CN110007077A - A kind of Porcine epidemic diarrhea virus agp antigen and its detection method for antibody test - Google Patents
A kind of Porcine epidemic diarrhea virus agp antigen and its detection method for antibody test Download PDFInfo
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Abstract
The invention discloses a kind of Porcine epidemic diarrhea virus agp antigen and its for the detection method of antibody test.The agp antigen is largely to be proliferated Porcine epidemic diarrhea virus SD plants of the 28th generation of poison disease vaccination Vero cell, collect viral cultures, freeze thawing 3 times, after Triton X-100 processing is added, centrifuging and taking supernatant, supernatant obtains Porcine epidemic diarrhea virus agp antigen through ultracentrifugation.Present invention process method is simple, scientific, and production is stablized, and low in cost, manufactured Porcine epidemic diarrhea virus agp antigen has the characteristics that sensibility height, high specificity, stability are good.Agp antigen of the present invention is Porcine epidemic diarrhea virus antibody agar gel diffusion test detection method for the detection method of Porcine epidemic diarrhea virus antibody.There is higher positive rate, and have the characteristics that experimental implementation requirement is low, testing cost is low, particularly suitable base's veterinary quarantine testing department and farm use.
Description
Technical field
The invention belongs to Disease Diagnosis of Veterinary technical field more particularly to a kind of Porcine epidemic diarrhea virus agp antigen and its systems
Preparation Method and application.
Background technique
Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) is a kind of enteric infection venereal disease viral disease, by
Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) causes, with watery diarrhea, vomiting and
Dehydration is characterized.This disease epidemic characteristic, clinical symptoms and pathological change are quite similar with transmissible gastroenteritis.Pig epidemic
It rushes down and Britain occurs within 1971 for the first time, ground was separated and identified about PEDV successively since early 1980s in China
Report.Winter is occurred mainly in, summer can also occur, and China is with December to next year 2 months for this disease high-incidence season.
PEDV belongs to coronaviridae coronavirus genus member, the pig at various ages can infection morbidity, suckling pig, frame
Up to 100%, the especially aggrieved most serious of suckling pig, sow disease incidence is 15%~90% for pigling or the disease incidence of growing and fattening pigs.PEDV
The incubation period of oral artificial challenge is 1~2 day, but natually morbid incubation period may be longer.Mainly through transmission, also have
Report through respiratory infectious.The aggrieved most serious of suckling pig, occurs vomitting and watery diarrhea；Chang Fasheng after newborn piglet infection
Serious dehydration and death, case fatality rate average out to 50%；There are lasting 4~6 days watery diarrheas in wean pig and growing and fattening pigs, most of
Sick pig rehabilitation, but growth and development is impacted, and the weight for fattening the later period detracts；Sow morbidity is with spiritual tired, anorexia and holds
With the characteristics of continuous 1 week diarrhea.This disease can spread all over entire pig farm during 4~5 weeks.
This disease is for a long time, popular in numerous pig-raising countries including China.Since the end of the year 2010, pig epidemic
Diarrhea virus variant is in China's eruption and prevalence, and successively in U.S., Canada, Mexico, Japan, South Korea, Colombia, secret
The outburst of the countries and regions such as Shandong, Ukraine, the Dominica Republica, this time epidemic situation duration is long, coverage is wide, caused by
Loss is big, brings serious disaster to various countries' pig breeding industry, is the hot spot of global pig breeding industry concern in recent years
AGP test antibody detection method has the characteristics that experimental implementation requirement is low, testing cost is low, particularly suitable base animal doctor
Quarantine and examination department and farm use.In addition the potency such as Porcine epidemic diarrhea virus serum antibody, milk antibody, Yolk antibody
The needs of detection and Efficacy evaluation, the simple and efficient method for creating a kind of Porcine epidemic diarrhea virus antibody very must
It wants and urgent.
Summary of the invention
It is of the invention technical problem to be solved by the present invention lies in providing a kind of Porcine epidemic diarrhea virus agp antigen
Another object is to provide the detection side that a kind of Porcine epidemic diarrhea virus agp antigen is used for Porcine epidemic diarrhea virus antibody
Method.
A kind of Porcine epidemic diarrhea virus agp antigen is obtained by following methods:
(1) virus liquid of the 28th generation Porcine epidemic diarrhea virus of 1mL, tissue culture infective dose (Tissue
Culture infective dose, TCID50) value be 10-7.5, it is inoculated into 25cm2The non-of single layer is grown up in Tissue Culture Flask
On continent green monkey kidney cell (VERO cell), set 37 DEG C, cultivate 72 hours in 5%CO2 incubator, connect malicious VERO cell occur it is typical
Tissue Culture Flask is set -20 DEG C so that down toward freezing completely, taking-up is melted under room temperature by cytopathy, and start aggregation and fall off,
Freeze thawing 3 times repeatedly, collect viral cultures, and the viral cultures include cell and culture solution；
Porcine epidemic diarrhea virus SD plants of 28th generation is deposited in China typical culture collection on March 29th, 2019
Center, preservation address: China, Wuhan, Wuhan University；Entitled epidemic diarrhea virus SD plants of its preservation, deposit number are as follows:
CCTCC NO: V201915；
(2) formaldehyde is added by the amount of final concentration 2 ‰ in viral cultures to be uniformly mixed, is inactivated 24 hours in 37 DEG C of shaking tables；
(3) in the viral cultures of freeze thawing 3 times and inactivation, polyethylene glycol octyl is added by the amount of viral cultures volume 1/20
Phenyl ether (TritonX-100), shaken at room temperature 20 minutes, revolving speed 6000r/min, under the conditions of 4 DEG C of temperature, high speed centrifugation 20min,
Collect supernatant；
(4) take supernatant under the conditions of revolving speed 45000r/min, 4 DEG C of temperature, ultracentrifugation 120min；Liquid is discarded supernatant, is precipitated
It is sufficiently dissolved with the phosphate buffer (phosphate buffer saline, PBS) of 0.01mol/L pH value 7.4, PBS is slow
The dosage of fliud flushing is the amount of viral cultures volume 1/15, both obtains Porcine epidemic diarrhea virus agp antigen, and -20 DEG C of packing are protected
It deposits.
A kind of Porcine epidemic diarrhea virus agp antigen is that pig is flowed for the detection method of Porcine epidemic diarrhea virus antibody
Row diarrhea virus antibody agar gel diffusion test detection method.
Specific detection operating procedure is as follows:
(1) matched with concentration 0.01mol/L, the phosphate buffer (phosphate buffer saline, PBS) that pH value is 7.4
The Ago-Gel of concentration 1% processed prepares fine jade and expands plate, and Ago-Gel is with a thickness of between 5~6mm, with Seven-hole plum punch
Punching；
(2) agp antigen is added to the interstitial hole in seven apertures in the human head, test antibodies is done into proportional diluted, extension rate is respectively 2-1、
2-2、2-3、2-4、2-5、2-6, it is added sequentially in 6 holes around, the amount that agp antigen and test antibodies is added is 30 holes μ L/, will
Fine jade expands plate and reacts for 24 hours in 37 DEG C of warm boxes, reads fine jade and expands antibody titer；Fine jade expands antibody titer >=1:2, has precipitation line to occur
It is judged to pig prevalence diarrhea virus antibody positive, what no precipitation line occurred is judged to feminine gender.
Advantageous effects of the invention embody in the following areas:
1. Porcine epidemic diarrhea virus agp antigen of the invention is to purify highly enriched antigen, the antigentic specificity is good, stability
Height, sensibility is good, yield is high, agglutination image clearly；It cannot be only used for the Efficacy evaluation of Porcine Epidemic Diarrhea, it is non-to exempt from pig
Infect And Diagnose, it may also be used for the titration of Porcine epidemic diarrhea virus Yolk antibody product, with good application prospect.
2. Porcine epidemic diarrhea virus antibody agar gel diffusion test detection method of the present invention has higher positive rate,
The Swine serum and colostrum sample of pig epidemic diarrhea seedling was immunized in measurement, and antibody total positives rate is 92.86%；Sow colostrum antibody
Fine jade expand detection with commercial kit IgA antibody ELISA detect, antibody positive and negative coincidence rate be 100%, and have experiment behaviour
It is required the features such as low, testing cost is low, particularly suitable base's veterinary quarantine testing department and farm use.
Detailed description of the invention
Fig. 1 be Swine serum antibody titer be 1:16 when fine jade expand result.
Specific embodiment
Explanation is further explained to technical solution of the present invention below by way of specific embodiment.
Embodiment 1
The preparation of Porcine epidemic diarrhea virus agp antigen
(1) Porcine epidemic diarrhea virus SD plants of the present invention are Husbandry & Veternity Research Inst. of Anhui Prov. Agriculture Science Academy animal doctor research department point
From.Porcine epidemic diarrhea virus SD is deposited in China typical culture collection center on March 29th, 2019, preservation
Location: China, Wuhan, Wuhan University；Entitled epidemic diarrhea virus SD plants of its preservation, deposit number are as follows: CCTCC NO:
V201915。
(2) with the sugared culture solution of the DMEM high containing 10% calf serum, (it is 100U/mL, chain that penicillin concn is added in culture solution
Mycin concentration is 0.1mg/mL) VERO cell is cultivated in 37 DEG C, 5%CO2 incubator to being paved with Tissue Culture Flask bottom
80% or so, culture solution is discarded, is washed Tissue Culture Flask 3 times with D-HANKS liquid.
(3) in 25cm2In Tissue Culture Flask, the 28th generation Porcine epidemic diarrhea virus SD strain virus liquid (TCID is added50Value is
10-7.5) 1mL, 0.25% trypsin solution, 4 μ L, 100 × penicillin and streptomycin mixed liquor, 20 μ L(penicillin concn is 10000U/mL, chain
Mycin concentration is 10mg/mL), it is uniformly mixed, 37 DEG C, effect 2 hours in 5%CO2 incubator, with D-HANKS liquid by cell culture
Bottle is washed 2 times.
(4) D-HANKS liquid, 25cm are discarded2The DMEM high sugar culture solution 7mL(culture of serum-free is added in Tissue Culture Flask
It is 100U/mL that penicillin concn is added in liquid, and streptomysin concentration is 0.1mg/mL), 0.25% trypsin solution, 15 μ L is uniformly mixed,
It sets 37 DEG C, continuously cultivate 72 hours in 5%CO2 incubator.
(5) it is cultivated 72 hours after connecing poison, the VERO cell of the 28th generation Porcine epidemic diarrhea virus SD strain virus liquid of inoculation goes out
Existing typical cells lesion, and start aggregation and fall off, Tissue Culture Flask is set -20 DEG C to take out room temperature condition down toward freezing completely
Cell bottle inner virus culture is collected in lower thawing, repeatedly freeze thawing 3 times (containing cell and culture solution).
(6) by freeze thawing 3 times viral cultures, formaldehyde is added by final concentration 2 ‰ and is uniformly mixed, is inactivated in 37 DEG C of shaking tables
24 hours.
(7) it takes freeze thawing 3 times and inactivation of viruses culture, the Triton X-100 of 1/20 volume is added
(TritonX-100), shaken at room temperature 20 minutes, 6000r/min, 4 DEG C of high speed centrifugation 20min collect supernatant.
(8) supernatant is taken, 45000r/min, 4 DEG C of ultracentrifugation 120min discard supernatant liquid, and precipitating uses 0.01mol/L
The PBS that PH is 7.4 dissolves, and PBS usage amount is viral cultures volume 1/15, both obtains Porcine epidemic diarrhea virus agp antigen.
(9) protein concentration is measured using Bradford method, with bovine serum albumin(BSA) (Albumin from bovine
Serum, BSA) standard items compound concentration be 100,250,500,750,1000 μ g/mL solution draw standard curve, with nucleic acid egg
White analyzer surveys the concentration of Porcine epidemic diarrhea virus agp antigen, and measuring its protein concentration is 6.5mg/mL.
Embodiment 2
The foundation of Porcine epidemic diarrhea virus antibody test agar diffusion test method
By the inactivation of viruses culture of 1 the method for embodiment preparation Porcine epidemic diarrhea virus, 1:1 is pressed with Freund's complete adjuvant
Ratio is emulsified, and 3 more than monthly age health SPF chickens are immunized, and immunizing dose is 1mL/, and interval is immunized for 15 days again, so exempts from
Epidemic disease 4 times, 4 immune rear 15 days acquisition chicken bloods extract serum, are prepared into positive serum；Acquire the 3 more than monthly age nonimmune health
SPF chicken blood extracts serum, is prepared into negative serum.
The Ago-Gel that 1% is prepared with the PBS that 0.01mol/L PH is 7.4 prepares fine jade and expands plate, and Ago-Gel is thick
Degree is punched with Seven-hole plum punch between 5 ~ 6mm, agar in hole is gently chosen, back cover is slowly heated on flame, is kept away
Liquid is leaked from bottom hole after exempting from sample-adding.
Fine jade is added in undiluted positive serum and expands plate centre bore, the positive serum amount of addition is 30 μ L, and embodiment 1 is made
Standby ultracentrifugal viral cultures precipitating, with 0.01mol/L PH be 7.4 PBS by provirus culture volume 1/5,
1/15, it 1/30,1/60,1/120,1/240 is diluted, sequentially adds in 6 holes around, the amount of addition is 30 holes μ L/, by fine jade
Expansion plate reacts in 37 DEG C of warm boxes to be observed afterwards for 24 hours, is read fine jade and is expanded as a result, determining that optimal Porcine epidemic diarrhea virus fine jade expansion is anti-
Former dilution is 1/15.
The acquisition pig farm Duo Jia was immunized pig epidemic diarrhea seedling more than twice and (uses pig epidemic diarrhea and pig transmissible stomach and intestine
Scorching Combined vaccine or pig epidemic diarrhea, transmissible gastroenteritis of swine and porcine rotavirus disease triple vaccine) 30 parts of Swine serum and sow
40 parts of colostrum, (by the 1/15 of provirus culture volume, the PBS that 0.01mol/L PH is 7.4 is added in the agp antigen of preparation
Dissolved dilution obtains, and measuring protein concentration is 6.5mg/mL) it is added to the interstitial hole in seven apertures in the human head, by test antibodies, (pig is popular
Serum or colostrum after diarrhea seedling is immune) proportional diluted is done, extension rate is respectively 2-1、2-2、2-3、2-4、2-5、2-6, successively plus
Enter around in 6 holes, the amount that agp antigen and test antibodies are added is 30 holes μ L/, and fine jade expansion plate is reacted in 37 DEG C of warm boxes
It observes afterwards for 24 hours, reads fine jade and expand potency (as shown in Figure 1).
1 agar diffusion test statistical result of table
By 40 parts of sow colostrums, with commercial goods Porcine epidemic diarrhea virus IgA antibody ELISA detection kit (selection
It is the Porcine epidemic diarrhea virus IgA antibody ELISA detection kit of BIONOTE company, batch number EB4410PO) it carries out
Detection, testing result show that IgA antibody number positive is 38 parts (positive rates 95.00%), and expand feminine gender, the positive of method measurement with fine jade
Sample coincidence rate is 100%.
As can be seen from Table 1, the fine jade expanding method established with Porcine epidemic diarrhea virus agp antigen prepared by the present invention,
The Swine serum and colostral antibody potency of pig epidemic diarrhea seedling was immunized in measurement, and antibody total positives rate is 92.86%, antibody titer
It focuses mostly between 1/4~1/16, the percentage that Zhan always detects sample is 70.00%, illustrates Porcine epidemic diarrhea virus of the present invention
Antibody test agar diffusion test method has higher positive rate, the detection suitable for Porcine epidemic diarrhea virus antibody.With this
The fine jade expanding method that the Porcine epidemic diarrhea virus agp antigen of invention preparation is established, to sow colostrum sample, pig epidemic
Feminine gender, positive test symbol and the commercial goods Porcine epidemic diarrhea virus IgA antibody ELISA detection reagent of diarrhea virus antibody
The coincidence rate of box testing result is 100%.Illustrate that Porcine epidemic diarrhea virus agp antigen made of the present invention has sensibility
The features such as height, high specificity, stability are good, process for making science, and it is low in cost, operating method is easy, very suitable
Preferably used in base animal doctor and farm.
Claims (3)
1. a kind of Porcine epidemic diarrhea virus agp antigen, it is characterised in that: obtained by following methods:
(1) virus liquid of the 28th generation Porcine epidemic diarrhea virus of 1mL, TCID50Value is 10-7.5, it is inoculated into 25cm2Cell culture
Grow up on the VERO cell of single layer in bottle, has set 37 DEG C, cultivates 72 hours in 5%CO2 incubator, connect malicious VERO cell and allusion quotation occur
Type cytopathy, and start aggregation and fall off, Tissue Culture Flask is set -20 DEG C so that down toward freezing completely, taking-up is melted under room temperature
Change, freeze thawing 3 times repeatedly, collect viral cultures, the viral cultures include cell and culture solution；
Porcine epidemic diarrhea virus SD plants of 28th generation was deposited in China typical culture collection on March 29th, 2019
The heart, preservation address: China, Wuhan, Wuhan University；Entitled epidemic diarrhea virus SD plants of its preservation, deposit number are as follows:
CCTCC NO: V201915；
(2) formaldehyde is added by the amount of final concentration 2 ‰ in viral cultures to be uniformly mixed, is inactivated 24 hours in 37 DEG C of shaking tables；
(3) in the viral cultures of freeze thawing 3 times and inactivation, polyethylene glycol octyl is added by the amount of viral cultures volume 1/20
Phenyl ether, shaken at room temperature 20 minutes, revolving speed 6000r/min, under the conditions of 4 DEG C of temperature, high speed centrifugation 20min collected supernatant；
(4) take supernatant under the conditions of revolving speed 45000r/min, 4 DEG C of temperature, ultracentrifugation 120min；Liquid is discarded supernatant, is precipitated
It is sufficiently dissolved with the PBS buffer solution of 0.01mol/L pH value 7.4, the dosage of PBS buffer solution is viral cultures volume 1/15
Amount, both obtains Porcine epidemic diarrhea virus agp antigen, and -20 DEG C of packing save.
2. a kind of Porcine epidemic diarrhea virus agp antigen according to claim 1 is used for Porcine epidemic diarrhea virus antibody
Detection method, it is characterised in that: the Porcine epidemic diarrhea virus antibody detection method be Porcine epidemic diarrhea virus antibody
Agar gel diffusion test detection method.
3. detection method according to claim 2, it is characterised in that operating procedure is as follows:
(1) with concentration 0.01mol/L, the Ago-Gel for the PBS buffer solution compound concentration 1% that pH value is 7.4, it is flat to prepare fine jade expansion
Plate, Ago-Gel are punched with a thickness of between 5~6mm with Seven-hole plum punch；
(2) agp antigen is added to the interstitial hole in seven apertures in the human head, test antibodies is done into proportional diluted, extension rate is respectively 2-1、
2-2、2-3、2-4、2-5、2-6, it is added sequentially in 6 holes around, the amount that agp antigen and test antibodies is added is 30 holes μ L/, will
Fine jade expands plate and reacts for 24 hours in 37 DEG C of warm boxes, reads fine jade and expands antibody titer；Fine jade expands antibody titer >=1:2, has precipitation line to occur
It is judged to pig prevalence diarrhea virus antibody positive, what no precipitation line occurred is judged to feminine gender.
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Cited By (1)
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| CN111686246A (en) * | 2020-05-29 | 2020-09-22 | 安徽省农业科学院畜牧兽医研究所 | Antigen-antibody complex vaccine for porcine epidemic diarrhea virus and preparation method thereof |
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