CN101819209A - Method for detecting foot and mouth disease (FMD) non-structural protein 3AB antibody by using dot immuno-gold filtration assay (DIGFA) - Google Patents
Method for detecting foot and mouth disease (FMD) non-structural protein 3AB antibody by using dot immuno-gold filtration assay (DIGFA) Download PDFInfo
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Abstract
The invention discloses a method for detecting a foot and mouth disease (FMD) non-structural protein 3AB antibody by using dot immuno-gold filtration assay (DIGFA). The method specifically comprises the following steps of: a) synthesizing a foot and mouth disease(FMD) non-structural protein 3AB; b) preparing SPA colloidal gold; c) manufacturing a reaction kit; and d) applying the reaction kit as a carrier, placing antigen sites of the 3AB non-structural protein synthesized in the step a) on an NC membrane, sealing the NC membrane and adding a sample to be detected, and after washing the NC membrane, using the SPA prepared in the step b) as the colloidal gold to mark the protein for detecting the FMD non-structural protein 3AB antibody. The method has the characteristics of simple, convenient and fast operation, no need of special instrument, bright spot color, strong specificity, high sensitivity, easy judgment of results and preservable detected samples.
Description
Technical field
The present invention relates to a kind of spot immune gold percolation (DIGFA) that utilizes and detect aftosa non-structural protein 3AB detection of antibodies method.
Background technology
(Foot and mouth disease FMD) is a kind of acute, hot, the height contagious disease of being suffered from altogether by the artiodactyl that the foot and mouth disease virus infection causes to aftosa.This disease is with mucous membrane of mouth, lingual surface, lip, asoscope, hoof and skin of breast generation blister and to fester be feature.OIE (OIE) classifies this disease first of the category-A zoonosis as.Because this disease incidence of disease height, velocity of propagation is fast, and popular scope is wide, in case break out then wayward and eliminate, has a strong impact on animal husbandry production and international trade, causes the tremendous economic loss, and the popular history of this disease is permanent, and it is very wide to distribute all over the world.The most of developing countries that comprise China mainly are the method prevention FMD by the injection inactivated vaccine.Therefore, the foundation antidiastole method that can distinguish inactivated vaccine immune animal and infection animal is very necessary.Ripe antidiastole method can reflect the infection state of FMDV in an area or the drove really, also is that OIE judges that an area or country have or not one of foundation of FMD.
The cause of disease of aftosa is foot and mouth disease virus (FMDV), belongs to the Picornaviridae Hostis.Virus has A, C, O, SAT I, SAT II, SAT III and seven principal modes of Asia I, hypotype is arranged again in each type, also has the strain of antigenic specificity in the hypotype, various between the antigenicity difference, and almost do not have cross-immunity, this has brought great difficulty for the diagnosis of FMD and anti-system.The FMDV genome contains a big open reading frame, the big polyprotein of encoding, polyprotein maturation step by step is cracked into virus structural protein (VP1, VP2, VP3, VP4) and non-structural protein (NSP:L, 2A, 2B, 2C, 3A, 3B, 3ABC, 3D etc.).The most of developing countries that comprise China prevent aftosa by the vaccine injection immunity, and also can detect foot-and-mouth disease antibody in the animal blood serum after the inoculation, utilizing the detected foot-and-mouth disease antibody of traditional foot-and-mouth disease virus antigen bag to fail to reach and distinguishing is wild virus infection animal or the purpose of inactivated vaccine immune animal.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide a kind of spot immune gold percolation that utilizes to detect aftosa non-structural protein 3AB detection of antibodies method, the present invention has easy and simple to handle quick, do not need special instruments and equipment, bright spot color, high specificity, sensitivity height, the result is easy to judge, and detects the characteristics that sample can be preserved.
For reaching above-mentioned purpose, the present invention adopts following technical scheme:
A kind of spot immune gold percolation that utilizes detects aftosa non-structural protein 3AB detection of antibodies method, specifically comprises the steps:
A) aftosa FMD 3AB non-structural protein is synthetic;
B) preparation of SPA collaurum;
C) making of reaction kit;
D) the application response kit is as carrier, earlier 3AB non-structural protein antigen synthetic in the step a) is put on the NC film, add testing sample after the sealing, washing back with the SPA for preparing in the step b) as colloid gold label Protein Detection aftosa non-structural protein 3AB antibody.
The synthesis step of described aftosa FMD 3AB non-structural protein is: at first through the GenBank retrieval analysis, choose the 3AB gene with reference to the Asia strain (AY687333) of having announced, degeneracy and colibacillary codon hobby property principle according to codon, under the prerequisite that does not influence amino acid sequence, revise the part rare codon in the 3AB sequence, it is divided into 9 long primers, 12 complementary seriess of design between each long primer, obtain 2 3AB genes through overlapping PCR method, the nucleotide sequence of gene is respectively:
SEQIDNO.1：
P?3AB 5’NdeI 5’-GGAATTC
CATATGATCAGCATTCCG-3’；
SEQIDNO.2：
P?3AB 3’SalI 5’-ACGC
GTCGACCTCAGT?GACAATCAA?3’；
The restriction enzyme site of introducing in above-mentioned 2 3AB genes is NdeI and SalI then; The expression vector PET-28 (a+) that is introduced (purchase the company in Novagen, the commission merchant is the grand biotechnology Development Co., Ltd of Xiamen aigret); Utilize expression vector PET-28 (a+) to make up recombinant expression plasmid, be transformed in the bacillus coli DH 5 alpha competent cell, screening recombinant plasmid, called after PET-28-3AB; Then carry out the abduction delivering of recombinant plasmid; Carry out purifying and renaturation after then the thalline behind the abduction delivering being utilized the Ultrasonic Cell Disruptor fragmentation.
The prescription of the ultrasonication damping fluid that adopts during described ultrasonication is: 20mMTris-HCl, 0.5M NaCl, 1Mm EDTA PH8.0,1mg/ml lysozyme (BBI is original-pack, the agency of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).
The inclusion body cleansing solution prescription that adopts in the described purge process is: 20mM Tris-HClPH8.0,0.5M NaCl, 2M carbonyl diamide (Urea), 2% Triton X-100 (Triton); The inclusion body sex change buffer formulation that adopts is: 20mM Tris-HCl PH8.0,0.5M NaCl, 8M carbonyl diamide (Urea), 100mM beta-mercaptoethanol, 2% Triton X-100 (Triton); The renaturation buffer prescription that adopts in the described renaturation process is: 20mM Tris-HCl PH8.0,50Mm NaCl, 1% glycerine.
Described SPA is for being staphylococcus aureus reorganization A albumen (purchasing in Beijing Vector Gene Technology Co., Ltd).
The preparation of described SPA collaurum comprises adopts trisodium citrate reduction method to prepare colloidal gold solution, dropwise adds staphylococcus aureus reorganization A albumen then in the colloidal gold solution; The resuspended damping fluid that is wherein adopted is for containing 1% casein, 0.05% Macrogol 6000 (PEG6000), 0.05% Tween-20, the PBS damping fluid of 0.01mol/L pH7.2; The collaurum dilution that is adopted is for containing 1% casein, 0.05% Macrogol 6000 (PEG6000), 5% sucrose, 0.05% Tween-20, the PBS damping fluid of 0.01mol/L pH7.2.
Described reaction kit is the square capsules of plastics, and size is 4.8cm * 3cm * 0.7cm, divides the end and lid two parts, and capping has diameter 0.5cm circular hole, has two-layerly in the box, and ground floor is nitrocellulose filter (a NC film), is close to the second layer under the NC film and is the suction paper washer.
Described step d) is: point two points on the NC film in the reaction kit circular hole are aftosa FMD 3AB antigen 1 ul a bit, as check point; The porcine blood serum 1uL that another point is known aftosa FMD3AB antibody positive is as the Quality Control point; Add the 100uL confining liquid after the drying at room temperature, infiltrate dry back and add serum 100uL to be checked; Infiltrate the back and add the 100uL flushing; Infiltrate the back and add the good SPA collaurum 80uL of mark; Add cleansing solution 100uL after waiting to infiltrate again, the unconjugated SPA collaurum of flush away; In the 5min if on film check point and Quality Control point punctation all to occur promptly positive, then negative as if having only the Quality Control point punctation to occur, and punctation does not all appear in check point and Quality Control point, illustrates that then reagent is invalid.
Described confining liquid is the PBS damping fluid that contains the 0.01mol/L pH 7.2 of 1% casein and 0.05% Tween-20; Described cleansing solution is the PBS damping fluid that contains the 0.01mol/LpH 7.2 of 0.05% Tween-20; Described PBS buffer formulation is: NaH
2PO
4H
2O 0.276g, anhydrous Na
2HPO
41.136g, NaCl 8.76g, KCl 0.187g is settled to 1000mL after adding an amount of dissolved in distilled water.
Concentration is 0.0875mg/mL during described aftosa FMD 3AB antigen protein point sample.
The invention has the beneficial effects as follows: at first in the production of vaccine process, the non-structural protein of the overwhelming majority is eliminated, so behind the immune animal, does not produce non-structural protein antibody in the body; And the expression of non-structural protein is arranged in the natural infected animal body, can stimulate the generation corresponding antibody.The present invention is as antigen with recombinant expressed aftosa 3AB non-structural protein, utilize the characteristic of SPA collaurum, research colloidal gold immunity percolation technology fast detecting aftosa non-structural protein 3AB antibody is set up a kind of method for quick that can distinguish aftosa vaccine immune animal and wild virus infection.Main application miillpore filter (NC film) is made the immunoassay technology of carrier, earlier antigen is put on the NC film, adds testing sample after the sealing, and corresponding antibodies is detected with colloidal gold probe in the washing back.Amplify immune response system by gold grain, reaction result is shown on solid phase carrier NC film.This method is easy and simple to handle, quick, does not need special instruments and equipment, and bright spot color is easy to judge, and detects sample and can preserve.The present invention is that the foot and mouth disease virus 3AB non-structural protein of purifying is antigen coated on nitrocellulose membrane, utilizes colloid gold label SPA colour developing, sets up a kind of method for quick that can distinguish aftosa vaccine immunity and wild virus infection animal.Simultaneously 150 parts of porcine blood serum are detected by using DIGFA method and ELISA method, DIGFA method positive rate is 6% as a result, and ELISA method positive rate is 5.3%, and both reach 99.3% by coincidence rate.The foot and mouth disease virus 3AB non-structural protein antigentic specificity that the present invention selects for use is good, be used to detect foot and mouth disease virus 3AB non-structural protein antibody with the SPA composition detection kit of colloid gold label, has high specificity, the sensitivity height, simple to operate, weak point consuming time, the result is easy to judge, and with aftosa immune swine serum, pig parvoviral, swine fever, pig blue-ear disease and PRV positive serum cross reaction does not take place.The successful development of this method can judge whether infect foot and mouth disease virus in the animal quickly and easily, will play a significant role for effectively controlling this disease.So especially to raising pigs and China of cowboying big country, set up easy, to distinguish aftosa vaccine antibody fast and accurately still be the detection of antibodies method that wild virus infection produces so that be widely applied to numerous raisers more, detect at any time; Animal doctor department of basic unit regularly carries out large tracts of land generaI investigation and monitoring and port one line department carries out the clearance that speeds passage through customs, and this plays effect very energetically for the propagation of controlling aftosa effectively, can provide diagnosis basis for preventing these infectious diseases.
Description of drawings
Fig. 1 is the selection experimental result picture of the best point sample antigen concentration of FMD of the present invention;
Fig. 2 is a kind of prescription of the present invention, the selection experimental result picture of the confining liquid of different amounts;
Fig. 3 is the another kind of prescription of the present invention, the selection experimental result picture of the confining liquid of different amounts;
Fig. 4 is the selection experimental result picture of colloid gold label SPA optium concentration of the present invention.
Embodiment
The present invention is further detailed explanation below in conjunction with accompanying drawing.
1, the at first synthetic FMD 3AB non-structural protein of present embodiment
Expressing the used antigen of primer and recombinant expression structure is the aftosa 3AB albumen of prokaryotic expression, synthesizing of FMD non-structural protein 3AB gene through the GenBank retrieval analysis, choose the 3AB gene with reference to the Asia strain (AY687333) of having announced, degeneracy and colibacillary codon hobby property principle according to codon, under the prerequisite that does not influence amino acid sequence, revise the part rare codon in the 3AB sequence, it is divided into 9 long primers, 12 complementary seriess of design between each long primer, obtain 2 3AB genes through overlapping PCR method, the nucleotide sequence of gene is respectively:
SEQIDNO.1：
P?3AB 5’NdeI 5’-GGAATTC
CATATGATCAGCATTCCG-3’；
SEQIDNO.2：
P?3AB 3’SalI 5’-ACGC
GTCGACCTCAGT?GACAATCAA?3’。
Introduce restriction enzyme site NdeI and SalI expression vector PET-28 (a
+) make up recombinant expression plasmid, be transformed into DH5 α competence, screening recombinant plasmid, called after PET-28-3AB.
The expression of the recombinant plasmid correct expression plasmid that will check order is transformed into expression strain BL21 (DE3) competence, the positive single bacterium colony of picking is in the LB nutrient culture media that contains kanamycins, by be inoculated at 1: 100 the LB medium culture that contains 50 μ g/ml kanamycins o'clock add to OD0.6～0.8 IPTG to final concentration be 1mmol/L, collect thalline behind the abduction delivering 6h, sampling 12%SDS-PAGE observation analysis result.
The purifying of expressing protein, the renaturation thalline after with abduction delivering is with ultrasonication damping fluid (20mM Tris-HCl, 0.5M NaCl, 1Mm EDTA, PH8.0, the 1mg/ml lysozyme) presses the resuspended precipitation of 20ml/g thalline, carry out ultrasonication, collect cleer and peaceful precipitation in the ultrasonication respectively with Ultrasonic Cell Disruptor, sampling is carried out SDS-PAGE and is analyzed observation destination protein expression.Ultrasound precipitation is through inclusion body cleansing solution (20mM Tris-HCl PH8.0,0.5M NaCl, 2M Urea, 2%Triton) washing back inclusion body sex change damping fluid (20mM Tris-HCl PH8.0,0.5MNaCl, 8M Urea 100mM beta-mercaptoethanol, 2%Triton) resuspended, 4 ℃ of stirrings are spent the night.Inclusion body after the dissolving carries out purifying with the nickel ion affinity chromatograph post, and purification process carries out with reference to instructions.Albumen renaturation buffer (20mM Tris-HCl PH8.0 with the purifying wash-out, 50MmNaCl, 1% glycerine) after one times of the dilution again with renaturation buffer to 4 renaturation of ℃ dialysing, change liquid once every 6～8h, the 24h that dialyses altogether is at last with ultrapure water or with the 5mM Tris-HClPH8.0 secondary of dialysing.Can concentrate with sucrose or PEG20000 for improving protein concentration, the albumen after concentrating is measured protein content with ultraviolet spectrophotometer, and-80 ℃ of preservations are standby.
2, the preparation of SPA collaurum
1) preparation of colloidal gold solution: prepare with trisodium citrate reduction method, getting 1% gold chloride 1mL joins in the 99mL ultrapure water, gained chlorauric acid solution concentration is 0.01%, be heated to boiling with water-bath magnetic agitation pot, under magnetic stirring apparatus constantly stirs with certain rotating speed, add 1% trisodium citrate aqueous solution of 2mL rapidly, continue the about 15min of agitating heating and stablize constant to solution colour.The collaurum that this method makes is vinicolor, surveys its absorption peak at the 520nm place through ultraviolet spectrophotometer, and the colloid gold particle diameter that obtains is about about 20nm, and the rearmounted 4 ℃ of refrigerators of cooling keep in Dark Place.
2) SPA mark: get the colloidal gold solution that needs preparation amount, use 0.1mol/L K
2CO
3Transfer its pH to 6.0 with the HCl of 0.1mol/L.Under magnetic agitation, dropwise add SPA (every mL colloidal gold solution adds 6ug SPA) then, continue to add 5%PEG (MW6000) to final concentration 0.1% behind the magnetic agitation 30min, continue again to stir 15min, place 4 ℃ of refrigerators at last, standing over night.The centrifugal 10min of SPA collaurum 4000r/min that next day is good with mark gets the centrifugal 30min of supernatant 12000r/min, and careful the suction abandoned supernatant.Precipitation returns to original volume with resuspended damping fluid (containing 1% casein, 0.05%PEG6000,0.05%Tween-20, the PBS damping fluid of 0.01mol/L pH7.2).The centrifugal 30min of 12000r/min abandons supernatant.Precipitation returns to 1/10th of original volume with collaurum dilution (containing 1% casein, 0.05%PEG6000,5% sucrose, 0.05%Tween-20, the PBS damping fluid of 0.01mol/L pH7.2), and it is standby to put 4 ℃ of refrigerators.The PBS buffer formulation is: NaH
2PO
4H
2O 0.276g, anhydrous Na
2HPO
41.136g, NaCl 8.76g, KCl 0.187g is settled to 1000mL after adding an amount of dissolved in distilled water.
3, the making of reaction kit
Reaction box is the square capsules of plastics, and size is 4.8cm * 3cm * 0.7cm, divides the end and lid two parts, and capping has diameter 0.5cm circular hole, has two-layerly in the box, is nitrocellulose filter (NC film) towards the ground floor of lid, is close to second layer suction paper washer under the NC film.
4, the application response kit is as carrier, earlier 3AB non-structural protein antigen synthetic in the step a) is put on the NC film, add testing sample after the sealing, washing back with the SPA for preparing in the step b) as colloid gold label Protein Detection aftosa non-structural protein 3AB antibody.
1) point two points on the NC film of concrete steps in circular hole are FMD 3AB antigen 1 ul a bit, as check point; The porcine blood serum 1uL that another point is known FMD 3AB antibody positive is as the Quality Control point; Adding 100uL confining liquid (the PBS damping fluid that contains the 0.01mol/L pH 7.2 of 1% casein and 0.05% Tween-20) PBS buffer formulation after the drying at room temperature is: NaH
2PO
4H
2O 0.276g, anhydrous Na
2HPO
41.136g, NaCl 8.76g, KCl0.187g is settled to 1000mL after adding an amount of dissolved in distilled water.Infiltrate dry back and add serum 100uL to be checked; Infiltrate the back and add 100uL cleansing solution (the PBS damping fluid that contains the 0.01mol/LpH 7.2 of 0.05% Tween-20) flushing (can according to circumstances wash 1 time～3 times); Infiltrate the back and add the good SPA collaurum 80uL of mark; Add cleansing solution 100uL (1 time～3 times) after waiting to infiltrate again, the unconjugated SPA collaurum of flush away.In the 5min if on film check point and Quality Control point punctation all to occur promptly positive, then negative as if having only the Quality Control point punctation to occur, and punctation does not all appear in check point and Quality Control point, illustrates that then reagent is invalid.
2) selection of best point sample antigen concentration is carried out doubling dilution with the FMD 3AB antigen of recombinant expressed purifying with the 0.05mol/L carbonate buffer solution of pH 9.6, then according to concentration height point sample successively on the NC film, carry out the DIGFA experiment respectively, as shown in Figure 1, the experiment done of present embodiment is: No. 1 plate is for detecting FMDV 3AB positive serum result; To be followed successively by concentration be 0.35,0.175,0.0875,0.044 and the reorganization 3AB antigen of 0.022mg/ml in order to detect FMDV 3AB negative serum result 1～5. for No. 2 plates; C. Quality Control point.As seen from Figure 1: point sample antigen amount minimum and the most clear antigen concentration of experimental result spot colors are as best point sample antigen concentration.Experimental result shows, FMDV 3AB albumen is during with the 0.0875mg/mL point sample, and antigen coated amount minimum and spot colors are clear.Therefore select the FMDV 3AB albumen point sample 1 μ L of 0.0875mg/ml as the antigen coated amount of the best.
3) selection of confining liquid is carried out the DIGFA experiment with the confining liquid sealing NC film of different formulations, as shown in Figures 2 and 3, the experiment done of present embodiment is: the used confining liquid of Fig. 2 is: (1) contains the PBS of the 0.01mol/L pH7.2 of 1% casein, 0.5%Tween-20; (2) contain the PBS of the 0.01mol/L pH7.2 of 0.5% casein, 0.5%Tween-20; (3) contain the PBS of the 0.01mol/L PH7.2 of 1% casein, 0.05%Tween-20; (4) contain the PBS of the 0.01mol/L PH7.2 of 0.5% casein, 0.05%Tween-20; The used confining liquid of Fig. 3 is: (1) contains the PBS of the 0.01mol/L pH7.2 of 1%BSA, 0.5%Tween-20; (2) contain the PBS of the 0.01mol/L pH7.2 of 0.5%BSA, 0.5%Tween-20; (3) contain the PBS of the 0.01mol/L pH7.2 of 1%BSA, 0.05%Tween-20; (4) contain the PBS of the 0.01mol/L pH7.2 of 0.5%BSA, 0.05%Tween-20.And experimental result compared, select spot colors clear and background is more shallow one group as the suitableeest confining liquid.The result shows, best with the PBS sealing effect of the 0.01mol/L pH7.2 of the PBS of the 0.01mol/L pH7.2 that contains 1% casein, 0.05%Tween-20 and 0.5% casein, 0.05%Tween-20, the spot colour developing is clear, and background is more shallow.
4) selection of colloid gold label SPA optium concentration is done the NC membrane interaction that FMDV 3AB antigen is arranged with point behind the doubling dilution with the SPA collaurum, selects shallow and the extension rate that effect is best of background color as colloid gold label SPA concentration.As shown in Figure 4, the experiment that present embodiment is done is: 1～No. 4 plate is respectively the SPA collaurum that dilutes by original volume 1/15,1/10,1/5,1/2.5, and the result shows that the extension rate effect of pressing original volume 1/10 is better, and background color is shallow.
Claims (10)
1. one kind is utilized spot immune gold percolation to detect aftosa non-structural protein 3AB detection of antibodies method, it is characterized in that specifically comprising the steps:
A) aftosa FMD 3AB non-structural protein is synthetic;
B) preparation of SPA collaurum;
C) making of reaction kit;
D) the application response kit is as carrier, earlier 3AB non-structural protein antigen synthetic in the step a) is put on the NC film, add testing sample after the sealing, washing back with the SPA for preparing in the step b) as colloid gold label Protein Detection aftosa non-structural protein 3AB antibody.
2. a kind of spot immune gold percolation that utilizes as claimed in claim 1 detects aftosa non-structural protein 3AB detection of antibodies method, the synthesis step that it is characterized in that described aftosa FMD 3AB non-structural protein is: at first through the GenBank retrieval analysis, choose the 3AB gene with reference to the Asia strain (AY687333) of having announced, degeneracy and colibacillary codon hobby property principle according to codon, under the prerequisite that does not influence amino acid sequence, revise the part rare codon in the 3AB sequence, it is divided into 9 long primers, 12 complementary seriess of design between each long primer, obtain 2 3AB genes through overlapping PCR method, the nucleotide sequence of gene is respectively:
SEQIDNO.1：
P?3AB?5’NdeI?5’-GGAATTC
CATATGATCAGCATTCCG-3’；
SEQIDNO.2：
P?3AB?3’SalI?5’-ACGC
GTCGACCTCAGT?GACAATCAA?3’；
The restriction enzyme site of introducing in above-mentioned 2 3AB genes is NdeI and SalI then; The expression vector PET-28 (a+) that is introduced; Utilize expression vector PET-28 (a+) to make up recombinant expression plasmid, be transformed in the bacillus coli DH 5 alpha competent cell, screening recombinant plasmid, called after PET-28-3AB; Then carry out the abduction delivering of recombinant plasmid; Carry out purifying and renaturation after then the thalline behind the abduction delivering being utilized the Ultrasonic Cell Disruptor fragmentation.
3. the preparation method of ocean as claimed in claim 2 ballstone phycovirus shell recombinant protein prokaryotic expression, the prescription of the ultrasonication damping fluid that adopts when it is characterized in that described ultrasonication is: 20mM Tris-HCl, 0.5M NaCl, 1Mm EDTA PH8.0,1mg/ml lysozyme.
4. the preparation method of ocean as claimed in claim 2 ballstone phycovirus shell recombinant protein prokaryotic expression, it is characterized in that the inclusion body cleansing solution prescription that adopts in the described purge process is: 20mM Tris-HCl PH8.0,0.5M NaCl, the 2M carbonyl diamide, 2% Triton X-100; The inclusion body sex change buffer formulation that adopts is: 20mM Tris-HCl PH8.0,0.5M NaCl, 8M carbonyl diamide, 100mM beta-mercaptoethanol, 2% Triton X-100; The renaturation buffer prescription that adopts in the described renaturation process is: 20mM Tris-HClPH8.0,50Mm NaCl, 1% glycerine.
5. the preparation method of ocean as claimed in claim 1 ballstone phycovirus shell recombinant protein prokaryotic expression is characterized in that described SPA is for being staphylococcus aureus reorganization A albumen.
6. as the preparation method of claim 1 or 5 described ocean ballstone phycovirus shell recombinant protein prokaryotic expressions, the preparation that it is characterized in that described SPA collaurum comprises that the employing trisodium citrate reduction method prepares colloidal gold solution, dropwise adds staphylococcus aureus reorganization A albumen then in the colloidal gold solution; The resuspended damping fluid that is wherein adopted is for containing 1% casein, 0.05% Macrogol 6000,0.05% Tween-20, the PBS damping fluid of 0.01mol/L pH7.2; The collaurum dilution that is adopted is for containing 1% casein, 0.05% Macrogol 6000,5% sucrose, 0.05% Tween-20, the PBS damping fluid of 0.01mol/L pH7.2.
7. the preparation method of ocean as claimed in claim 1 ballstone phycovirus shell recombinant protein prokaryotic expression, it is characterized in that: described reaction kit is the square capsules of plastics, size is 4.8cm * 3cm * 0.7cm, divide the end and lid two parts, capping has diameter 0.5cm circular hole, have two-layerly in the box, ground floor is a nitrocellulose filter, is close to the second layer under the NC film and is the suction paper washer.
8. the preparation method of ocean as claimed in claim 1 ballstone phycovirus shell recombinant protein prokaryotic expression, it is characterized in that described step d) is: point sample on the NC film in the reaction kit circular hole, be aftosa FMD 3AB antigen 1 ul a bit, as check point; The porcine blood serum 1uL that another point is known aftosa FMD 3AB antibody positive is as the Quality Control point; Add the 100uL confining liquid after the drying at room temperature, infiltrate dry back and add serum 100uL to be checked; Infiltrate the back and add the 100uL flushing; Infiltrate the back and add the good SPA collaurum 80uL of mark; Add cleansing solution 100uL after waiting to infiltrate again, the unconjugated SPA collaurum of flush away; Promptly positive in the 5min if punctation on film, occurs, otherwise negative.
9. the preparation method of ocean as claimed in claim 8 ballstone phycovirus shell recombinant protein prokaryotic expression, it is characterized in that: described confining liquid is the PBS damping fluid that contains the 0.01mol/L pH 7.2 of 1% casein and 0.05% Tween-20; Described cleansing solution is the PBS damping fluid that contains the 0.01mol/LpH 7.2 of 0.05% Tween-20; The PBS buffer formulation is: NaH
2PO
4H
2O 0.276g, anhydrous Na
2HPO
41.136g, NaCl 8.76g, KCl0.187g is settled to 1000mL after adding an amount of dissolved in distilled water.
10. the preparation method of ocean as claimed in claim 8 ballstone phycovirus shell recombinant protein prokaryotic expression is characterized in that: concentration is 0.0875mg/mL during described aftosa FMD 3AB antigen protein point sample.
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| CN102393455A (en) * | 2011-08-26 | 2012-03-28 | 厦门出入境检验检疫局检验检疫技术中心 | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus |
| CN102393455B (en) * | 2011-08-26 | 2014-01-29 | 厦门出入境检验检疫局检验检疫技术中心 | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus |
| CN103792351A (en) * | 2014-01-23 | 2014-05-14 | 新疆纳秒脉冲技术应用研究所 | Method for rapidly detecting gram bacteria as well as kit |
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