CN106546742A - A kind of gold mark detection test paper bar based on goat capripoxviruses and preparation method thereof - Google Patents
A kind of gold mark detection test paper bar based on goat capripoxviruses and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of gold mark detection test paper bar based on goat capripoxviruses, it includes PVC offset plates, nitrocellulose filter, gold standard pad, sample pad and absorbent paper.One end of PVC offset plates is stained with sample pad, and its other end is stained with the absorbent paper, is stained with gold standard pad, nitrocellulose filter in the middle part of which successively；One end of gold standard pad is mutually adhered to sample pad, and its other end is mutually adhered to nitrocellulose filter, and the nitrocellulose filter is mutually adhered to absorbent paper；Gold standard pad has been coated with the anti-GTPV P32 protein monoclonal antibodies of colloid gold label；It is sequentially provided with the detection line of the anti-GTPV ORF122 protein monoclonal antibodies of the linear coating of the concentration of 1mg/mL, with the nature controlling line of the linear coating goat anti-mouse igg antibody of the concentration of 1mg/mL along sample flow direction on nitrocellulose filter.Meanwhile, the invention also discloses the preparation method of the test strips.Test strips of the present invention, with high specificity, good stability, it is simple to operate, detect it is quick the characteristics of, be suitable for animal epidemic occur Site Detection.
Description
Technical field
The present invention relates to virus detection techniques field, more particularly to a kind of gold mark detection test paper bar based on goat capripoxviruses
And preparation method thereof.
Background technology
Goat capripoxviruses（GTPV）It is goatpox（Goat Pox , GTP）Pathogen, main harm goat and sheep,
Ill sheep occurs to generate heat, varioliform exanthema, respiratory tract and injury to alimentary tract occur on skin and mucosa and lymphadenectasis is characterized
Symptom, is a kind of acute, hot, contagious disease.The disease is in endemicity, popular in China and countries in the world, outburst
Or huge loss is caused to local sheep husbandry and economy when popular.
Goat capripoxviruses Genome Size is 143 ~ 147 kb.P32 genes are located at 64 ~ 65kb of GTPV genomes.P32
With antigenic specificity, P32 contains an antigenic determinant, and the envelope protein being made up of 319 ~ 324 aminoacid, c-terminuses have
One membrane spaning domain, to the toxic effect of cell.The structural protein of goat capripoxviruses P32, with immunogenicity.ORF122 eggs
It is the glycoprotein on GTPV EEV cyst membranes in vain, with vaccinia viruss A33R albumen homology, it may be possible to the principal immune protectiveness of GTPV
One of antigen, may play a significant role in the immunne response of GTPV.Detection GTPV conventional method mainly includes at present：Disease
The methods such as malicious isolation identification method, indirect hemagglutination test method, AGP methods, immunofluorescence assay, neutralization test method, PCR detection sides.
Virus Isolation method is can to do tentative diagnosis by epidemiology, pathological changes and clinical symptoms, and further making a definite diagnosis needs
Laboratory separation and Culture identifying virus, isolation identification virus are wanted typically to adopt cell culture processes.The method is at present more
General method, is characterized in accurate, simple to operate, but needs the time longer, resultant error is larger, manpower and materials spend compared with
Greatly, the quick detection needs being not suitable with practical application.Indirect hemagglutination test method, it is red using rabbit-anti goat capripoxviruses IgG sensitization
The reverse indirect hemagglutination assay that cell is set up, has specificity and sensitivity to goat capripoxviruses antigen, with other animal viruss
There is no cross reaction in antigen, more sensitive than AGP method, and sensitized erythrocyte is coagulation stable, be it is a kind of it is sensitive, simple to operate,
Quick detection method, but need a large amount of preparations before testing, needs professional to operate, and needs particular instrument, and result
Error is also easy to produce in judgement.AGP methods are presently the most conventional detection method, simple to operate, but need the long period, resultant error
Larger, sensitivity is low, is not suitable for their early stage diagnosis.Immunofluorescence assay, the combination resisted by fluorescein labelling two will
Signal amplifies, and is identified by fluorescence microscope, improves the sensitivity of detection to a certain extent, but in immunofluorescence
Conventional fluorescent antibody is not sufficiently stable, and judges upper subjective, cross reaction easily occurs, while needing the spies such as fluorescence microscope
Different equipment, using when receive many conditionalities.Neutralization test method is cumbersome, and accuracy is high, but needs the long period, needs special
Industry human users, need to spend substantial amounts of manpower.PCR methods are the primers by designing conserved sequence on GTPV genomes, then are led to
Cross PCR amplifications, detected, the method detection sensitivity is high, reliable results, but assay method is loaded down with trivial details, time-consuming, efficiency is low, into
This height, is disturbed by other compositions in sample, needs special testing equipment, while operator need Jing professional trainings, it is difficult to general
And promote.
Immunochromatography technique is a kind of immunology detection technology of simple and fast developed in recent years, is with gold colloidal
For the tachysynthesises binding tests of label, antigen, antibody are reacted on NC films.The technology has high specificity, sensitive
Degree height, good stability, easy to operate, quick, visual result, without instrument and equipment the advantages of, operator are without the need for professional training
It is i.e. operable, with extensive using value.
The content of the invention
The technical problem to be solved is to provide a kind of quick, easy gold mark detection based on goat capripoxviruses
Test strips and preparation method thereof.
Another technical problem to be solved by this invention is to provide the gold mark detection test paper bar based on goat capripoxviruses
Preparation method.
To solve the above problems, a kind of gold mark detection test paper bar based on goat capripoxviruses of the present invention, its feature
It is：It includes PVC offset plates, nitrocellulose filter, gold standard pad, sample pad and absorbent paper；One end of the PVC offset plates is stained with
The sample pad, its other end are stained with the absorbent paper, are stained with the gold standard pad, the celluloid in the middle part of which successively
Film；One end of the gold standard pad is mutually adhered to the sample pad, and its other end is mutually adhered to the nitrocellulose filter, the nitric acid
Cellulose membrane is mutually adhered to the absorbent paper；The gold standard pad has been coated with the anti-GTPV-P32 protein monoclonals of colloid gold label and has resisted
Body；It is sequentially provided with along sample flow direction with the anti-GTPV- of the linear coating of the concentration of 1mg/mL on the nitrocellulose filter
The detection line of ORF122 protein monoclonal antibodies, with the nature controlling line of the linear coating goat anti-mouse igg antibody of the concentration of 1mg/mL.
The anti-GTPV-P32 protein monoclonal antibodies and the anti-GTPV-ORF122 protein monoclonal antibodies are by hybridization
Oncocyte secretion is produced.
The detection line is with the nature controlling line at intervals of 0.5cm.
A kind of preparation method of the gold mark detection test paper bar based on goat capripoxviruses as above, comprises the following steps：
(1) anti-GTPV-P32 protein monoclonal antibodies are prepared：
P 32 gene of goat capripoxvirus fragment is connected in pET28a (+) prokaryotic expression carrier, Jing IPTG are induced and expressed, P32
Albumen obtains high efficient expression in e. coli bl21, extracts albumen as immunizing antigen；
Goat capripoxviruses P32 protein immunizations BALB/C mice is prepared into anti-goatpox according to conventional monoclonal anti Antibody Production Techniques
Viral P32 protein monoclonal antibodies, and purification is carried out, obtain final product anti-goat capripoxviruses P32 protein monoclonal antibodies after purification；
(2) anti-GTPV-ORF122 protein monoclonal antibodies are prepared：
Goat capripoxviruses ORF122 genetic fragments are connected in pET32a (+) prokaryotic expression carrier, Jing IPTG are induced and expressed,
ORF122 albumen obtains high efficient expression in e. coli bl21, extracts albumen as immunizing antigen；
Goat capripoxviruses ORF122 protein immunizations BALB/C mice is prepared into anti-mountain according to conventional monoclonal anti Antibody Production Techniques
Capripox viruses ORF122 protein monoclonal antibodies, and purification is carried out, obtain final product anti-goat capripoxviruses ORF122 albumen lists after purification
Clonal antibody；
(3) the anti-goat capripoxviruses ORF122 protein monoclonal antibodies after purification are coated onto into the nitrocellulose filter after processing
On, a region of the nitrocellulose filter after coating process obtains detection line；The goat anti-mouse igg antibody of purification is coated with
Another region of nitrocellulose filter after process, obtains nature controlling line；
(4) by the nitrocellulose filter of the step (3) gained room temperature envelope in 0.05moL/L and in the Tris-HCL solution of pH=8.5
10 ~ 60min is closed, then with 0.01M and after the PBST rinsings twice of pH=8.5, in 4 DEG C of drying for standby；
(5) the nitrocellulose filter of the step (4) gained is adhered to into the middle part of PVC offset plates；
(6) colloid gold label protein solution is prepared：
1. glass drying oven used is cleaned up, is immersed in 24h in washing liquid；Rinsed with tap water after taking-up, distilled water flushing 3
It is secondary, glass drying oven used is soaked in the chloroformic solution containing the dichlorodimethylsilane that mass concentration is 5% after drying
1min, deionized water are rinsed, drying for standby；
2. the gold chloride that 100mL mass concentrations are 0.01% is added in the glass drying oven used after 1. processing to the step described in,
After on magnetic stirring apparatuss, agitating heating is boiled, it is 1% sodium citrate solution newly matched somebody with somebody to be rapidly added 2.5mL mass concentrations, continues to add
Heat boils 5 ~ 10min, and solution colour is gradually changed into orange red, lovely luster, stops boiling, and moisturizing is to volume by navy blue
For 100mL, colloidal gold solution is obtained；The colloidal gold solution sediments microscope inspection, makes the gold grain of the colloidal gold solution of preparation big
It is little consistent, uniformly, 4 DEG C closed keep in dark place it is standby；
3. with the K of 0.1moL/L2CO3The pH value of the colloidal gold solution is adjusted to into 8.5, magnetic agitation is transferred in lucifuge condition
Device is slowly stirred, and takes the anti-goat capripoxviruses P32 protein monoclonal antibodies after purification and is added dropwise to final concentration of 50 μ g/
ML, continues stirring 30min, stands 10min, add stabilizer BSA so as to which mass concentration reaches 1%, then is slowly stirred 30min,
Obtain final product gold mark monoclonal antibody solution；
4. the gold is marked into monoclonal antibody solution in 4 DEG C of speed centrifugation 10min with 2000r/min, removes gold colloidal polymerization therein
Thing, obtains supernatant A；The supernatant A in 4 DEG C of speed centrifugation 60min with 12000r/min, is now centrifuged thing and is divided into three again
Layer：Supernatant B contains still unconjugated monoclonal antibody, and nethermost atrouss speckle is still unconjugated colloid gold particle polymerization
Thing, it is middle that the gold mark monoclonal antibody complex being well combined is in mauve flocculent deposit；Collection aubergine flocculent deposit, and
The PBS suspensions of the 10mmoL/L and pH=7.2 of supernatant A volume described in 1/10 are added to dissolve in the aubergine flocculent deposit, 4
DEG C keep in dark place, obtain final product colloid gold label protein solution；
(7) glass fibre element film is immersed in into 30min in the Tris-HCL solution of pH=5.2 and 0.02moL/L first, 4 DEG C are overnight done
It is dry；Again glass fibre membrane is immersed in the anti-GTPV-P32 protein monoclonal antibodies solution of colloid gold label, Jing is impregnated with, dries,
Obtain final product gold standard pad；The gold standard pad is dried to constant weight at 37 DEG C, is adhered on the nitrocellulose filter away from the matter
One end of control line；
(8) sample pad is immersed in into 30min in the Tris-HCL solution of pH=5.2 and 0.02moL/L, 4 DEG C of dried overnights are obtained
Sample pad after process；
(9) the sample pad in one end of the gold standard pad after the process is stained with, its other end adhere to described step (4) gained
Nitrocellulose filter；Sample pad after the process adheres to one end of the PVC offset plates；Absorbent paper and the step (4) gained
Nitrocellulose filter adhesion, and adhere to the other end of the PVC offset plates.
(4) the Tris-HCl solution of middle 0.05mol/L and pH=8.5 is referred to and is contained in 100mL Tris solution the step
The Tris solution of 1g BSA, 0.2mL Tween-20.
1. middle washing liquid is referred to potassium dichromate 1000g with distilling after water dissolution on a small quantity the step, is slowly added along chamber wall
Enter concentrated sulphuric acid 2500mL, after mixing, add distilled water and mix to 10000mL and obtain final product.
4. the PBS suspensions of middle 10mmol/L and pH=7.2 are referred to and contain 1g in 100mL PBS solutions the step
The PBS solution of BSA, 1gPVP, 0.02g sodium azide.
(7) the step is each meant in 100mL with the Tris-HCL solution of the step (8) middle pH=5.2 and 0.02mol/L
Tris solution containing 3g BSA, 0.1mL Tween-20 in Tris solution.
The present invention has advantages below compared with prior art：
1st, the present invention builds goat capripoxviruses P32 albumen pronucleus expression plasmid pET28a-P32 and goat capripoxviruses ORF122 first
Albumen pronucleus expression plasmid pET32a-ORF122, is converted respectively to BL21 competent cells high efficient expression, ELISA and
Western bLot results show that two kinds of albumen are respectively provided with good immunogenicity.With the P32 albumen and ORF122 albumen of expression
Respectively immune mouse prepares monoclonal antibody, and Jing specific tests show, anti-goat capripoxviruses P32 albumen monoclonal antibody and anti-goatpox
The features such as virus O RF122 albumen monoclonal antibodies are respectively provided with susceptiveness height, high specificity, good stability.
2nd, gold label test strip of the invention has simple to operate, overcomes in traditional detection method to the numerous of sample early stage process
General labourer's skill, to operator without the need for special professional technique training, without the need for especial equipment requirements, testing cost is low.
3rd, the gold label test strip detection of the present invention is quick, and result of determination by only needing 5 ~ 10 minutes is adapted to Site Detection.
4th, the gold label test strip of detection GTPV of the invention, with high specificity, viral with sheep of virus, EDS-76,
Escherichia coli, Salmonella, Columba livia Newcastle virus no cross reaction；Good stability, the gold label test strip matter between batch and in batch
Amount is stable.Meanwhile, gold label test strip of the present invention is applied in the goatpox morbidity sheep of artificial challenge and clinic,
Prove that its high specificity, accuracy are high, can be used for the method application of Clinical detection goat capripoxviruses.
(1) the gold label test strip effectiveness of goat capripoxviruses is detected：
It is random to extract 10 from batch test agent bar, 100 μ L normal saline are added in well, wait 5 ~ 10min observations
In the middle part of detector bar, but 20min reading results are may not exceed, detection line occurs without reaction when reaction occurs in nature controlling line in two lines
When, can determine that test strips are effective；When only detection line appearance reaction occur reaction or in two lines simultaneously, and nature controlling line does not react
When, can determine that the test strips are invalid.
As a result：There are red stripes in the nature controlling line of 10 test strips, and band does not occur in detection line, shows prepared
Test strips it is effective.
(2) the gold label test strip susceptiveness test of goat capripoxviruses is detected：
The GTPV concentration poison of known content is diluted to into the virus liquid of different content, is entered with same batch of gold-labeled strip to be measured of trial-production
Row detection, it is determined that the minimum limitation of detection GTPV.
As a result show, this can detect GTPV minimum contents for 100 μ g/mL.
(3) the gold label test strip specific test of goat capripoxviruses is detected：
Goat capripoxviruses culture fluid, goatpox skin pathological material of disease, sore mouth virus are tested respectively with same batch of gold-labeled strip to be measured of trial-production
Virus-culturing fluid, chicken egg-decreasing syndrome（EDS-76）Virus-culturing fluid, Pigeon newcastle disease（PPMV-1-PL）Virus liquid, escherichia coli
And Salmonella, whether aubergine occurs at observation C, T line.
As a result show, this can detect goat capripoxviruses culture fluid, goatpox skin pathological material of disease treatment fluid, detection line occurs
Red bar line, is positive；And with sheep of virus culture fluid, chicken egg-decreasing syndrome（EDS-76）Virus-culturing fluid, Pigeon newcastle disease
（PPMV-1-PL）There is not red bar line, are negative in the effect of virus liquid, escherichia coli and Salmonella, detection line.Illustrate to be ground
The gold-labeled strip no cross reaction of system and false positive, specificity are good.
(4) the gold label test strip stability test of goat capripoxviruses is detected：
The random same batch of gold label test strip to be measured from trial-production extracts the test strips of q.s, places different time at 37 DEG C and 4 DEG C,
Detector bar is randomly selected, test is carried out with the virus liquid of standard and is observed its stability.
As a result show, test strips are placed placement in 6 days and 4 DEG C 12 and monthly can detect GTPV, nature controlling line and inspection at 37 DEG C
Occur red stripes at survey line, it is still 100% to further relate to manufactured experimently test strips homogeneity when positive quality control product is detected.
(5) the gold label test strip elaboration test of goat capripoxviruses is detected：
Random 10 ELISA test strips of extraction each from 3 batches of test strips to be measured are with portion goat capripoxviruses positive blood sample, each reagent paper
Uniformity is answered in detection line and nature controlling line colour developing between bar, is carried out coefficient of variation statistics to testing result between 3 batches, is pressed
The coefficient of variation between batch is calculated according to formula（CV values）.
CV=(maximum variance-minimum variance)/total number of samples × 100%
As a result：Detection same sample concentration positive quality control product when, the detection line of 3 batch detector bars and nature controlling line colour developing result
Uniformity, CV=0.
(6) goodness of fit test：
Same measuring samples are carried out with gold-labeled strip to be measured and the goat capripoxviruses reverse blood clotting test method set up
Detection, to determine the matching degree of two kinds of inspection methods.
1 comparative test result of table
As can be seen from Table 1, goat capripoxviruses gold-labeled strip and the goat capripoxviruses reverse indirect hemagglutination assay side for setting up
Testing result coincidence rate 100% of the method to different samples.Therefore, goat capripoxviruses gold-labeled strip can be used for Clinical detection goat
The method application of pox.
Description of the drawings
Below in conjunction with the accompanying drawings the specific embodiment of the present invention is described in further detail.
Fig. 1 is the structural representation of the present invention.
In figure：1-PVC offset plate 2-nitrocellulose filter, 3-gold standard pad, 4-sample pad, 5-absorbent paper, 6-detection
7-nature controlling line of line.
Specific embodiment
As shown in figure 1, a kind of gold mark detection test paper bar based on goat capripoxviruses, it includes PVC offset plates 1, cellulose nitrate
Plain film 2, gold standard pad 3, sample pad 4 and absorbent paper 5.
One end of PVC offset plates 1 is stained with sample pad 4, and its other end is stained with absorbent paper 5, is stained with gold in the middle part of which successively
Mark pad 3, nitrocellulose filter 2；One end of gold standard pad 3 is mutually adhered to sample pad 4, and its other end is mutually glued with nitrocellulose filter 2
Attached, the nitrocellulose filter 2 is mutually adhered to absorbent paper 5；Gold standard pad 3 has been coated with the anti-GTPV-P32 albumen Dan Ke of colloid gold label
Grand antibody；It is sequentially provided with along sample flow direction with the anti-GTPV- of the linear coating of the concentration of 1mg/mL on nitrocellulose filter 2
The detection line 6 of ORF122 protein monoclonal antibodies, with the nature controlling line 7 of the linear coating goat anti-mouse igg antibody of the concentration of 1mg/mL.
Detection line 6 is with nature controlling line 7 at intervals of 0.5cm.
Wherein：Anti- GTPV-P32 protein monoclonal antibodies are produced by hybridoma secretion, and the hybridoma is named as
GTPV-P32 hybridoma cell strains；Anti- GTPV-ORF122 protein monoclonal antibodies are produced by hybridoma secretion, the hybridoma
Cell is named as GTPV-ORF122 hybridoma cell strains；GTPV-P32 hybridoma cell strains and GTPV-ORF122 hybridomies
Strain is deposited in Gansu Livestock and Veterinary Inst.'s bacterium kind holding room.
Should be comprised the following steps based on the preparation method of the gold mark detection test paper bar of goat capripoxviruses：
(1) anti-GTPV-P32 protein monoclonal antibodies are prepared：
P 32 gene of goat capripoxvirus fragment is connected in pET28a (+) prokaryotic expression carrier, Jing IPTG are induced and expressed, P32
Albumen obtains high efficient expression in e. coli bl21, extracts albumen as immunizing antigen.Concrete preparation method is as follows：
The structure of GTPV P32 prokaryotic expression vectors：
A. the design of GTPV P32 gene expressions primer：
Using DNASTAR softwares according to P 32 gene of goat capripoxvirus primers, and introduce I enzyme action position of Nco I and Xho
Point, amplified production size are 882bp, and its sequence is as follows：
Upstream：5 '-ATCCATGGCAGATATCCCATTATATG-3 ',
Downstream：5 '-ATCTCGAGTCTATGAGCCATCCATTTTC-3 ',
Synthesized by Nanjing Genscript Biotechnology Co., Ltd. and Jing PAGE Purified in electrophoresis, ultrapure water dissolution, concentration are used using front
For 25 μm of oL/L, -20 DEG C save backup.
B. the PCR amplifications of purpose fragment
Amplification template processes as follows：
Take 200 μ L of goat capripoxviruses culture fluid and 10 min, 12000r/min centrifugation 10min are boiled in 30mL tetra- steams water, take
Clearly as template.
In PCR reaction tubes, reaction system is as follows：
5 μ L of template
4 μ L of dNTP mixture
10×PCR buffer 5μL
Each (50pmoL/ μ L) the 1 μ L of upstream and downstream primer
0.25 μ L of EX Taq enzymes
Sterilizing ddH2O is mended to 50 μ L.
To be positioned in PCR amplification instrument after liquid blending brief centrifugation in PCR pipe, the system of reaction is as follows：
94℃ 5min 1 Cycles
72℃ 10min 1 Cycles
After the completion of amplification, agarose gel of the 5 μ L of PCR primer in 10g/L is taken（Containing 0.5 μ g/mL ethidium bromides, EB）In carry out
Electrophoresis observation result.
C. the structure of P 32 gene of goat capripoxvirus prokaryotic expression carrier
With the pcr amplification product and prokaryotic expression carrier pET28a (+) of I double digestion P 32 gene of goat capripoxvirus of Xho I and Nco,
Gel-purified reclaims purpose fragment, the P32 genes of goat capripoxviruses is connected with pET28a (+), construction recombination plasmid pET28a-
P32, is transformed into escherichia coli DH5a competent cell.Extract positive transformant plasmid DNA, Jing PCR identification and enzyme action identify and
After sequencing, save backup.
The abduction delivering of restructuring P32 albumen and identification
A. recombinate P32 albumen abduction delivering
P 32 gene of goat capripoxvirus recombiant plasmid pET28a-P32 is transformed in e. coli bl21 (DE3) competent cell,
After picking single bacterium colony carries out positive identification, LB fluid mediums are inoculated with（Containing 50 μ g/mL of ampicillin）In, 230r/min,
37 DEG C of culture 12h activation.Activation bacterium is pressed into 1：50 ratio is inoculated in fluid medium（Containing 50 μ g/mL of ampicillin）In,
230r/min, 37 DEG C of cultures, carry out abduction delivering, when culture is to 150min using the IPTG of final concentration of 1mmoL/L 37
DEG C abduction delivering 6h, while setting the expression bacterium containing pET28a (+) empty carrier as control.
5min is centrifuged with 10000r/min, collects thalline precipitation abandons supernatant；5mL is added in bacterial sediment
0.01MpH7.4PBS, mixes；Ultrasonic Treatment thalline（Ice bath interruption is ultrasonic, ultrasonic 8s, interval 6s, common 30min, and power is
80W）, 20min is centrifuged with 12000 r/min at 4 DEG C, collects supernatant precipitation respectively.It is separately sampled to carry out SDS-PAGE electrophoresis,
Analysis purpose protein expression situation.
B. the Western bLot identifications of recombiant protein
After the fusion protein S DS-PAGE electrophoresis that recombinant bacterium pET28a-P32 is expressed, film adopts half dry type printing transferring method, Jing
15V 15min are transferred to nitrocellulose membrane（NC films）On.By 4 DEG C of closings in NC films TBST buffer of the immersion containing 50g/L BSA
Overnight, after being washed with TBST, by NC films and 1:37 DEG C of effect 2h of GTPV mices positive serum of 100 dilutions.TBST is fully washed
With 1 after film:37 DEG C of reaction 2h of rabbit anti-mouse IG-HRP of 4000 dilutions, TBST is washed 3 times, with substrate benzidine ammonia
（DAB）And hydrogen peroxide（6mgDAB,0.01mmoL Tris-HCL 9mL,0.03%CoCL21mL,30%H2O210uL）Colour developing.
The antigen of restructuring P32 albumen is prepared and purification
A. recombinate P32 albumen purification
Expressed using optimal expression condition, harvested thalline, freeze thawing 3 times is precipitated with 0.01M pH7.4PBS suspension thallines,
4 DEG C, 12000r/min centrifugation 10min, inclusion body precipitation 3moL/L urea liquids wash away most of protein precipitation, 4 DEG C,
4500r/min centrifugation 10min, inclusion body precipitation lysate room-temperature dissolution 1hs of the pH8.0 containing 8moL/L carbamide, 4 DEG C,
12000r/min is centrifuged 20min, takes supernatant and Ni2+- NTA fillers combine 1h, respectively with the 8moL/L carbamide of pH8.0,6.3,5.9
After solution washing foreigh protein removing, with the 8moL/L urea liquid eluting destination proteins of pH4.5, the destination protein for collecting eluting is
The destination protein of purification；By SDS-PAGE purification Identification albumen, and with ultraviolet-uisible spectrophotometer determine OD260 and
OD280, after calculating its concentration, -20 DEG C save backup.
B. the immunogenicity detection of restructuring P32 albumen
By 6 ~ 8 week old BALB/c mouse 30,4 groups are randomly divided into, 10 per group, male and female half and half.Respectively：A group normal saline,
0.1mL/ is only；B groups pET28a（+）Matched group, 100 μ g/ are only；C groups restructuring P32 albumen+Freund's complete adjuvant, 100 μ g/ are only；D
Group goatpox live vaccine, 1 part/only.Each group is injected in mice quadriceps femoris.After first immunisation, 15d is added with same dose
Strong immunity 1 time.After immunity, 0d, 7d, 14d, 21d, 28d docking blood sampling, separates serum, detects each group with indirect ELISA method
Serum antibody.
Goat capripoxviruses P32 protein immunizations BALB/C mice is prepared into anti-goatpox according to conventional monoclonal anti Antibody Production Techniques
Viral P32 protein monoclonal antibodies, and purification is carried out, obtain final product anti-goat capripoxviruses P32 protein monoclonal antibodies after purification.Tool
Preparation is as follows：
Choosing by 6 ~ 8 week old female BAl BIcs/C white mice, with the P32 recombiant proteins of purification as immunogen, immunity 3 times, every time between
Every 2 weeks, first time antigen added equivalent Freund's complete adjuvant, 4 points of injections of Jing emulsifyings dorsal sc point, co-injection 0.2mL；Second
Plus the incomplete Freund's adjuvant of equivalent, method is the same；Third time is not added with the injection of adjuvant dorsal sc branch, while tail vein blood
Serum titer is determined, in first three day booster immunization is merged when reaching requirement, with the equivalent amount of antigen lumbar injection without adjuvant.Take and add
The splenocyte and SP2/0 myeloma cell's cell fusion of strong immune mouse, filters out the anti-GTPV-P32 albumen Dan Ke of stably excreting
The positive cell strain of grand antibody, and wherein 1 plant of monoclonal antibody amplification culture of potency highest, injection cell induce abdomen in mice body
Water；Oils and fatss and precipitation are centrifuged off, that is, obtain mouse ascites, then purified monoclonal antibody is carried out with caprylic acid-ammonium, that is, obtain pure
Anti- GTPV-P32 protein monoclonal antibodies after change, comprise the following steps that：4 DEG C of ascites Jing, 10000r/min centrifugations first
15min, except degreasing and little particle precipitate, adds the acetate buffer of 2 times of volumes in the filtrate of ascites supernatant
（0.06moL/L、pH5.0）, pH to 4.5 is adjusted with hydrochloric acid, it is sad to be added dropwise over 33 μ L under stirring at room temperature in 30min, and 4 DEG C stand
2h, 12000r/min be centrifuged 30min, abandon precipitation and take supernatant, add in supernatant 1/10 volume 0.1moL/L PB (pH7.4,
8.5%NaCL), and with the NaOH of 1moL/L pH to 7.4 is adjusted, 0.277g/mL ammonium sulfate is added in 30min under 4 DEG C of ice baths,
To 45% saturation, stand more than 1h, 12000r/min centrifugations 30min at 4 DEG C abandons supernatant, precipitation be dissolved in 0.01moL/L and
In the PBS of pH7.4, load bag filter, fully dialysed, the inspection of 0.5moL/L barium chloride solutions does not measure BaSO4, that is, obtain pure
The antibody of change.
(2) anti-GTPV-ORF122 protein monoclonal antibodies are prepared：
Goat capripoxviruses ORF122 genetic fragments are connected in pET32a (+) prokaryotic expression carrier, Jing IPTG are induced and expressed,
ORF122 albumen obtains high efficient expression in e. coli bl21, extracts albumen as immunizing antigen.Concrete preparation method is as follows：
The structure of GTPV ORF122 prokaryotic expression vectors：
A. the design of GTPV ORF122 gene expressions primer：
Primer is designed according to goat capripoxviruses ORF122 gene orders using DNASTAR softwares, and introduces I enzyme action of BamH I and SaL
Site, amplified production size are 390bp, and its sequence is as follows：
Upstream：5 '-AGGATCCAATAATAATACATGTGA-3 ',
Downstream：5 '-CGTCGACAAAAAAAGATCTTACACAG -3 ',
Synthesized by Nanjing Genscript Biotechnology Co., Ltd. and Jing PAGE Purified in electrophoresis, ultrapure water dissolution, concentration are used using front
For 25 μm of oL/L, -20 DEG C save backup.
B. the PCR amplifications of purpose fragment：
Amplification template processes as follows：
Take 200 μ L of goat capripoxviruses culture fluid and 10 min, 12000r/min centrifugation 10min are boiled in 30mL tetra- steams water, take
Clearly as template.
In PCR reaction tubes, reaction system is as follows：
5 μ L of template
4 μ L of dNTP mixture
10×PCR buffer 5μL
Each (50pmoL/ μ L) the 1 μ L of upstream and downstream primer
0.25 μ L of EX Taq enzymes
Sterilizing ddH2O is mended to 50 μ L.
To be positioned in PCR amplification instrument after liquid blending brief centrifugation in PCR pipe, the system of reaction is as follows：
94℃ 5min 1 Cycles
72℃ 10min 1 Cycles
After the completion of amplification, agarose gel of the 5 μ L of PCR primer in 10g/L is taken（Containing 0.5 μ g/mL ethidium bromides, EB）In carry out
Electrophoresis observation result.
C. the structure of goat capripoxviruses ORF122 prokaryotic expression vectors：
With the pcr amplification product and prokaryotic expression carrier pET32a of I double digestion goat capripoxviruses ORF122 genes of BamH I and SaL
(+), gel-purified reclaim purpose fragment, the ORF122 genes of goat capripoxviruses are connected with pET32a (+), construction recombination plasmid
PET32a-ORF122, is transformed into escherichia coli DH5a competent cell.Extract positive transformant plasmid DNA, Jing PCR identifications, enzyme
After cutting identification and being sequenced, save backup.
The abduction delivering of restructuring ORF122 albumen and identification
A. recombinate OR122 albumen abduction delivering：
Goat capripoxviruses ORF122 gene recombination plasmid pET32a-P32 are transformed into into e. coli bl21 (DE3) competent cell
In, after picking single bacterium colony carries out positive identification, it is inoculated with LB fluid mediums（Containing 50 μ g/mL of ampicillin）In, 230r/
Min, 37 DEG C of culture 12h activation.Activation bacterium is pressed into 1：50 ratio is inoculated in fluid medium（Containing 50 μ g/mL of ampicillin）
In, 230r/min, 37 DEG C of cultures carry out abduction delivering, when culture is to 150min using the IPTG of final concentration 1mmoL/L 37
DEG C abduction delivering 6h, while setting the expression bacterium containing pET28a (+) empty carrier as control.
5min is centrifuged with 10000r/min, collects thalline precipitation abandons supernatant；5mL 0.01M are added in bacterial sediment
PH7.4PBS, mixes；Ultrasonic Treatment thalline（Ice bath interruption ultrasound, ultrasonic 8s, interval 6s, common 30min, power are 80W）,
20min is centrifuged with 12000 r/min at 4 DEG C, collects supernatant precipitation respectively.It is separately sampled to carry out SDS-PAGE electrophoresis, analysis
Destination protein expression.
B. the Western bLot identifications of recombiant protein：
After the fusion protein S DS-PAGE electrophoresis that recombinant bacterium pET32a-ORF122 is expressed, film adopts half dry type printing transferring method,
Jing 15V 15min are transferred to nitrocellulose membrane（NC films）On.By 4 DEG C of envelopes in NC films TBST buffer of the immersion containing 50g/L BSA
Close overnight, after being washed with TBST, by NC films and 1:37 DEG C of effect 2h of GTPV mices positive serum of 100 dilutions.TBST is fully washed
Wash after film with 1:37 DEG C of reaction 2h of rabbit anti-mouse IG-HRP of 4000 dilutions, TBST is washed 3 times, with substrate benzidine ammonia
（DAB）And hydrogen peroxide（6mgDAB,0.01mmoL Tris-HCL 9mL,0.03%CoCL21mL,30%H2O210uL）Colour developing.
The antigen of restructuring ORF122 albumen is prepared and purification
A. recombinate ORF122 albumen purification：
Expressed using optimal expression condition, harvested thalline, freeze thawing 3 times is precipitated with 0.01M pH7.4PBS suspension thallines,
4 DEG C, 12000r/min centrifugation 10min, inclusion body precipitation 3moL/L urea liquids wash away most of protein precipitation, 4 DEG C,
4500r/min centrifugation 10min, inclusion body precipitation lysate room-temperature dissolution 1hs of the pH8.0 containing 8moL/L carbamide, 4 DEG C,
12000r/min is centrifuged 20min, takes supernatant and Ni2+- NTA fillers combine 1h, respectively with the 8moL/L carbamide of pH8.0,6.3,5.9
After solution washing foreigh protein removing, with the 8moL/L urea liquid eluting destination proteins of pH4.5, the destination protein for collecting eluting is
The destination protein of purification；By SDS-PAGE purification Identification albumen, and with ultraviolet-uisible spectrophotometer determine OD260 and
OD280, after calculating its concentration, -20 DEG C save backup.
B. the immunogenicity detection of restructuring ORF122 albumen：
By 6 ~ 8 week old BALB/c mouse 30,4 groups are randomly divided into, 10 per group, male and female half and half.Respectively：A group normal saline,
0.1mL/ is only；B groups pET32a（+）Matched group, 100 μ g/ are only；C groups restructuring ORF122 albumen+Freund's complete adjuvant, 100 μ g/ are only；
D group goatpox live vaccine, 1 part/only.Each group is injected in mice quadriceps femoris.After first immunisation, 15d is with same dose
Booster immunization 1 time.After immunity, 0d, 7d, 14d, 21d, 28d docking blood sampling, separates serum, each with indirect ELISA method detection
The serum antibody of group.
Goat capripoxviruses ORF122 protein immunizations BALB/C mice is prepared into anti-mountain according to conventional monoclonal anti Antibody Production Techniques
Capripox viruses ORF122 protein monoclonal antibodies, and purification is carried out, obtain final product anti-goat capripoxviruses ORF122 albumen lists after purification
Clonal antibody.Concrete grammar is as follows：
Select 6 ~ 8 week old female BAl BIcs/C white mice, it is with the ORF122 recombiant proteins of purification as immunogen, immune 3 times, every time
It is spaced 2 weeks, first time antigen adds equivalent Freund's complete adjuvant, it is subcutaneous that Jing emulsifying branches are injected in mouse back, co-injection
0.2mL；Second plus the incomplete Freund's adjuvant of equivalent, the injection of Jing emulsifyings pneumoretroperitoneum, co-injection 0.2mL；Third time is not added with assistant
Agent dorsal sc branch is injected, while tail vein blood determines serum titer, in merging first three day booster immunization when reaching requirement,
With the equivalent amount of antigen lumbar injection without adjuvant.The splenocyte and SP2/0 myeloma cell's cell fusion of booster immunization mice are taken,
Filter out the positive cell strain of the anti-GTPV-ORF122 protein monoclonal antibodies of stably excreting, and wherein 1 plant of monoclonal antibody of potency highest
Amplification culture, injection cell induce ascites in mice body；Oils and fatss and precipitation are centrifuged off, that is, are obtained mouse ascites, then is used
Caprylic acid-ammonium carries out purified monoclonal antibody, that is, obtain anti-GTPV-ORF122 protein monoclonal antibodies after purification, concrete steps
It is as follows：4 DEG C of ascites Jing, 10000r/min are centrifuged 15min first, except degreasing and little particle precipitate, to ascites supernatant
The acetate buffer of 2 times of volumes is added in filtrate（0.06moL/L、pH5.0）, pH to 4.5 is adjusted with hydrochloric acid, under stirring at room temperature
33 μ L are added dropwise in 30min sad, 4 DEG C of standing 2h, 12000r/min centrifugation 30min abandon precipitation and take supernatant, add in supernatant
Enter the 0.1moL/L PB (pH7.4,8.5%NaCL) of 1/10 volume, and pH to 7.4 is adjusted with the NaOH of 1moL/L, under 4 DEG C of ice baths
0.277g/mL ammonium sulfate is added in 30min, to 45% saturation, more than 1h is stood, 12000r/min centrifugations at 4 DEG C
30min, abandons supernatant, and precipitation is dissolved in the PBS of 0.01moL/L and pH7.4, is loaded bag filter, is fully dialysed, 0.5moL/
The inspection of L barium chloride solutions does not measure BaSO4, that is, obtain the antibody of purification.
(3) anti-goat capripoxviruses ORF122 protein monoclonal antibodies after purification are coated onto on nitrocellulose filter 2, are coated with
One region of nitrocellulose filter 2, obtains detection line 6；By the goat anti-mouse igg antibody coating nitrocellulose filter 2 of purification
Another region, obtain nature controlling line 7.Wherein：Goat anti-mouse igg antibody is purchased from Suo Laibao bio tech ltd.
(4) by the nitrocellulose filter 2 of step (3) gained room temperature in 0.05moL/L and in the Tris-HCL solution of pH=8.5
10 ~ 60min of closing, then with 0.01M and after the PBST rinsings twice of pH=8.5, in 4 DEG C of drying for standby.
Wherein：The Tris-HCl solution of 0.05mol/L and pH=8.5 refer in 100mL Tris solution containing 1g BSA,
The Tris solution of 0.2mL Tween-20.
(5) the nitrocellulose filter 2 of step (4) gained is adhered to into the middle part of PVC offset plates 1.
(6) colloid gold label protein solution is prepared：
1. glass drying oven used is cleaned up, is immersed in 24h in washing liquid；Rinsed with tap water after taking-up, distilled water flushing 3
It is secondary, glass drying oven used is soaked in the chloroformic solution containing the dichlorodimethylsilane that mass concentration is 5% after drying
1min, deionized water are rinsed, drying for standby.
Wherein：Washing liquid is referred to is distilled potassium dichromate 1000g after water dissolution with a small amount of, is slowly added to concentrated sulphuric acid along chamber wall
2500mL, after mixing, adds distilled water and mixes to 10000mL and obtain final product.
2. the gold chloride that 100mL mass concentrations are 0.01% is added in the glass drying oven used Jing after 1. step is processed,
After on magnetic stirring apparatuss, agitating heating is boiled, it is 1% sodium citrate solution newly matched somebody with somebody to be rapidly added 2.5mL mass concentrations, continues to add
Heat boils 5 ~ 10min, and solution colour is gradually changed into orange red, lovely luster, stops boiling, and moisturizing is to volume by navy blue
For 100mL, colloidal gold solution is obtained；The colloidal gold solution sediments microscope inspection, makes the gold grain of the colloidal gold solution of preparation big
It is little consistent, uniformly, 4 DEG C closed keep in dark place it is standby.
3. with the K of 0.1moL/L2CO3The pH value of colloidal gold solution is adjusted to into 8.5, magnetic agitation is transferred in lucifuge condition
Device is slowly stirred, and the anti-goat capripoxviruses P32 protein monoclonal antibodies for taking after purification are added dropwise to final concentration of 50 μ g/mL,
Continue stirring 30min, stand 10min, add stabilizer BSA so as to which mass concentration reaches 1%, then is slowly stirred 30min, obtains final product
Gold mark monoclonal antibody solution.
4. gold mark monoclonal antibody solution is removed into gold colloidal polymerization therein in 4 DEG C of speed centrifugation 10min with 2000r/min
Thing, obtains supernatant A；Supernatant A in 4 DEG C of speed centrifugation 60min with 12000r/min, is now centrifuged thing and is divided into three layers again：
Supernatant B contains still unconjugated monoclonal antibody, and nethermost atrouss speckle is still unconjugated colloid gold particle polymer, in
Between in mauve flocculent deposit be well combined gold mark monoclonal antibody complex；Aubergine flocculent deposit is collected, and in the purple
The PBS suspension dissolvings of the 10mmoL/L and pH=7.2 of 1/10 supernatant A volume, 4 DEG C of lucifuges is added to protect in red flocculent precipitation
Deposit, obtain final product colloid gold label protein solution.
Wherein：The PBS suspensions of 10mmol/L and pH=7.2 refer in 100mL PBS solutions containing 1g BSA,
The PBS solution of 1gPVP, 0.02g sodium azide.
(7) glass fibre element film is immersed in into 30min in the Tris-HCL solution of pH=5.2 and 0.02moL/L, 4 DEG C of mistakes first
Night is dried；Again glass fibre membrane is immersed in the anti-GTPV-P32 protein monoclonal antibodies solution of colloid gold label, Jing is impregnated with, dries in the air
It is dry, obtain final product gold standard pad 3；Gold standard pad 3 at 37 DEG C be dried to constant weight, adhere to step (4) gained nitrocellulose filter 2 on
Away from one end of nature controlling line 7.
Wherein：The Tris-HCL solution of pH=5.2 and 0.02mol/L each means and contains 3g in 100mL Tris solution
The Tris solution of BSA, 0.1mL Tween-20.
(8) sample pad 4 is immersed in into 30min in the Tris-HCL solution of pH=5.2 and 0.02moL/L, 4 DEG C of dried overnights,
Sample pad 4 after being processed.
Wherein：The Tris-HCL solution of pH=5.2 and 0.02mol/L each means and contains 3g in 100mL Tris solution
The Tris solution of BSA, 0.1mL Tween-20.
(9), in one end of gold standard pad 3 in the sample pad 4 being stained with after processing, the nitric acid of its other end adhering step (4) gained is fine
The plain film 2 of dimension；Sample pad 4 after process adheres to one end of PVC offset plates 1；The nitrocellulose filter of absorbent paper 5 and step (4) gained
2 adhesions, and adhere to the other end of PVC offset plates 1.
The wide test strips of 4mm are cut into cutting machine, the test strips for cutting are put in the packaging bag equipped with desiccant, sealed
It is standby.
The present invention reagent strip directly can take morbidity sheep or sheep skin of dying of illness on varioliform exanthema, crust block as detection sample
Product.
Take, shredded in mortar with the shears of sterilizing, 1 is made into normal saline：2
（W/V）Tissue suspension, after multigelation 3 times, 3000r/min centrifugation 30min take supernatant and are detected.
Result judgement：Positive findingses, nature controlling line, detection line are presented red stripes, illustrate in sample with the presence of GTPV.It is cloudy
Property result, nature controlling line is presented red stripes, and detection line occurs without red stripes, illustrates no GTPV in sample.If detection
Line, nature controlling line occur without red stripes or only red stripes occurs in detection line, then illustrate product failure.
Claims (8)
1. a kind of gold mark detection test paper bar based on goat capripoxviruses, it is characterised in that：It includes PVC offset plates（1）, cellulose nitrate
Plain film（2）, gold standard pad（3）, sample pad（4）And absorbent paper（5）；The PVC offset plates（1）One end be stained with the sample pad
（4）, its other end is stained with the absorbent paper（5）, the gold standard pad in the middle part of which, is stained with successively（3）, the celluloid
Film（2）；The gold standard pad（3）One end and the sample pad（4）Mutually adhere to, its other end and the nitrocellulose filter（2）
Mutually adhere to, the nitrocellulose filter（2）With the absorbent paper（5）Mutually adhere to；The gold standard pad（3）It has been coated with colloid gold label
Anti- GTPV-P32 protein monoclonal antibodies；The nitrocellulose filter（2）On be sequentially provided with along sample flow direction with 1mg/mL
The anti-GTPV-ORF122 protein monoclonal antibodies of the linear coating of concentration detection line（6）, with the linear coating sheep of the concentration of 1mg/mL
The nature controlling line of anti-mouse IgG antibody（7）.
2. a kind of gold mark detection test paper bar based on goat capripoxviruses as claimed in claim 1, it is characterised in that：It is described anti-
GTPV-P32 protein monoclonal antibodies and the anti-GTPV-ORF122 protein monoclonal antibodies are produced by hybridoma secretion
It is raw.
3. a kind of gold mark detection test paper bar based on goat capripoxviruses as claimed in claim 1, it is characterised in that：The detection
Line（6）With the nature controlling line（7）At intervals of 0.5cm.
4. a kind of preparation method of the gold mark detection test paper bar based on goat capripoxviruses as claimed in claim 1, including it is following
Step：
(1) anti-GTPV-P32 protein monoclonal antibodies are prepared：
P 32 gene of goat capripoxvirus fragment is connected in pET28a (+) prokaryotic expression carrier, Jing IPTG are induced and expressed, P32
Albumen obtains high efficient expression in e. coli bl21, extracts albumen as immunizing antigen；
Goat capripoxviruses P32 protein immunizations BALB/C mice is prepared into anti-goatpox according to conventional monoclonal anti Antibody Production Techniques
Viral P32 protein monoclonal antibodies, and purification is carried out, obtain final product anti-goat capripoxviruses P32 protein monoclonal antibodies after purification；
(2) anti-GTPV-ORF122 protein monoclonal antibodies are prepared：
Goat capripoxviruses ORF122 genetic fragments are connected in pET32a (+) prokaryotic expression carrier, Jing IPTG are induced and expressed,
ORF122 albumen obtains high efficient expression in e. coli bl21, extracts albumen as immunizing antigen；
Goat capripoxviruses ORF122 protein immunizations BALB/C mice is prepared into anti-mountain according to conventional monoclonal anti Antibody Production Techniques
Capripox viruses ORF122 protein monoclonal antibodies, and purification is carried out, obtain final product anti-goat capripoxviruses ORF122 albumen lists after purification
Clonal antibody；
(3) the anti-goat capripoxviruses ORF122 protein monoclonal antibodies after purification are coated onto into the nitrocellulose filter after processing
（2）On, the nitrocellulose filter after coating process（2）A region, obtain detection line（6）；By the goat anti-mouse igg of purification
Nitrocellulose filter after the process of antibody coating（2）Another region, obtain nature controlling line（7）；
By the step (3) gained nitrocellulose filter（2）Room in 0.05moL/L and in the Tris-HCL solution of pH=8.5
Temperature 10 ~ 60min of closing, then with 0.01M and after the PBST rinsings twice of pH=8.5, in 4 DEG C of drying for standby；
By the step (4) gained nitrocellulose filter（2）Adhere to PVC offset plates（1）Middle part；
(6) colloid gold label protein solution is prepared：
1. glass drying oven used is cleaned up, is immersed in 24h in washing liquid；Rinsed with tap water after taking-up, distilled water flushing 3
It is secondary, glass drying oven used is soaked in the chloroformic solution containing the dichlorodimethylsilane that mass concentration is 5% after drying
1min, deionized water are rinsed, drying for standby；
2. the gold chloride that 100mL mass concentrations are 0.01% is added in the glass drying oven used after 1. processing to the step described in,
After on magnetic stirring apparatuss, agitating heating is boiled, it is 1% sodium citrate solution newly matched somebody with somebody to be rapidly added 2.5mL mass concentrations, continues to add
Heat boils 5 ~ 10min, and solution colour is gradually changed into orange red, lovely luster, stops boiling, and moisturizing is to volume by navy blue
For 100mL, colloidal gold solution is obtained；The colloidal gold solution sediments microscope inspection, makes the gold grain of the colloidal gold solution of preparation big
It is little consistent, uniformly, 4 DEG C closed keep in dark place it is standby；
3. with the K of 0.1moL/L2CO3The pH value of the colloidal gold solution is adjusted to into 8.5, magnetic agitation is transferred in lucifuge condition
Device is slowly stirred, and takes the anti-goat capripoxviruses P32 protein monoclonal antibodies after purification and is added dropwise to final concentration of 50 μ g/
ML, continues stirring 30min, stands 10min, add stabilizer BSA so as to which mass concentration reaches 1%, then is slowly stirred 30min,
Obtain final product gold mark monoclonal antibody solution；
4. the gold is marked into monoclonal antibody solution in 4 DEG C of speed centrifugation 10min with 2000r/min, removes gold colloidal polymerization therein
Thing, obtains supernatant A；The supernatant A in 4 DEG C of speed centrifugation 60min with 12000r/min, is now centrifuged thing and is divided into three again
Layer：Supernatant B contains still unconjugated monoclonal antibody, and nethermost atrouss speckle is still unconjugated colloid gold particle polymerization
Thing, it is middle that the gold mark monoclonal antibody complex being well combined is in mauve flocculent deposit；Collection aubergine flocculent deposit, and
The PBS suspensions of the 10mmoL/L and pH=7.2 of supernatant A volume described in 1/10 are added to dissolve in the aubergine flocculent deposit, 4
DEG C keep in dark place, obtain final product colloid gold label protein solution；
(7) glass fibre element film is immersed in into 30min in the Tris-HCL solution of pH=5.2 and 0.02moL/L first, 4 DEG C are overnight done
It is dry；Again glass fibre membrane is immersed in the anti-GTPV-P32 protein monoclonal antibodies solution of colloid gold label, Jing is impregnated with, dries,
Obtain final product gold standard pad（3）；The gold standard pad（3）It is dried to constant weight at 37 DEG C, adheres to the nitrocellulose filter（2）It is upper remote
From the nature controlling line（7）One end；
(8) by sample pad（4）30min in the Tris-HCL solution of pH=5.2 and 0.02moL/L is immersed in, 4 DEG C of dried overnights are obtained
Sample pad to after process（4）；
(9) in the gold standard pad（3）Sample pad of the one end after the process is stained with（4）, its described step of other end adhesion is (4)
The nitrocellulose filter of gained（2）；Sample pad after the process（4）Adhere to the PVC offset plates（1）One end；Absorbent paper
（5）With the step (4) gained nitrocellulose filter（2）Adhesion, and adhere to the PVC offset plates（1）The other end.
5. a kind of preparation method of the gold mark detection test paper bar based on goat capripoxviruses as claimed in claim 4, its feature exist
In：(4) the Tris-HCl solution of middle 0.05mol/L and pH=8.5 is referred to and contains 1g in 100mL Tris solution the step
The Tris solution of BSA, 0.2mL Tween-20.
6. a kind of preparation method of the gold mark detection test paper bar based on goat capripoxviruses as claimed in claim 4, its feature exist
In：1. middle washing liquid is referred to potassium dichromate 1000g with distilling after water dissolution on a small quantity the step, is slowly added to dense sulfur along chamber wall
Sour 2500mL, after mixing, adds distilled water and mixes to 10000mL and obtain final product.
7. a kind of preparation method of the gold mark detection test paper bar based on goat capripoxviruses as claimed in claim 4, its feature exist
In：The PBS suspensions of the step 4. middle 10mmol/L and pH=7.2 refer in 100mL PBS solutions containing 1g BSA,
The PBS solution of 1gPVP, 0.02g sodium azide.
8. a kind of preparation method of the gold mark detection test paper bar based on goat capripoxviruses as claimed in claim 4, its feature exist
In：(7) the step is each meant in 100mL Tris with the Tris-HCL solution of the step (8) middle pH=5.2 and 0.02mol/L
Tris solution containing 3g BSA, 0.1mL Tween-20 in solution.
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| CN107525923A (en) * | 2017-04-01 | 2017-12-29 | 北京康华源科技发展有限公司 | One kind centrifuges immunochromatography detection method and device |
| CN108101995A (en) * | 2017-12-27 | 2018-06-01 | 中国动物疫病预防控制中心 | Recombinate capripox virus fusion protein and its application |
| CN108164603A (en) * | 2017-12-27 | 2018-06-15 | 中国动物疫病预防控制中心 | Recombinate the solubility expression method of capripox virus fusion protein and its biomaterial used |
| CN108912226A (en) * | 2018-07-27 | 2018-11-30 | 中国农业科学院兰州兽医研究所 | One kind being based on the anti-goat capripoxvirus VHH-4-2 of two-humped camel and purposes |
| CN108912225A (en) * | 2018-07-27 | 2018-11-30 | 中国农业科学院兰州兽医研究所 | One kind being based on the anti-goat capripoxvirus VHH-4-1 of two-humped camel and purposes |
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| CN108164603A (en) * | 2017-12-27 | 2018-06-15 | 中国动物疫病预防控制中心 | Recombinate the solubility expression method of capripox virus fusion protein and its biomaterial used |
| CN108164603B (en) * | 2017-12-27 | 2020-05-08 | 中国动物疫病预防控制中心 | Soluble expression method of recombinant capripoxvirus fusion protein and biological material used by same |
| CN108101995B (en) * | 2017-12-27 | 2020-05-12 | 中国动物疫病预防控制中心 | Recombinant capripoxvirus fusion protein and application thereof |
| CN108912226A (en) * | 2018-07-27 | 2018-11-30 | 中国农业科学院兰州兽医研究所 | One kind being based on the anti-goat capripoxvirus VHH-4-2 of two-humped camel and purposes |
| CN108912225A (en) * | 2018-07-27 | 2018-11-30 | 中国农业科学院兰州兽医研究所 | One kind being based on the anti-goat capripoxvirus VHH-4-1 of two-humped camel and purposes |
| CN108912226B (en) * | 2018-07-27 | 2020-06-09 | 中国农业科学院兰州兽医研究所 | Bactrian camel-based anti-goatpox virus VHH-4-2 and application |
| CN108912225B (en) * | 2018-07-27 | 2020-06-09 | 中国农业科学院兰州兽医研究所 | Bactrian camel-based anti-goatpox virus VHH-4-1 and application |
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Application publication date: 20170329 |