CN103342749B - Triple egg yolk antibody against newcastle disease virus, avian influenza virus and infectious bronchitis virus - Google Patents
Triple egg yolk antibody against newcastle disease virus, avian influenza virus and infectious bronchitis virus Download PDFInfo
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Abstract
The invention discloses a triple egg yolk antibody against newcastle disease virus, avian influenza virus and infectious bronchitis virus and a preparation method and application thereof. The preparation method of the egg yolk antibody comprises the following steps of: (1) selecting a hen; (2) performing triple inactivation immunization of the hen obtained by the step (1) by use of newcastle disease virus, infectious bronchitis virus and avian influenza virus; (3) taking the egg laid by the hen and separating the egg yolk liquid; and (4) separating and purifying an egg yolk antibody from the egg yolk liquid obtained by the step (3). The triple egg yolk antibody disclosed by the invention is of great value in treating and preventing newcastle disease, avian influenza and infectious bronchitis.
Description
Technical field
The present invention relates to a kind of three yolk antibodies for newcastle disease virus, avian influenza virus and avian infectious bronchitis virus and its preparation method and application.
Background technology
Newcastle disease is the high degree in contact sexually transmitted disease that newcastle disease virus causes, also known as philippine fowl disease or pseudo-checken pest, principal character be expiratory dyspnea, just rare, nervous disorders, mucous membrane and serous coat are hemorrhage, mortality ratio is high, causes harm seriously to poultry husbandry.
Bird flu is a kind of communicable disease caused by avian influenza virus, is decided to be category A infectious disease by International Office of Epizootics, also known as fowl plague or European checken pest.The common sympton of bird flu: sick chicken spirit is depressed, feed consumption reduces, and becomes thin; The broodiness of hen strengthens, and egg productivity declines; Slightly until serious respiratory symptom, comprise cough, sneeze and shed tears in a large number; Head and face's oedema, nervous disorders and diarrhoea.
Chicken infectious bronchitis is the acute high degree in contact respiratory infectious disease of one of the chicken caused by infectious bronchitis virus, and it faces examines feature and be expiratory dyspnea, send rale, cough, mouth breathing, sneeze.Infectious bronchitis virus, mainly through airborne transmission, also can pass through the propagation such as feed, drinking-water, bedding and padding.
For a long time, the morbidity bird in bird feedlot with above-mentioned three kinds of viral coinfections or secondary infection in the majority.Control at present for above-mentioned disease depends on vaccine and microbiotic.Microbiotic can cause drug residue in animal body and develop immunity to drugs.The effective constituent of existing vaccine is inactivation of viruses, bird feedlot is the immunity when chicken 1-2 age in week usually, produce antibody playing a role the needs time of about 14 days in animal body, so within chicken all periods in age in 1 week age to 4, be easier to the outburst occurring one or more diseases above-mentioned.
Summary of the invention
The object of this invention is to provide a kind of three yolk antibodies for newcastle disease virus, avian influenza virus and avian infectious bronchitis virus and its preparation method and application.
The method preparing yolk antibody provided by the invention, comprises the steps:
(1) hen is chosen by following principle: without artificial immunization; Open and produced and lay eggs normal; Be that Avian pneumo-encephalitis virus is negative, avian influenza virus is negative and avian infectious bronchitis virus negative after testing;
(2) with the hen that newcastle disease virus, infectious bronchitis virus, avian influenza virus three deactivation vaccine immunity step (1) obtain;
(3), after completing steps (2), the egg getting hen production is also separated and obtains egg yolk liquid;
(4) egg yolk liquid that step (3) obtains is got, mix with equal-volume distilled water, 650rpm vibrates 10min, then in sealed states 65 DEG C leave standstill 10 minutes, mix with the volume ratio of 1:4 with distilled water after being cooled to 10 DEG C, adjust pH to 5.2,4 DEG C leave standstill 6 hours, then centrifugal 20 minutes of 5000rpm, gets supernatant liquor;
(5) added by the equal-volume saturated ammonium sulphate aqueous solution in the supernatant liquor that step (4) obtains, 4 DEG C leave standstill 12 hours, and centrifugal 20 minutes of 8000rpm, gets precipitation;
(6) dissolve with 3 mass parts distilled water the precipitation that 1 mass parts step (5) obtains, then adding the volume ratio that n-caprylic acid makes it in system is 1 ‰, mixing;
(7) the 1 parts by volume saturated ammonium sulphate aqueous solution and 2 parts by volume steps (6) are obtained solution to mix, 4 DEG C leave standstill 12 hours, and centrifugal 20 minutes of 8000rpm, gets precipitation;
(8) precipitation that obtains of aseptic by 3 mass parts, pH7.2,0.01M PBS buffer solution 1 mass parts step (7);
(9) solution 100Ku ultra-filtration membrane bag step (8) obtained carries out desalination and concentrates, and obtains concentrated solution;
(10) 1 parts by volume 40% formalin is added in the concentrated solution that 399 parts by volume steps (9) obtain, then room temperature leaves standstill 2 hours in sealed states, then filter step by step with the millipore filtration of 0.45 micron, 0.22 micron and 0.1 micron successively, obtain filtrate, be yolk antibody.
Hen in described step (1) can when chick immunity chicken interferon α.Described chicken interferon α is specifically as shown in the sequence 1 of sequence table.Chicken during 7 age in days is specifically can be during described chick.The immunizing dose of described chicken interferon α is every chicken 15U.
In described step (2), described immunity comprises the steps:
Fundamental immunity: described three deactivation vaccines of every chicken immune l0 immunizing dose;
After booster immunization: fundamental immunity l0 days, described three deactivation vaccines of every chicken immune l0 immunizing dose;
Reinforced immunological: after booster immunization l0 days, described three deactivation vaccines of every chicken immune 20 immunizing doses;
Maintain immunity: reinforced immunological is after 10 days, described three deactivation vaccines of every chicken immune l0 immunizing dose.
In described step (2), described newcastle disease virus, infectious bronchitis virus, avian influenza virus three deactivation vaccine specifically can be newcastle disease virus (La Sota strain), infectious bronchitis virus (M41 strain), avian influenza virus (H9 hypotype, HL strain) three deactivation vaccines, more specifically can be following vaccine: Luoyang Pulaike Biological Engineering Co., Ltd.'s product, veterinary drug new word (2006) 160022124.
Also comprise the steps: in described step (3) to choose and described step (4) is carried out to the HI of newcastle disease virus (specifically can be chicken Newcastle disease live vaccine La Sota strain), infectious bronchitis virus (specifically can be chicken infectious bronchitis M41 strain), avian influenza virus (specifically the can be H9N2 subtype avian influenza virus YB strain) egg yolk liquid being all greater than 1024 of tiring.
In described step (10), the HI of described yolk antibody to newcastle disease virus (specifically can be chicken Newcastle disease live vaccine LaSota strain), infectious bronchitis virus (specifically can be chicken infectious bronchitis M41 strain), avian influenza virus tires and is all greater than 128.
The yolk antibody that the present invention also protects above arbitrary described method to prepare.
The present invention also protects described yolk antibody to prevent and/or treat the application in the triple vaccine of Newcastle disease, bird flu and chicken infectious bronchitis in preparation.
The present invention also protects a kind of triple vaccine preventing and/or treating Newcastle disease, bird flu and chicken infectious bronchitis, containing described yolk antibody.
The immunity system of the chick within 20 ages in days is not also grown completely, and in the 5-28 age in days stage, the maternal antibody level of chick reduces, and autoantibody generates not yet in a large number.Adopt yolk antibody provided by the invention immunity, chick can be protected not by the invasion and attack of the viruses such as newcastle disease, bird flu, chicken infectious bronchitis, and to by the chick of above-mentioned virus attack, being there is therapeutic action.
Hen produces immune response after stimulating by immunogen, the yolk ripening stage in uterine tube, IgG in blood selectively transfers in yolk, and becomes immunoglobulins unique in yolk, and this IgG antibody-like entering yolk liquid by blood transfer is yolk antibody.Yolk antibody has specific combination and deactivation to pathogenic micro-organism, thus can treat and/or prevent disease.
The invention provides three yolk antibodies and preparation method thereof for newcastle disease virus, avian influenza virus and avian infectious bronchitis virus, for the treatment of Newcastle disease, avian influenza and chicken infectious bronchitis and prevention, there is substantial worth.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.In embodiment, the aqueous solution of each material all adopts distilled water.
Three deactivation vaccines used in embodiment are newcastle disease virus (La Sota strain), infectious bronchitis virus (M41 strain), avian influenza virus (H9 hypotype, HL strain) three deactivation vaccines, Luoyang Pulaike Biological Engineering Co., Ltd.'s product, veterinary drug new word (2006) 160022124.Chicken Newcastle disease live vaccine (La Sota strain) is purchased from China Veterinery Drug Inspection Office.H9N2 subtype avian influenza virus YB strain: Kingsoft, Wei Jin Feng Liyuling village Deng Jun Hua Baichao is brave, the isolation identification of a strain H9N2 subtype avian influenza virus (YB strain), " animal doctor's guide " the 7th phase in 2012,69-71 page.Chicken infectious bronchitis M41 strain: Zhao Bao Huama is with locking Yang Hong, and clone and the eukaryotic expression of chicken infectious bronchitis M41 strain S1 gene are studied, animal medicine is in progress, 2005,26(1), 71-75.
The preparation of embodiment 1, egg yolk liquid
Chicken interferon α (chicken genetic engineering interferon): purchased from Beijing Ka Ruinuo Bioisystech Co., Ltd; For the protein shown in the sequence 1 of sequence table; Each 6ml(that is packaged as is containing 30000U chicken interferon α), use front normal saline dilution to 200ml, every chicken injection 0.1ml.
One, egg yolk liquid is prepared
1, the hen of 7 ages in days is got, neck subcutaneous injection chicken interferon α, every chicken 15U.
2, choose according to following principle from the hen of step 1: without artificial immunization; Open and produced and lay eggs normal; Be that Avian pneumo-encephalitis virus is negative, avian influenza virus is negative and avian infectious bronchitis virus negative after testing.
3, hen step 2 chosen carries out following immunity:
Fundamental immunity: neck subcutaneous injection three deactivation vaccine, every chicken l0 immunizing dose (10 plumage part);
After booster immunization: fundamental immunity l0 days, neck subcutaneous injection three deactivation vaccine, every chicken l0 immunizing dose (10 plumage part);
Reinforced immunological: after booster immunization l0 days, neck subcutaneous injection three deactivation vaccine, every chicken 20 immunizing doses (20 plumage part);
Maintain immunity: reinforced immunological is after 10 days, subcutaneous three deactivation vaccines of neck, every chicken 10 immunizing doses (10 plumage part).
4, after completing steps 3, the egg of hen is collected every day, 4 DEG C of preservations.
5, the egg of step 4 is got, with clear water wiping to remove the dirt on eggshell, then 0.1% bromogeramine solution soaking disinfection is used, taking-up is aseptically separated egg white and yolk after drying, yolk surface (as far as possible removing egg white) is repeatedly rinsed with sterile purified water, use punctures membrane of yolk, collect egg yolk liquid.
Two, egg yolk liquid is prepared
Do not inject the step of chicken interferon α, other is with step one.
Three, the titration of egg yolk liquid
The egg yolk liquid that the egg yolk liquid that obtains of detecting step one or step 2 obtain respectively is tired to H9N2 subtype avian influenza virus YB strain, chicken Newcastle disease live vaccine (La Sota strain) and the HI of chicken infectious bronchitis M41 strain.
1, the preparation of 1% chicken erythrocyte suspension
The blood of 2-6 monthly age chicken is taked to mix with equivalent A Shi liquid, then use brine 3-4 time (the centrifugal 5-10 minute of each 1500r/min), the red corpuscle normal saline of precipitation is become 1% chicken erythrocyte suspension (namely 1 μ l erythroprecipitin adds 99 μ l physiological saline).
2, the titration (HA test) of virus
96 hole micro-reaction plates carry out, and every hole adds 25 μ L physiological saline; The 1st hole, left side adds 25 μ L virus liquids, inhales 25 μ L to the 2nd hole after mixing from the 1st hole, and doubling dilution is to the 11st hole (the 11st hole is inhaled and abandoned 25 μ L) successively; Every hole adds 25 μ l physiological saline and 25 μ l1% chicken erythrocyte suspensions, and vibrate 15s on the oscillator, and room temperature leaves standstill observations after 20min.
The whole aggegation of red corpuscle, is sunken at the bottom of hole, and tiling in netted, is 100% aggegation (++++).Be the HA-HI test of this virus liquid with the virus liquid greatest dilution of 100% aggegation, be an aggegation unit, or claim a HAU.
3, the titration (HI test) of egg yolk liquid
96 hole micro-reaction plates carry out, and every hole adds 25 μ L physiological saline; The 1st hole, left side adds 25 μ L egg yolk liquids, inhales 25 μ L to the 2nd hole after mixing from the 1st hole, and doubling dilution is to the 11st hole (the 11st hole is inhaled and abandoned 25 μ L) successively; Every hole adds the virus liquid of 25 μ l, tetra-HAUs, vibration 30s, and then room temperature leaves standstill 30min; Every hole adds 25 μ l1% chicken erythrocyte suspensions, and concussion 15s, room temperature leaves standstill observations after 20min.
The blood clotting being this egg yolk liquid with the greatest dilution of the tested egg yolk liquid of 100% suppression aggegation (not aggegation completely) suppresses to tire, and namely HI tires.
The egg yolk liquid getting 20 eggs from the egg yolk liquid that step one obtains at random carries out above-mentioned titration, and the egg yolk liquid of 18 eggs is tired to three kinds of viral HI and is more than 1024.
The egg yolk liquid getting 20 eggs from the egg yolk liquid that step 2 obtains at random carries out above-mentioned titration, and the egg yolk liquid of 6 eggs is tired to three kinds of viral HI and is more than 1024.
Result shows, chick injects chicken interferon α period, obtains the probability of the egg with the egg yolk liquid that high HI tires after significantly can increasing immune three deactivation vaccines.
The preparation of embodiment 2, yolk antibody
1, the step one of Example 1 prepare and by the method for the step 3 of embodiment 1 measure to three kinds of viral HI tire be more than 1024 egg yolk liquid, mix with equal-volume distilled water, 650rpm vibrates 10min, then in sealed states 65 DEG C leave standstill 10 minutes, be cooled to rapidly 10 DEG C, then mix with the volume ratio of 1:4 with distilled water, pH to 5.2 is adjusted with the 1M HCl aqueous solution, 4 DEG C leave standstill 6 hours, and then centrifugal 20 minutes of 5000rpm, gets supernatant liquor.
2, slowly added by isopyknic saturated ammonium sulphate aqueous solution in the supernatant liquor that step 1 obtains (stirring of limit edged), then 4 DEG C leave standstill 12 hours, and centrifugal 20 minutes of 8000rpm, gets precipitation.
3, dissolve with 3 mass parts distilled water the precipitation that 1 mass parts step 2 obtains, then adding the volumetric concentration that n-caprylic acid makes it in system is 1 ‰, stirs.
4, the 1 parts by volume saturated ammonium sulphate aqueous solution slowly being added 2 parts by volume steps 3 obtains in solution, and limit edged stirs, and then 4 DEG C leave standstill 12 hours, and centrifugal 20 minutes of 8000rpm, gets precipitation.
5, the precipitation that obtains of aseptic by 3 mass parts, pH7.2,0.01M PBS buffer solution 1 mass parts step 4.
6, (interception is 100ku to the solution 100Ku ultra-filtration membrane bag 1000 milliliters of steps 5 obtained, the full name of pillar is Hollow Fiber Ultrafiltration post, purchased from Ai Yan bio tech ltd, Shanghai) and by specification carry out desalination concentrate, obtain 100 milliliters of concentrated solutions.
7, by 1 parts by volume 40%(volume ratio) formalin dropwise adds in the concentrated solution that 399 parts by volume steps 6 obtain (stirring of limit edged), then room temperature leaves standstill 2 hours in sealed states, then filter step by step with the millipore filtration of 0.45 micron, 0.22 micron and 0.1 micron successively, obtain filtrate, filtrate 4 DEG C is saved backup.
The HI of the filtrate 8, adopting the method detecting step 7 of step 3 of embodiment 1 to obtain tires, and HI tires the yolk antibody that filtrate being that be greater than 128 prepares.
The application of embodiment 3, yolk antibody
One, the application of yolk antibody
Get the sick chicken of 10 ages in days in poulty house, wherein make a definite diagnosis generation Newcastle disease (first group) for 20,20 have confirmed bird flu (second group) occurs, make a definite diagnosis for 20 and chicken infectious bronchitis (the 3rd group) has occurred, made a definite diagnosis for 20 and Newcastle disease, bird flu and chicken infectious bronchitis (the 4th group) occur simultaneously.The yolk antibody that neck subcutaneous injection embodiment 2 prepares, every chicken injects 1.5 milliliters.Injection yolk antibody after 5 days, to be the curative ratio of 95%, second group be that the curative ratio of the 95%, three group is that the curative ratio of the 100%, four group is 95% for the curative ratio of first group.
Two, the application of yolk antibody
In Beijing, multiple poulty house is applied, if there is a chicken to there occurs Newcastle disease and/or bird flu and/or chicken infectious bronchitis in this poulty house, this feedlot is morbidity feedlot, single immunization is carried out to all 5-20 age in days chickens be wherein in same feedlot with morbidity chicken, namely the yolk antibody for preparing of every chicken injection neck subcutaneous injection 1.5 milliliters of embodiments 2, adds up the sickness rate of Newcastle disease and/or bird flu and/or chicken infectious bronchitis in the chicken of all Immune Yolk Antibodies for 10 days afterwards.The results are shown in Table 1.
The each feedlot of table 1 uses yolk antibody situation statistics
| Date | Poulty house is numbered | Use the chicken number of yolk antibody | Chicken age in days | Sickness rate |
| 2013.04.15 | SYC001 | 3000 | 13 | 2% |
| 2013.04.20 | SYC002 | 6000 | 8 | 2% |
| 2013.05.05 | SYC003 | 2500 | 9 | 0% |
| 2013.05.25 | SYC004 | 3000 | 10 | 2% |
| 2013.05.27 | SYC005 | 2700 | 11 | 2% |
| 2013.06.04 | SYC006 | 4300 | 12 | 2% |
| 2013.06.08 | SYC007 | 2000 | 6 | 0% |
| 2013.06.10 | SYC008 | 5000 | 10 | 2% |
| 2013.06.20 | SYC009 | 2300 | 5 | 0% |
Claims (5)
1. prepare a method for yolk antibody, comprise the steps:
(1) hen is chosen by following principle: without artificial immunization; Open and produced and lay eggs normal; Be that Avian pneumo-encephalitis virus is negative, avian influenza virus is negative and avian infectious bronchitis virus negative after testing; Hen in described step (1) is immunity chicken interferon α when chick;
(2) with the hen that newcastle disease virus, infectious bronchitis virus, avian influenza virus three deactivation vaccine immunity step (1) obtain;
In described step (2), described immunity comprises the steps:
Fundamental immunity: described three deactivation vaccines of every chicken immune l0 immunizing dose;
After booster immunization: fundamental immunity l0 days, described three deactivation vaccines of every chicken immune l0 immunizing dose;
Reinforced immunological: after booster immunization l0 days, described three deactivation vaccines of every chicken immune 20 immunizing doses;
Maintain immunity: reinforced immunological is after 10 days, described three deactivation vaccines of every chicken immune l0 immunizing dose;
(3), after completing steps (2), the egg getting hen production is also separated and obtains egg yolk liquid;
(4) egg yolk liquid that step (3) obtains is got, mix with equal-volume distilled water, 650rpm vibrates 10min, then in sealed states 65 DEG C leave standstill 10 minutes, mix with the volume ratio of 1:4 with distilled water after being cooled to 10 DEG C, adjust pH to 5.2,4 DEG C leave standstill 6 hours, then centrifugal 20 minutes of 5000rpm, gets supernatant liquor;
(5) added by the equal-volume saturated ammonium sulphate aqueous solution in the supernatant liquor that step (4) obtains, 4 DEG C leave standstill 12 hours, and centrifugal 20 minutes of 8000rpm, gets precipitation;
(6) dissolve with 3 mass parts distilled water the precipitation that 1 mass parts step (5) obtains, then adding the volume ratio that n-caprylic acid makes n-caprylic acid in system is 1 ‰, mixing;
(7) the 1 parts by volume saturated ammonium sulphate aqueous solution and 2 parts by volume steps (6) are obtained solution to mix, 4 DEG C leave standstill 12 hours, and centrifugal 20 minutes of 8000rpm, gets precipitation;
(8) precipitation that obtains of aseptic by 3 mass parts, pH7.2,0.01M PBS buffer solution 1 mass parts step (7);
(9) solution 100Ku ultra-filtration membrane bag step (8) obtained carries out desalination and concentrates, and obtains concentrated solution;
(10) 1 parts by volume 40% formalin is added in the concentrated solution that 399 parts by volume steps (9) obtain, then room temperature leaves standstill 2 hours in sealed states, then filter step by step with the millipore filtration of 0.45 micron, 0.22 micron and 0.1 micron successively, obtain filtrate, be yolk antibody.
2. the method for claim 1, is characterized in that: also comprise the steps: in described step (3) to choose and carry out described step (4) to the HI of newcastle disease virus, infectious bronchitis virus, the avian influenza virus egg yolk liquid being all greater than 1024 of tiring.
3. the yolk antibody for preparing of method described in claim 1 or 2.
4. yolk antibody described in claim 3 prevents and/or treats the application in the triple vaccine of Newcastle disease, bird flu and chicken infectious bronchitis in preparation.
5. prevent and/or treat a triple vaccine for Newcastle disease, bird flu and chicken infectious bronchitis, containing yolk antibody described in claim 3.
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| CN107787914A (en) * | 2017-10-26 | 2018-03-13 | 绿春县农业园林生态有限责任公司 | The method for breeding of Lvchun County coptis chicken |
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| CN103585628B (en) * | 2013-11-21 | 2016-08-17 | 青岛润达生物科技有限公司 | A kind of immunoglobulin preparation preventing Newcastle disease and preparation method thereof |
| CN104383538A (en) * | 2014-12-14 | 2015-03-04 | 郑州后羿制药有限公司 | Tetragenous egg yolk antibody oral liquid and preparation method for same |
| CN104524570A (en) * | 2014-12-14 | 2015-04-22 | 郑州后羿制药有限公司 | Newcastle disease and avian influenza dual egg yolk antibody oral solution and preparation method thereof |
| CN105367653A (en) * | 2015-12-08 | 2016-03-02 | 天津瑞普生物技术股份有限公司 | Extraction method of infectious bronchitis egg yolk antibodies |
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| CN106310252A (en) * | 2016-08-25 | 2017-01-11 | 青岛润达生物科技有限公司 | Preparing method of triple egg yolk antibody oral liquid |
| CN106267194A (en) * | 2016-08-25 | 2017-01-04 | 青岛润达生物科技有限公司 | A kind of yolk antibody and Swine spleen transfer factor joint product |
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| CN102584990A (en) * | 2011-12-19 | 2012-07-18 | 李昌仁 | Method for extracting binary high-immunity yolk antibodies against newcastle disease and avian influenza |
| CN102558347A (en) * | 2012-03-01 | 2012-07-11 | 广东紫金正天药业有限公司 | Yolk antibody for treating bird flu and infectious bronchitis and preparation method thereof |
| CN102827275B (en) * | 2012-09-12 | 2014-03-05 | 青岛易邦生物工程有限公司 | Method for preparing duck virus hepatitis divalent refined egg yolk antibody |
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