ holes, colour developing after stopping.Result is presented at every hole bag by 50ng recombinant protein c oronin and the antibody still can effectively monitored under the positive serum condition of dilute according to 1:50 in Infection of Toxoplasma Gondii positive dog serum, as shown in table 1.
Table 1 utilizes the antibody in the positive dog serum of recombinant protein c oronin detection Infection of Toxoplasma Gondii
Note: * represents antigen coated concentration and antibody dilution ratio the best.
As can be seen from Table 1, when recombinant protein is according to 50ng/ hole bag quilt, during serum sample to be checked 50 times dilution, ELISA Detection results is optimum.
Effect example 3
Heavier histone Coronin and import reagent box detect the effect of Infection of Toxoplasma Gondii acute infection
For the clinical detection effect of inspection Coronin recombinant protein, the serum of 90 of clinical acquisitions portions of dogs is detected, and with import reagent box (ID
toxoplasmosis indirect multi-speciesELISA kit, IDVET, France) testing result compares, as shown in table 2.
Table 2 utilizes recombinant protein and import reagent box to detect dog arch insect infection result
As can be seen from Table 2, the specificity utilizing recombinant protein c oronin to detect dog arch insect infection reaches 95.71%, susceptibility is 90.00%, observing concordance rate (Po) is 94.44%, opportunity concordance rate (Pe) is 64.82%, к=(Po-Pe)/(1-Pe)=0.83, к > 0.6 expression utilizes the result of recombinant protein c oronin detection dog arch insect infection unanimously reliable.
Result shows that this recombinant protein detects dog arch insect infection and has higher Sensitivity and Specificity, high with the consistance of import reagent box.
Effect example 4
Improvement double crush syndrome carries out the detection of dog Infection of Toxoplasma Gondii acute infection
1) Coronin rabbit polyvalent antibody adds elisa plate to get 100 μ l after 1: 500 (v/v) dilution proportion, and 4 DEG C of bags, by 8h, establish positive and negative (500ng/ml recombinant protein) control wells, the repetition of two, each sample simultaneously;
2) discard serum, wash 3 times with PBST cleansing solution, the 37 DEG C of closed 2-3h of the gelatin with 1%;
3) discard confining liquid, wash 3 times with PBST cleansing solution, add 1: 100 (v/v) and dilute serum to be checked, hatch 1.5h for 37 DEG C;
4) discard confining liquid, wash 3 times with PBST cleansing solution, add the Coronin mouse polyvalent antibody that 1: 1000 (v/v) dilutes, hatch 1.5h for 37 DEG C;
5) discard antibody diluent, wash 3 times, add enzyme conjugates with PBST cleansing solution, press working concentration dilution with dilution, 100 μ l/ holes, hatch 1 hour for 37 DEG C;
6) discard enzyme labelled antibody, wash 6 times with cleansing solution, dry;
7) nitrite ion is added: in each reacting hole, add TMB nitrite ion 100 μ l/ hole, 37 DEG C, lucifuge 3-5 minute;
8) add stop buffer in every reacting hole, 50 μ l/ holes, microplate reader OD=450nm detects light absorption value,
9) result judges: detect aperture OD value/negative hole OD value >=2.1 are judged to be Infection of Toxoplasma Gondii acute infection.
Table 3 utilizes the acute infection of recombinant protein c oronin antibody test Infection of Toxoplasma Gondii
As can be seen from Table 3, acute infection dog OD value/negative control hole OD value > 2.1, effectively can detect the acute infection of dog Infection of Toxoplasma Gondii.
Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications correlated condition of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (11)
1. a method for the detection Infection of Toxoplasma Gondii acute infection of non-diseases diagnosis or therapeutic purposes, it is characterized in that, described method comprises the steps: the antibody of sample to be detected and Infection of Toxoplasma Gondii Coronin albumen or described Infection of Toxoplasma Gondii Coronin albumen is reacted.
2. detect the method for Infection of Toxoplasma Gondii acute infection as claimed in claim 1, it is characterized in that, the preparation method of described Infection of Toxoplasma Gondii Coronin albumen is Prokaryotic expression, purification method; And/or the preparation method of the antibody of described Infection of Toxoplasma Gondii Coronin albumen comprises the steps: the antibody of described Infection of Toxoplasma Gondii Coronin albumen obtained after Infection of Toxoplasma Gondii Coronin protein immune animal.
3. detect the method for Infection of Toxoplasma Gondii acute infection as claimed in claim 2, it is characterized in that, the preparation method of the antibody of described Infection of Toxoplasma Gondii Coronin albumen comprises the steps: described Infection of Toxoplasma Gondii Coronin protein immune animal, gathers blood and be separated the serum containing described antibody after secondary immunity.
4. as described in Claims 2 or 3, detect the method for Infection of Toxoplasma Gondii acute infection, it is characterized in that, described animal is rabbit or mouse; And/or described immunity for be expelled to described Infection of Toxoplasma Gondii Coronin albumen in described animal body together with Freund's adjuvant.
5. as described in claim 3 or 4, detect the method for Infection of Toxoplasma Gondii acute infection, it is characterized in that, the interval time of described secondary immunity is 2 weeks; And/or described collection blood is the tail venous collection from described animal.
6. detect the method for Infection of Toxoplasma Gondii acute infection as claimed in claim 1, it is characterized in that, described reaction comprises the steps:
(1) antibody of described Infection of Toxoplasma Gondii Coronin albumen or described Infection of Toxoplasma Gondii Coronin albumen is coated in matrix;
(2) matrix being coated with the antibody of described Infection of Toxoplasma Gondii Coronin albumen or described Infection of Toxoplasma Gondii Coronin albumen in step (1) adds sample to be detected, the antibody of itself and described Infection of Toxoplasma Gondii Coronin albumen or described Infection of Toxoplasma Gondii Coronin albumen is reacted.
7. detect the method for Infection of Toxoplasma Gondii acute infection as claimed in claim 6, it is characterized in that, described reaction also comprises step (3): the result of detecting step (2) described reaction.
8. detect an antibody for Infection of Toxoplasma Gondii acute infection, it is characterized in that, it is obtained by the preparation method by comprising the steps: by described antibody obtained after Infection of Toxoplasma Gondii Coronin protein immune animal.
9. detect the antibody of Infection of Toxoplasma Gondii acute infection as claimed in claim 8, it is characterized in that, it is according to any one of claim 2-5 obtained by method.
10. detect a kit for Infection of Toxoplasma Gondii acute infection, it is characterized in that, it antibody comprising as described in any one of claim 1-2 Coronin albumen and/or detect Infection of Toxoplasma Gondii acute infection as described in any one of claim 8-9.
11. kits detecting Infection of Toxoplasma Gondii acute infection as claimed in claim 10, it is characterized in that, it comprises immune response confining liquid, immune response stop buffer, coupling have the antibody of the anti-igg of chromogenic reaction enzyme, Coronin albumen and/or detect the antibody of Infection of Toxoplasma Gondii acute infection as described in any one of claim 8-9 as described in any one of claim 1-2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110108628A (en) * | 2019-06-04 | 2019-08-09 | 扬州大学 | The method that a kind of pair of toxoplasma tachyzoite directly counts |
| WO2023206773A1 (en) * | 2022-04-25 | 2023-11-02 | 中国科学院深圳先进技术研究院 | Protein rod-shaped body marker and use thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837688A (en) * | 2014-03-27 | 2014-06-04 | 吉林农业大学 | Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies |
| CN104155443A (en) * | 2014-08-06 | 2014-11-19 | 浙江大学 | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit based on toxoplasma gondii matrix protein 1 |
| CN104502605A (en) * | 2014-12-12 | 2015-04-08 | 天津拓瑞医药科技有限公司 | ELISA kit for detecting canine toxoplasmosis IgM antibody and preparation method of ELISA kit |
-
2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837688A (en) * | 2014-03-27 | 2014-06-04 | 吉林农业大学 | Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies |
| CN104155443A (en) * | 2014-08-06 | 2014-11-19 | 浙江大学 | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit based on toxoplasma gondii matrix protein 1 |
| CN104502605A (en) * | 2014-12-12 | 2015-04-08 | 天津拓瑞医药科技有限公司 | ELISA kit for detecting canine toxoplasmosis IgM antibody and preparation method of ELISA kit |
Non-Patent Citations (1)
| Title |
|---|
| JULIEN SALAMUN, ET AL.: "Structure of Toxoplasma gondii coronin, an actin-binding protein that relocalizes to the posterior pole of invasive parasites and contributes to invasion and egress", 《THE FASEB JOURNAL • RESEARCH COMMUNICATION》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110108628A (en) * | 2019-06-04 | 2019-08-09 | 扬州大学 | The method that a kind of pair of toxoplasma tachyzoite directly counts |
| WO2023206773A1 (en) * | 2022-04-25 | 2023-11-02 | 中国科学院深圳先进技术研究院 | Protein rod-shaped body marker and use thereof |
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