CN104984337B - A kind of newcastle disease, avian influenza antigen antibody complex inactivated vaccine and preparation method thereof - Google Patents
A kind of newcastle disease, avian influenza antigen antibody complex inactivated vaccine and preparation method thereof Download PDFInfo
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- CN104984337B CN104984337B CN201410848495.3A CN201410848495A CN104984337B CN 104984337 B CN104984337 B CN 104984337B CN 201410848495 A CN201410848495 A CN 201410848495A CN 104984337 B CN104984337 B CN 104984337B
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Abstract
The present invention relates to a kind of newcastle disease, the preparation methods of avian influenza antigen antibody complex vaccine comprising following steps：It prepares avian influenza inactivation antigen, prepare newcastle disease inactivation antigen and avian influenza inactivation Yolk antibody, the ewcastle disease antigen inactivated, avian influenza antigen and avian influenza egg yolk antibodies semi-finished product are emulsified and mixed respectively.The present invention, which provides, a kind of can be suitably used for all chickens group's; safely and effectively and vaccine immunity protection especially can be enhanced to chick and diseased chicken; reach and antigen is delayed to discharge, the good purity of safety is high, and can save a kind of newcastle disease of production cost, avian influenza antigen antibody complex vaccine.
Description
Technical field
The invention belongs to biological new medicine field, be related to a kind of newcastle disease, avian influenza antigen antibody complex vaccine and
Preparation method.
Technical background
Newcastle disease (Newcastle Disease, ND) be by newcastle disease virus (Newcastle Disease Virus,
NDV the infectious disease of the acute high degree in contact of chicken caused by) and a variety of birds has very high incidence and case fatality rate.Fowl is flowed
It is to betide the acute infectious disease of various poultry and wild bird caused by influenza A to feel (Avian Influenza, AI).
The influenza A of bird flu is caused to be called avian influenza virus (Avian Influenza Virus, AIV), which has been in the whole world
Property distribution, there are a pathogenetic report in many countries and regions, and global bird flu, which is very popular, have been occurred repeatedly, provisions fowl
Industry brings heavy strike.
Currently, the main means of prevention ND and AI are vaccine immunity, and assist antibiotic and antiviral treatment.But it lacks
Point is that artificial infection labor intensity is big and poultry is big to the stress reaction of immunity inoculation, thus study and application bigeminy vaccine and
Multiple vaccines have become the inexorable trend of live vaccine industry development.
Avian influenza virus is easy to happen variation, therefore cannot use attenuated live vaccines.Inactivated vaccine mainly stimulates body to produce
Raw humoral immunity after animal body is by the stimulation of antigenic substance, is converted into thick liquid cell by bone-marrow-derived lymphocyte and generates and can resist with corresponding
The immunoglobulin of primary raw specific binding reaction, i.e. specific antibody.And the bursa of farbricius of chicken is bone-marrow-derived lymphocyte generation and divides
Change important place, and the development of the bursa of farbricius of chick is not completely, occurs and the ability of differentiation bone-marrow-derived lymphocyte is limited therefore existing
Multi-joint inactivated vaccine it is not high for the Immune efficiency of chick.And using the high special yolk antibody exempted from, chick can be made to obtain
Passive immunity is the effective means that chick spends the bird flu Blank immunization phase.Therefore, it develops one kind and can provide and passively exempt from
Epidemic disease, but can provide active immunity novel avian influenza antigen antibody complex bigeminy or multiple vaccines it is significant.
Antigen-antibody complex vaccine is paid close attention to by domestic and foreign scholars, and has carried out correlative study.Currently, newcastle disease
Compound vaccine research with the poultry dieases such as gumboro disease has been achieved with certain achievement.He Hongxuan (the patent No.s：
ZL200810226023.9 it) is prepared for a kind of avian influenza antigen antibody complex, but its kind poison used is that H 5 N 1 avian influenza is sub-
Type virus, and immune effect and conventional vaccine difference is not notable.Sun Ruiqin exists《Avian influenza virus antigen-antibody complex
Preparation and its immunogenicity》In (Sun Ruiqin etc., Products in China magazine, the 6th phase page 588 of volume 22 in 2009) text
It discloses using the HA albumen of H5 subtype avian influenza virus as antigen, the high-immunity yolk of virus is antibody, and antigen and antibody are pressed
Different proportion mix, the method for preparing avian influenza virus antigen antibody complex vaccine, antibody level and attack malicious protection with
H5 subtype avian influenza oil emulsion inactivated vaccines are compared nothing and are significantly improved.The reason is that its Yolk antibody potency is not high, dosage is inadequate, no
It is enough to provide strong passive immunity.In addition Yolk antibody is added in vaccine directly increase production of vaccine cost, broiler chicken economy
Value is not high, and expensive vaccine is difficult to be received.
Invention content
The purpose of the present invention is to provide a kind of can be suitably used for all chickens group, especially have safely to chick and diseased chicken
It imitates and the vaccine immunity blank phase can be shortened, enhance vaccine protection, newcastle disease, the bird flu for reducing production of vaccine cost are anti-
Original antibody compound inactivated vaccine and preparation method thereof.
In order to realize that above-mentioned purpose, the present invention adopt the following technical scheme that：
The preparation method of the present invention includes the following steps：
1) avian influenza antigen is prepared：Exempt from chicken embryo cleaning and sterilizing by non-, after hatching, H9N2 hypotypes are inoculated in its allantoic cavity
HP plants of avian influenza virus, then proceed to hatch, and aseptically harvest chicken embryo liquid, and carry out purifying and be concentrated to give new chicken embryo
Liquid, and new chicken embryo hydroful foot avian influenza virus content >=108.7EID50/ 0.1mL, the chicken embryo liquid first that then will newly obtain
Aldehyde inactivates；
2) ewcastle disease antigen and avian influenza egg yolk antibodies are prepared：First exempted from HP plants of avian influenza virus inactivated vaccines of H9N2 hypotypes
Epidemic disease breeder, detection chicken embryo H9N2 subclass antibodies are horizontal, select the high-immunity egg that potency reaches 9log2 or more；Then it will select
Avian influenza virus high-immunity egg carries out cleaning and sterilizing, and is hatched；When through hatching to 9~10 age in days, inoculation is new in allantoic cavity
LaSota plants of city epidemic disease poison, then hatches, and aseptically harvests chicken embryo liquid and yolk liquid respectively, and carry out purifying concentration,
Chicken embryo hydroful foot newcastle disease virus content >=10 for obtaining new chicken embryo liquid and yolk liquid, and newly obtaining8.9EID50/ 0.1mL,
Yolk hydroful foot Yolk antibody HI values >=9log2, then by the chicken embryo liquid newly obtained and yolk liquid formalin-inactivated；
3) it emulsifies：By the ewcastle disease antigen inactivated, avian influenza antigen and avian influenza egg yolk antibodies semi-finished product respectively into
Row emulsification adjusts newcastle disease, the HLB value of avian influenza antigen emulsifier obtains oil phase for 3~6, then enables water phase：Oil phase is equal to 1:3；
It adjusts the HLB value of Yolk antibody emulsifier and obtains oil phase for 6~10, then enable water phase：Oil phase is equal to 1:1.5；
4) it mixes：Ewcastle disease antigen, avian influenza antigen and Yolk antibody after emulsification is pressed 1.5~3:1.5~3:1 ratio
Mixing obtains product.
It is non-in the step 1) to exempt to hatch required temperature after chicken embryo cleaning and sterilizing to be 37 DEG C, and need to hatch 9~10
Age；The chicken embryo of inoculated HP plants of avian influenza virus of H9N2 hypotypes continues to hatch required temperature to be 37 DEG C, and hatches and taken
Between be 24~96h.
The temperature needed for the hatching of bird flu high-immunity egg in the step 2) after cleaned disinfection is 37 DEG C, and needs to hatch
9~10 ages in days.
The temperature needed for hatching in the step 2) after LaSota plants of newcastle disease virus of inoculation is 37 DEG C, and is incubated
It is 60~96h the time required to changing.
The present invention is new using a kind of newcastle disease, the obtained Novel chicken of the preparation method of avian influenza antigen antibody complex vaccine
City epidemic disease, avian influenza antigen antibody complex inactivated vaccine.
The present invention uses above technical scheme, and it is anti-to exempt from chicken embryo production yolk using HP plants of avian influenza virus height of H9N2 hypotypes
Body, the Yolk antibody potency produced is high, and dosage foot is suitable for all chickens group, especially high to chick and the same safety of diseased chicken
Effect；By adjusting the HLB value of emulsifier, the i.e. HLB value 6~10 of Yolk antibody emulsifier, newcastle disease, avian influenza antigen emulsifier
HLB value 3~6 so that Yolk antibody hydrophily after emulsification is higher than ewcastle disease antigen and avian influenza antigen, and it is preferential to reach antibody
It is absorbed by organisms and plays a role, delayed antigen release, shorten the Blank immunization phase of vaccine, improve vaccine immunity protection；
Exempt from chicken embryo using bird flu height and prepare newcastle disease virus so that chicken embryo maximizes the use, and it is compound to greatly reduce antigen-antibody
The production cost of object vaccine；The present invention to Yolk antibody to carrying out purifying concentration, then with formalin-inactivated, and obtained Yolk antibody is not
Only purity is high, and safety is good.
Specific implementation mode
The preparation method of the present invention comprising following steps：
1) avian influenza antigen is prepared：Exempt from chicken embryo cleaning and sterilizing by non-, after hatching, H9N2 hypotypes are inoculated in its allantoic cavity
HP plants of avian influenza virus, then proceed to hatch, and aseptically harvest chicken embryo liquid, and carry out purifying and be concentrated to give new chicken embryo
Liquid, and new chicken embryo hydroful foot avian influenza virus content >=108.7EID50/ 0.1mL, the chicken embryo liquid first that then will newly obtain
Aldehyde is inactivated；
2) newcastle disease inactivation antigen and avian influenza inactivation Yolk antibody are prepared：First gone out with HP plants of avian influenza virus of H9N2 hypotypes
The breeder of chicken is immunized in live vaccine, and detection chicken embryo avian influenza antibody is horizontal, selects the high-immunity egg that potency reaches 9log2 or more；So
The avian influenza virus high-immunity egg selected is subjected to cleaning and sterilizing afterwards, and is hatched；In the high-immunity egg allantoic cavity after hatching
LaSota plants of newcastle disease virus of inoculation is then hatched, and aseptically harvests chicken embryo liquid and yolk liquid respectively, and purified
Concentration, chicken embryo hydroful foot newcastle disease virus content >=10 for obtaining new chicken embryo liquid and yolk liquid, and newly obtaining8.9EID50/
0.1mL, yolk hydroful foot Yolk antibody HI value >=9log2, then by the chicken embryo liquid newly obtained and yolk liquid formalin-inactivated；
3) it emulsifies：By the ewcastle disease antigen inactivated, avian influenza antigen and avian influenza egg yolk antibodies semi-finished product respectively into
Row emulsification adjusts newcastle disease, the HLB value of avian influenza antigen emulsifier obtains oil phase, then another water phase for 3~6：Oil phase is equal to 1:3；
It adjusts the HLB value of Yolk antibody emulsifier and obtains oil phase, then another water phase for 6~10：Oil phase is equal to 1:1.5；
4) it mixes：Ewcastle disease antigen, avian influenza antigen and Yolk antibody after emulsification is pressed 1.5~3:1.5~3:1 ratio
Mixing obtains product.
It is non-in the step 1) to exempt to hatch required temperature after chicken embryo cleaning and sterilizing to be 37 DEG C, and need to hatch 9~10
Age；The chicken embryo of inoculated HP plants of avian influenza virus of H9N2 hypotypes continues to hatch required temperature to be 37 DEG C, and hatches and taken
Between be 24~96h.
The temperature needed for the hatching of bird flu high-immunity egg in the step 2) after cleaned disinfection is 37 DEG C, and needs to hatch
9~10 ages in days.
The temperature needed for hatching in the step 2) after LaSota plants of newcastle disease virus of inoculation is 37 DEG C, and is incubated
It is 60~96h the time required to changing.
The present invention is new using a kind of newcastle disease, the obtained Novel chicken of the preparation method of avian influenza antigen antibody complex vaccine
City epidemic disease, avian influenza antigen antibody complex inactivated vaccine.
Embodiment 1
1, bird flu height exempts from the preparation of chicken embryo：
(1) immune programme of breeder：The antibody obtains after being immunized with HP plants of inactivated vaccines of H9N2 subtype avian influenzas
Yolk antibody.First, fundamental immunity is carried out to chicken embryo with the inactivated vaccine of 2 times of immunizing doses；After 7~10d, with 2~5 times of immunizing agents
Amount carries out booster immunization to chicken embryo；After 10d, then maintenance is carried out to chicken embryo with 2~5 times of immunizing doses and is immunized.
(2) the high harvest for exempting from chicken embryo：A HI is detected every 3d after remaining immune, is received when HI values are in 9log2 or more
Collection chicken embryo is used to prepare Yolk antibody and ewcastle disease antigen.
2, the preparation of ewcastle disease antigen：
(1) it sterilizes：The bird flu height of collection is exempted from into 0.1% bromogeramine solution cleaning and sterilizing of chicken embryo, is put into hatching
It is incubated to 9~10 ages in days for 37 DEG C in device；
(2) it is inoculated with：LaSota plants of production kind poison NDV is taken, 10 are diluted to sterile saline-2~10-4, it is inoculated into
37 DEG C are incubated in the allantoic cavity for exempting from chicken embryo to the bird flu height of 9~10 ages in days, per embryo 0.1mL, after being then placed in 37 DEG C of incubators
Hatch 60~96h；
(3) observation and harvest：Dead chicken embryo before 60h is discarded, every 4~6h is primary according to egg observation later, collects 60h in time
The dead afterwards and apparent chicken embryo of lesion, it is stored refrigerated, until 96h all takes out, it is placed in 2~8 DEG C of coolings 12~for 24 hours, sterile item
Chicken embryo liquid to be collected under part, with several chicken embryos for one group, is loaded on sterile chamber, every group of sampling carries out steriling test and hemagglutination test,
It discards pollution or HA values is less than 1:128 blastochyle indicates harvest date, Virus passages etc.；
Table 1：H9N2HP plant heights exempt from chicken embryo production ewcastle disease antigen potency situation table
As can be seen from the above table, the present invention is feasible using the method that H9N2HP plant heights exempt from chicken embryo production ewcastle disease antigen
, the antigenic virus content of output with it is non-exempt from chicken embryo compared with no significant difference.
3, the harvest of bird flu high immunity yolk antibody：
(1) collection of yolk liquid：Yolk liquid is synchronous with the collection of ewcastle disease antigen to carry out；It takes and has harvested ewcastle disease antigen
Chicken embryo, draw yolk liquid (or eggshell is smashed into picking and goes out yolk bag) with sterile No. 7 syringe needles, hold loaded on sterilizing
Device, and keep sample and carry out steriling test, it takes the yolk liquid of steriling test qualification to mix, measures its HI value；
(2) extraction of Yolk antibody：By yolk and sterile purified water with 1:5 ratio mixing, is sufficiently stirred mixing, uses salt
Acid for adjusting pH is to 5.0~5.4, and after standing 4~6h, 4000~6000r/min low-temperature centrifugations, obtained supernatant is that yolk is anti-
Body crude extract；
(3) purifying and concentration of Yolk antibody：The film packet for being 100KD with molecule interception, to Yolk antibody crude extract into
Row is concentrated by ultrafiltration.HI values are measured after concentration, answer >=9log2.
Table 2 tests Yolk antibody potency (the extraction and purification method all same of Yolk antibody) three times
| Number | Type | Quantity | Receipts amount (mL) | HI values |
| The present invention 1 | The height of inoculation NDV exempts from embryo | 200 | 1100 | 9log2 |
| The present invention 2 | The height of inoculation NDV exempts from embryo | 200 | 1200 | 10log2 |
| The present invention 3 | The height of inoculation NDV exempts from embryo | 200 | 1200 | 9log2 |
| Control 4 | Unhatched height exempts from embryo | 200 | 3000 | 9log2 |
From table 2 it can be seen that the present invention exempts from chicken embryo synchronous production ewcastle disease antigen and Yolk antibody using H9N2 high, with list
It solely uses and uses the non-obtained antibody HI value no significant differences of method exempted from chicken embryo and prepare Yolk antibody, it was demonstrated that side of the invention
Method is effective and feasible, and production cost can be greatly lowered.
4, the preparation of bird flu H9N2 hypotypes antigen
(1) it is inoculated with：Production kind poison is taken, 10 are diluted to sterile saline-4~10-5, it is low to be inoculated into 9~10 ages in days
Exempt from chicken embryo, per embryonic breeding kind 0.1mL, be placed in 37 DEG C of 24~96h of hatching of incubator,
(2) it observes：Dead chicken embryo before discarding for 24 hours, then primary according to egg every 6h, dead chicken after taking out for 24 hours in time
Embryo is stored refrigerated, until 96h, takes out all chicken embryos, is placed in 2~8 DEG C of coolings 12~for 24 hours；
(3) it harvests：The chicken embryo of refrigeration is taken out, chicken embryo liquid is collected under aseptic condition, with several chicken embryos for one group, loaded on sterile
Container, every group of sampling carry out steriling test and hemagglutination test, discard pollution or HA values are less than 1:128 blastochyle, inactivation preceding 2~8
It DEG C preserves, indicates harvest date, Virus passages etc.；
5, the concentration of ewcastle disease antigen and bird flu H9N2 hypotype antigens
Under aseptic condition, chicken embryo liquid is filtered with the sterile funnel equipped with 80 mesh filter screens, removes allantoic fluid residue and other
Coarse granule impurity is centrifuged off smaller impurity after 5000 revs/min, is then concentrated by ultrafiltration 3 times, and ewcastle disease antigen, which is selected, divides
The film packet and avian influenza antigen that sub- interception is 10KD select the film packet that molecule interception is 300KD, newcastle disease virus after concentration
Content >=108.9EID50/ 0.1mL and avian influenza virus content >=108.7EID50/ 0.1mL, by 2~8 DEG C of guarantors of antigen after concentration
It deposits；
6, the inactivation of bird flu H9N2 hypotypes Yolk antibody, ewcastle disease antigen and bird flu H9N2 hypotype antigens
Yolk antibody and two kinds of antigen liquids are respectively placed in sterilizing inactivation tank, 10% formalin is added, adjustment yolk is anti-
For the formaldehyde final concentration of body inactivation to 0.05%, 25 DEG C of inactivation (reaching 25 DEG C of beginning timing with temperature in bottle) 20~for 24 hours, adjustment is new
The formaldehyde final concentration of city epidemic disease antigens inactive to 0.1%, 37 DEG C inactivation 16h, the same newcastle disease of avian influenza antigen ablation method；
7, it emulsifies
The ewcastle disease antigen inactivated, avian influenza antigen and avian influenza egg yolk antibodies semi-finished product are emulsified respectively,
Adjust emulsifier HLB value, newcastle disease, avian influenza antigen emulsifier HLB value 3~6, water phase：Oil phase is equal to 1:3；Yolk is anti-
The HLB value 6~10 of body emulsifier, water phase：Oil phase is equal to 1:1.5, finally by the ewcastle disease antigen emulsified, avian influenza antigen and
Avian influenza egg yolk antibodies are according to 2:2:1 ratio mixing.
8, Efficacy evaluation
1, physical behavior
Appearance milky white emulsion
Dosage form water-in-oil type.A small amount of vaccine drop is drawn in cold water, is in oil droplet shape indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, is centrifuged 15 minutes with 3000 revs/min, the water phase that tube bottom is precipitated
Answer≤0.5ml
Viscosity 28cP
2, steriling test
According to《People's Republic of China's regulations》It tests, equal asepsis growth.
3, safety verification
Take 1~3 week old SPF chick 10, every intramuscular injection or 0.5~1mL be subcutaneously injected, observe 14d, do not occur because
The locally or systemically adverse reaction for vaccinating and causing.
3 groups of vaccine finished product safety verification results prepared by 3 present invention of table
| Vaccine group | Age in days | Injection dosage | Injection site | Adverse reaction | Death condition |
| 1 | 7 | 0.5ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
| 2 | 14 | 0.5ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
| 3 | 21 | 1ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
4, effect detects
(1) this patent product is inoculated with 7 age in days SPF chick, detection antibody level of taking a blood sample after 7d, 14d, 21d, with commercialization
Newcastle disease, bird flu (H9 hypotypes) bivalent inactivated vaccine (newly flowing bivalent inactivated vaccine) compare experiment.It can be with from table 4
Find out that the immune effect of new stream 7 Japanese instar chickling of bivalent inactivated vaccine pair of commercialization is poor, and 7 Japanese instar chickling of this patent product pair
Immune effect it is preferable, reach qualified horizontal.
4 this patent product of table is inoculated with the antibody level after 7 age in days SPF chick
(2) this patent product is inoculated with 10 age in days SPF chick, 7d, 14d, 21d blood sampling detection antibody level, knot after being immunized
Fruit is as shown in table 5, and the immune effect of new stream 10 Japanese instar chickling of bivalent inactivated vaccine pair of commercialization is poor, and this patent product pair
The immune effect of 10 Japanese instar chicklings equally reaches qualified horizontal.
5 this patent product of table is inoculated with the antibody level after 10 age in days SPF chick
(3) this patent product is inoculated with 30 age in days SPF chick, 7d, 14d, 21d blood sampling detection antibody level, knot after being immunized
Fruit is as shown in table 6, and the new stream bivalent inactivated vaccine of commercialization and the immune effect of 30 Japanese instar chickling of this patent product pair reach
It is qualified horizontal.
6 this patent product of table is inoculated with the antibody level after 30 age in days SPF chick
5, formaldehyde and the thimerosal determination of residual amount
According to《People's Republic of China's regulations》Examine the formaldehyde and thimerosal residual of this patent product
Amount, as a result shows：Formalin residual quantity 0.08%, thimerosal residual quantity 0.005%.It can show that this patent product compares peace
Entirely.
Embodiment 2：
1, bird flu height exempts from the preparation of chicken embryo：
(1) immune programme of chicken embryo：The antibody is the Yolk antibody obtained after immune with HP plants of inactivated vaccines.First,
Fundamental immunity is carried out to breeder with HP plants of inactivated vaccines of H9N2 hypotypes of 2 times of immunizing doses；After 7~10d, with 2~5 times of immunizing doses
Booster immunization is carried out to breeder；After 10d, then maintenance is carried out to breeder with 2~5 times of immunizing doses and is immunized.
(2) the high harvest for exempting from chicken embryo：A HI is detected every 3d after remaining immune, is received when HI values are in 9log2 or more
Collection chicken embryo is used to prepare Yolk antibody and ewcastle disease antigen.
2, the preparation of ewcastle disease antigen：
(1) it sterilizes：The bird flu height of collection is exempted from into 0.1% bromogeramine solution cleaning and sterilizing of chicken embryo, is put into hatching
It is incubated to 9~10 ages in days for 37 DEG C in device；
(2) it is inoculated with：LaSota plants of production kind poison NDV is taken, 10 are diluted to sterile saline-2~10-4, it is inoculated into
37 DEG C are incubated in the allantoic cavity for exempting from chicken embryo to the bird flu height of 9~10 ages in days, per embryo 0.1mL, after being then placed in 37 DEG C of incubators
Hatch 60~96h；
(3) observation and harvest：Chicken embryo dead before 60h after being inoculated with LaSota plants of NDV and having hatched is discarded, later every 4
~6h is primary according to egg observation, collects the dead and apparent chicken embryo of lesion after 60h in time, stored refrigerated, until 96h all takes out,
2~8 DEG C of coolings 12~for 24 hours are placed in, chicken embryo liquid is collected under aseptic condition, with several chicken embryos for one group, are loaded on sterile chamber, every group
Sampling carries out steriling test and hemagglutination test, discards pollution or HA values are less than 1:128 blastochyle indicates harvest date, seed culture of viruses generation
It is inferior；
Table 1：H9N2HP plant heights exempt from chicken embryo production ewcastle disease antigen potency situation table
As can be seen from the above table, the present invention is feasible using the method that H9N2HP plant heights exempt from chicken embryo production ewcastle disease antigen
, the antigenic virus content of output with it is non-exempt from chicken embryo compared with no significant difference.
3, the harvest of bird flu high immunity yolk antibody：
(1) collection of yolk liquid：Yolk liquid is synchronous with the collection of ewcastle disease antigen to carry out；It takes and has harvested ewcastle disease antigen
Chicken embryo, draw yolk liquid (or eggshell is smashed into picking and goes out yolk bag) with sterile No. 7 syringe needles, hold loaded on sterilizing
Device, and keep sample and carry out steriling test, it takes the yolk liquid of steriling test qualification to mix, measures its HI value；
(2) extraction of Yolk antibody：By yolk and sterile purified water with 1:5 ratio mixing, is sufficiently stirred mixing, uses salt
Acid for adjusting pH is to 5.0~5.4, and after standing 4~6h, 4000~6000r/min low-temperature centrifugations, obtained supernatant is that yolk is anti-
Body crude extract；
(3) purifying and concentration of Yolk antibody：The film packet for being 100KD with molecule interception, to Yolk antibody crude extract into
Row is concentrated by ultrafiltration.HI values are measured after concentration, answer >=9log2.
Table 2 tests Yolk antibody potency (the extraction and purification method all same of Yolk antibody) three times
From table 2 it can be seen that the present invention exempts from chicken embryo synchronous production ewcastle disease antigen and Yolk antibody using H9N2 high, with list
It solely uses and uses the non-obtained antibody HI value no significant differences of method exempted from chicken embryo and prepare Yolk antibody, it was demonstrated that side of the invention
Method is effective and feasible, and production cost can be greatly lowered.
4, the preparation of bird flu H9N2 hypotypes antigen
(1) it is inoculated with：Production kind poison is taken, 10 are diluted to sterile saline-4~10-5, it is low to be inoculated into 9~10 ages in days
Exempt from chicken embryo, per embryonic breeding kind 0.1mL, be placed in 37 DEG C of 24~96h of hatching of incubator,
(2) it observes：It discards and is inoculated with after HP plants of bird flu H9N2 hypotypes preceding dead chicken embryo for 24 hours, then shine egg one every 6h
Secondary, dead chicken embryo is stored refrigerated after taking out for 24 hours in time, until 96h, takes out all chicken embryos, be placed in 2~8 DEG C of coolings 12~
24h；
(3) it harvests：The chicken embryo of refrigeration is taken out, chicken embryo liquid is collected under aseptic condition, with several chicken embryos for one group, loaded on sterile
Container, every group of sampling carry out steriling test and hemagglutination test, discard pollution or HA values are less than 1:128 blastochyle, inactivation preceding 2~8
It DEG C preserves, indicates harvest date, Virus passages etc.；
5, the concentration of ewcastle disease antigen and bird flu H9N2 hypotype antigens
Under aseptic condition, chicken embryo liquid is filtered with the sterile funnel equipped with 80 mesh filter screens, removes allantoic fluid residue and other
Coarse granule impurity is centrifuged off smaller impurity after 5000 revs/min, is then concentrated by ultrafiltration 3 times, and ewcastle disease antigen, which is selected, divides
The film packet and avian influenza antigen that sub- interception is 10KD select the film packet that molecule interception is 300KD, newcastle disease virus after concentration
Content >=108.9EID50/ 0.1mL and avian influenza virus content >=108.7EID50/ 0.1mL, by 2~8 DEG C of guarantors of antigen after concentration
It deposits；
6, the inactivation of bird flu H9N2 hypotypes Yolk antibody, ewcastle disease antigen and bird flu H9N2 hypotype antigens
Yolk antibody and two kinds of antigen liquids are respectively placed in sterilizing inactivation tank, 10% formalin is added, adjustment yolk is anti-
For the formaldehyde final concentration of body inactivation to 0.05%, 25 DEG C of inactivation (reaching 25 DEG C of beginning timing with temperature in bottle) 20~for 24 hours, adjustment is new
The formaldehyde final concentration of city epidemic disease antigens inactive to 0.1%, 37 DEG C inactivation 16h, the same newcastle disease of avian influenza antigen ablation method；
7, it emulsifies
The ewcastle disease antigen inactivated, avian influenza antigen and avian influenza egg yolk antibodies semi-finished product are emulsified respectively,
Adjust emulsifier HLB value, newcastle disease, avian influenza antigen emulsifier HLB value 3~6, water phase：Oil phase is equal to 1:3；Yolk is anti-
The HLB value 6~10 of body emulsifier, water phase：Oil phase is equal to 1:1.5, finally by three according to 1.5:1.5:1 ratio mixing.
8, Efficacy evaluation
1, physical behavior
Appearance milky white emulsion
Dosage form water-in-oil type.A small amount of vaccine drop is drawn in cold water, is in oil droplet shape indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, is centrifuged 15 minutes with 3000 revs/min, the water phase that tube bottom is precipitated
Answer≤0.5ml
Viscosity 28cP
2, steriling test
According to《People's Republic of China's regulations》It tests, equal asepsis growth.
3, safety verification
Take 1~3 week old SPF chick 10, every intramuscular injection or 0.5~1mL be subcutaneously injected, observe 14d, do not occur because
The locally or systemically adverse reaction for vaccinating and causing.
3 groups of vaccine finished product safety verification results prepared by 3 present invention of table
| Vaccine group | Age in days | Injection dosage | Injection site | Adverse reaction | Death condition |
| 1 | 7 | 0.5ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
| 2 | 14 | 0.5ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
| 3 | 21 | 1ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
4, effect detects
(1) this patent product is inoculated with 7 age in days SPF chick, detection antibody level of taking a blood sample after 7d, 14d, 21d, with commercialization
Newcastle disease, bird flu (H9 hypotypes) bivalent inactivated vaccine (newly flowing bivalent inactivated vaccine) compare experiment.It can be with from table 4
Find out that the immune effect of new stream 7 Japanese instar chickling of bivalent inactivated vaccine pair of commercialization is poor, and 7 Japanese instar chickling of this patent product pair
Immune effect it is preferable, reach qualified horizontal.
4 this patent product of table is inoculated with the antibody level after 7 age in days SPF chick
(2) this patent product is inoculated with 10 age in days SPF chick, 7d, 14d, 21d blood sampling detection antibody level, knot after being immunized
Fruit is as shown in table 5, and the immune effect of new stream 10 Japanese instar chickling of bivalent inactivated vaccine pair of commercialization is poor, and this patent product pair
The immune effect of 10 Japanese instar chicklings equally reaches qualified horizontal.
5 this patent product of table is inoculated with the antibody level after 10 age in days SPF chick
(3) this patent product is inoculated with 30 age in days SPF chick, 7d, 14d, 21d blood sampling detection antibody level, knot after being immunized
Fruit is as shown in table 6, and the new stream bivalent inactivated vaccine of commercialization and the immune effect of 30 Japanese instar chickling of this patent product pair reach
It is qualified horizontal.
6 this patent product of table is inoculated with the antibody level after 30 age in days SPF chick
5, formaldehyde and the thimerosal determination of residual amount
According to《People's Republic of China's regulations》Examine the formaldehyde and thimerosal residual of this patent product
Amount, as a result shows：Formalin residual quantity 0.08%, thimerosal residual quantity 0.007%.It can show that this patent product compares peace
Entirely.
Embodiment 3：
1, bird flu height exempts from the preparation of chicken embryo：
(1) immune programme of chicken embryo：The antibody is the Yolk antibody obtained after immune with HP plants of inactivated vaccines.First,
Fundamental immunity is carried out to breeder with HP plants of inactivated vaccines of H9N2 hypotypes of 2 times of immunizing doses；After 7~10d, with 2~5 times of immunizing doses
Booster immunization is carried out to breeder；After 10d, then maintenance is carried out to breeder with 2~5 times of immunizing doses and is immunized.
(2) the high harvest for exempting from chicken embryo：A HI is detected every 3d after remaining immune, is received when HI values are in 9log2 or more
Collection chicken embryo is used to prepare Yolk antibody and ewcastle disease antigen.
2, the preparation of ewcastle disease antigen：
(1) it sterilizes：The bird flu height of collection is exempted from into 0.1% bromogeramine solution cleaning and sterilizing of chicken embryo, is put into hatching
It is incubated to 9~10 ages in days for 37 DEG C in device；
(2) it is inoculated with：LaSota plants of production kind poison NDV is taken, 10 are diluted to sterile saline-2~10-4, it is inoculated into
37 DEG C are incubated in the allantoic cavity for exempting from chicken embryo to the bird flu height of 9~10 ages in days, per embryo 0.1mL, after being then placed in 37 DEG C of incubators
Hatch 60~96h；
(3) observation and harvest：Chicken embryo dead before 60h after being inoculated with LaSota plants of NDV and having hatched is discarded, later every 4
~6h is primary according to egg observation, collects the dead and apparent chicken embryo of lesion after 60h in time, stored refrigerated, until 96h all takes out,
2~8 DEG C of coolings 12~for 24 hours are placed in, chicken embryo liquid is collected under aseptic condition, with several chicken embryos for one group, are loaded on sterile chamber, every group
Sampling carries out steriling test and hemagglutination test, discards pollution or HA values are less than 1:128 blastochyle indicates harvest date, seed culture of viruses generation
It is inferior；
Table 1：H9N2HP plant heights exempt from chicken embryo production ewcastle disease antigen potency situation table
As can be seen from the above table, the present invention is feasible using the method that H9N2HP plant heights exempt from chicken embryo production ewcastle disease antigen
, the antigenic virus content of output with it is non-exempt from chicken embryo compared with no significant difference.
3, the harvest of bird flu high immunity yolk antibody：
(1) collection of yolk liquid：Yolk liquid is synchronous with the collection of ewcastle disease antigen to carry out；It takes and has harvested ewcastle disease antigen
Chicken embryo, draw yolk liquid (or eggshell is smashed into picking and goes out yolk bag) with sterile No. 7 syringe needles, hold loaded on sterilizing
Device, and keep sample and carry out steriling test, it takes the yolk liquid of steriling test qualification to mix, measures its HI value；
(2) extraction of Yolk antibody：By yolk and sterile purified water with 1:5 ratio mixing, is sufficiently stirred mixing, uses salt
Acid for adjusting pH is to 5.0~5.4, and after standing 4~6h, 4000~6000r/min low-temperature centrifugations, obtained supernatant is that yolk is anti-
Body crude extract；
(3) purifying and concentration of Yolk antibody：The film packet for being 100KD with molecule interception, to Yolk antibody crude extract into
Row is concentrated by ultrafiltration.HI values are measured after concentration, answer >=9log2.
Table 2 tests Yolk antibody potency (the extraction and purification method all same of Yolk antibody) three times
| Number | Type | Quantity | Receipts amount (mL) | HI values |
| The present invention 1 | The height of inoculation NDV exempts from embryo | 200 | 1130 | 10log2 |
| The present invention 2 | The height of inoculation NDV exempts from embryo | 200 | 1100 | 9log2 |
| The present invention 3 | The height of inoculation NDV exempts from embryo | 200 | 1120 | 9log2 |
| Control 4 | Unhatched height exempts from embryo | 200 | 2900 | 10log2 |
From table 2 it can be seen that the present invention exempts from chicken embryo synchronous production ewcastle disease antigen and Yolk antibody using H9N2 high, with list
It solely uses and uses the non-obtained antibody HI value no significant differences of method exempted from chicken embryo and prepare Yolk antibody, it was demonstrated that side of the invention
Method is effective and feasible, and production cost can be greatly lowered.
4, the preparation of bird flu H9N2 hypotypes antigen
(1) it is inoculated with：Production kind poison is taken, 10 are diluted to sterile saline-4~10-5, it is low to be inoculated into 9~10 ages in days
Exempt from chicken embryo, per embryonic breeding kind 0.1mL, be placed in 37 DEG C of 24~96h of hatching of incubator,
(2) it observes：It discards and is inoculated with after HP plants of bird flu H9N2 hypotypes preceding dead chicken embryo for 24 hours, then shine egg one every 6h
Secondary, dead chicken embryo is stored refrigerated after taking out for 24 hours in time, until 96h, takes out all chicken embryos, be placed in 2~8 DEG C of coolings 12~
24h；
(3) it harvests：The chicken embryo of refrigeration is taken out, chicken embryo liquid is collected under aseptic condition, with several chicken embryos for one group, loaded on sterile
Container, every group of sampling carry out steriling test and hemagglutination test, discard pollution or HA values are less than 1:128 blastochyle, inactivation preceding 2~8
It DEG C preserves, indicates harvest date, Virus passages etc.；
5, the concentration of ewcastle disease antigen and bird flu H9N2 hypotype antigens
Under aseptic condition, chicken embryo liquid is filtered with the sterile funnel equipped with 80 mesh filter screens, removes allantoic fluid residue and other
Coarse granule impurity is centrifuged off smaller impurity after 5000 revs/min, is then concentrated by ultrafiltration 3 times, and ewcastle disease antigen, which is selected, divides
The film packet and avian influenza antigen that sub- interception is 10KD select the film packet that molecule interception is 300KD, newcastle disease virus after concentration
Content >=108.9EID50/ 0.1mL and avian influenza virus content >=108.7EID50/ 0.1mL, by 2~8 DEG C of guarantors of antigen after concentration
It deposits；
6, the inactivation of bird flu H9N2 hypotypes Yolk antibody, ewcastle disease antigen and bird flu H9N2 hypotype antigens
Yolk antibody and two kinds of antigen liquids are respectively placed in sterilizing inactivation tank, 10% formalin is added, adjustment yolk is anti-
For the formaldehyde final concentration of body inactivation to 0.05%, 25 DEG C of inactivation (reaching 25 DEG C of beginning timing with temperature in bottle) 20~for 24 hours, adjustment is new
The formaldehyde final concentration of city epidemic disease antigens inactive to 0.1%, 37 DEG C inactivation 16h, the same newcastle disease of avian influenza antigen ablation method；
7, it emulsifies
The ewcastle disease antigen inactivated, avian influenza antigen and avian influenza egg yolk antibodies semi-finished product are emulsified respectively,
Adjust emulsifier HLB value, newcastle disease, avian influenza antigen emulsifier HLB value 3~6, water phase：Oil phase is equal to 1:3；Yolk is anti-
The HLB value 6~10 of body emulsifier, water phase：Oil phase is equal to 1:1.5 finally by three according to 3:3:1 ratio mixing.
8, Efficacy evaluation
1, physical behavior
Appearance milky white emulsion
Dosage form water-in-oil type.A small amount of vaccine drop is drawn in cold water, is in oil droplet shape indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, is centrifuged 15 minutes with 3000 revs/min, the water phase that tube bottom is precipitated
Answer≤0.5ml
Viscosity 28cP
2, steriling test
According to《People's Republic of China's regulations》It tests, equal asepsis growth.
3, safety verification
Take 1~3 week old SPF chick 10, every intramuscular injection or 0.5~1mL be subcutaneously injected, observe 14d, do not occur because
The locally or systemically adverse reaction for vaccinating and causing.
3 groups of vaccine finished product safety verification results prepared by 3 present invention of table
| Vaccine group | Age in days | Injection dosage | Injection site | Adverse reaction | Death condition |
| 1 | 7 | 0.5ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
| 2 | 14 | 0.5ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
| 3 | 21 | 1ml | Muscle is subcutaneous | Nothing | 10/10 strong work |
4, effect detects
(1) this patent product is inoculated with 7 age in days SPF chick, detection antibody level of taking a blood sample after 7d, 14d, 21d, with commercialization
Newcastle disease, bird flu (H9 hypotypes) bivalent inactivated vaccine (newly flowing bivalent inactivated vaccine) compare experiment.It can be with from table 4
Find out that the immune effect of new stream 7 Japanese instar chickling of bivalent inactivated vaccine pair of commercialization is poor, and 7 Japanese instar chickling of this patent product pair
Immune effect it is preferable, reach qualified horizontal.
4 this patent product of table is inoculated with the antibody level after 7 age in days SPF chick
(2) this patent product is inoculated with 10 age in days SPF chick, 7d, 14d, 21d blood sampling detection antibody level, knot after being immunized
Fruit is as shown in table 5, and the immune effect of new stream 10 Japanese instar chickling of bivalent inactivated vaccine pair of commercialization is poor, and this patent product pair
The immune effect of 10 Japanese instar chicklings equally reaches qualified horizontal.
5 this patent product of table is inoculated with the antibody level after 10 age in days SPF chick
(3) this patent product is inoculated with 30 age in days SPF chick, 7d, 14d, 21d blood sampling detection antibody level, knot after being immunized
Fruit is as shown in table 6, and the new stream bivalent inactivated vaccine of commercialization and the immune effect of 30 Japanese instar chickling of this patent product pair reach
It is qualified horizontal.
6 this patent product of table is inoculated with the antibody level after 30 age in days SPF chick
5, formaldehyde and the thimerosal determination of residual amount
According to《People's Republic of China's regulations》Examine the formaldehyde and thimerosal residual of this patent product
Amount, as a result shows：Formalin residual quantity 0.06%, thimerosal residual quantity 0.004%.It can show that this patent product compares peace
Entirely.
Claims (5)
1. the preparation method of a kind of newcastle disease, avian influenza antigen antibody complex vaccine, it is characterised in that：It includes following step
Suddenly：
1）Prepare avian influenza antigen：Exempt from chicken embryo cleaning and sterilizing by low, after hatching, HP plants of H9N2 hypotypes are inoculated in its allantoic cavity
Avian influenza virus then proceedes to hatch, and aseptically harvests chicken embryo liquid, and carries out purifying and be concentrated to give new chicken embryo liquid,
And new chicken embryo hydroful foot avian influenza virus content >=108.7EID50/ 0.1mL, then by the chicken embryo liquid formaldehyde newly obtained into
Row inactivation；
2）Prepare newcastle disease inactivation antigen and avian influenza inactivation Yolk antibody：HP plants of avian influenza virus of H9N2 hypotypes are first used to inactivate epidemic disease
Seedling immunized embryo, detection chicken embryo H9N2 subclass antibodies are horizontal, select HI potency and reach the height of 9log2 or more and exempt from chicken embryo；Then
The avian influenza virus height selected is exempted from into chicken embryo and carries out cleaning and sterilizing, and is hatched；Exempt from chick embryo allantoic cavity in the height after hatching
LaSota plants of interior inoculation newcastle disease virus is then hatched, and aseptically harvests chicken embryo liquid and yolk liquid respectively, and carry out pure
Change concentration, chicken embryo hydroful foot newcastle disease virus content >=10 for obtaining new chicken embryo liquid and yolk liquid, and newly obtaining8.9EID50/
0.1mL, yolk hydroful foot Yolk antibody HI value >=9log2, then by the chicken embryo liquid newly obtained and yolk liquid formalin-inactivated；
3）Emulsification：The ewcastle disease antigen inactivated, avian influenza antigen and avian influenza egg yolk antibodies semi-finished product are subjected to breast respectively
Change, adjusts newcastle disease, the HLB value of avian influenza antigen emulsifier obtains oil phase for 3~6, then enables water phase：Oil phase is equal to 1:3；It adjusts
The HLB value of Yolk antibody emulsifier obtains oil phase for 6~10, then enables water phase：Oil phase is equal to 1:1.5；
4）Mixing：Ewcastle disease antigen, avian influenza antigen and Yolk antibody after emulsification is pressed 1.5~3:1.5~3:1 ratio is mixed
It is even, obtain product.
2. the preparation method of a kind of newcastle disease according to claim 1, avian influenza antigen antibody complex vaccine, special
Sign is：The step 1）In it is non-exempt to hatch required temperature after chicken embryo cleaning and sterilizing to be 37 DEG C, and need to hatch to 9~10 days
Age；The chicken embryo of inoculated HP plants of avian influenza virus of H9N2 hypotypes continues to hatch required temperature to be 37 DEG C, and hatches and taken
Between be 24~96h.
3. the preparation method of a kind of newcastle disease according to claim 1, avian influenza antigen antibody complex vaccine, special
Sign is：The step 2）In bird flu height after cleaned disinfection to exempt from the temperature needed for hatching be 37 DEG C, and need to incubate
Change to 9~10 ages in days.
4. the preparation method of a kind of newcastle disease according to claim 1, avian influenza antigen antibody complex vaccine, special
Sign is：The step 2）The temperature needed for hatching after middle LaSota plants of newcastle disease virus of inoculation is 37 DEG C, and is incubated
It is 60~96h the time required to changing.
5. utilizing any newcastle disease of Claims 1 to 44, the preparation method system of avian influenza antigen antibody complex vaccine
Novel newcastle disease, avian influenza antigen antibody complex inactivated vaccine.
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