CN103834620B - NDV, ewcastle disease inactivated vaccine and preparation method thereof - Google Patents
NDV, ewcastle disease inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses NDV, ewcastle disease inactivated vaccine and preparation method thereof. Wherein, NDV preserving number CGMCC? No.7805; be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center; this NDV has stronger virulence; immunogenicity is good, and cross-protection is strong, and malicious valency is higher; the vaccine safety that utilizes this NDV to prepare is high, and immunogenicity is good.
Description
Technical field
The present invention relates to biological technical field, be specifically related to NDV, ewcastle disease inactivated vaccine and preparation method thereof.
Background technology
Newcastle disease virus is the serious harm aviculture being caused by NDV (NewcastleDisease, ND)Height contagious disease. NDV belongs to Paramyxoviridae, the avian paramyxoviruses I type of fowl Rubulavirus. BloodSolidifying element (HA) and neuraminidase (NA) are two kinds of main protection antigen of virus.
Newcastle disease, first nineteen twenty-six is found in Indonesia, finds again soon at Britain's new city, and the world is each afterwardsAll there is popular record in state. Have velogen strain and low virulent strain two classes, virus is divided into strong virus force type, middle virulence type and low virulence type. PoultrySusceptible, young fowl is stronger than adult bird neurological susceptibility. The death rate is high, serious to poultry husbandry harm. There is no effective medicine, onlyCan rely on strict sterilization, isolation, the collaborative use immunization campaign of live vaccine and inactivated vaccine. Based on existing vaccine quality problem,Immunity inoculation methods, immune programme for children, chicken house feeding and management level, the very different present situation of chicken group antibody horizontal, make new cityEpidemic disease immuning failure is of common occurrence. For solving this difficult problem, the epidemic characteristic, the variation that depend on the strain popular to this area are specialPoint is studied and is screened and isolates suitable advantage vaccine strain, so that the prevention and control of ewcastle disease have more specific aim and effectiveProperty.
Summary of the invention
The present invention one of is intended to solve the problems of the technologies described above at least to a certain extent or a kind of useful business is at least providedIndustry is selected. For this reason, one object of the present invention is to propose a kind of NDV, ewcastle disease inactivated vaccine and preparation side thereofMethod.
In a first aspect of the present invention, the present invention proposes a kind of NDV, this NDV deposit numberCGMCCNo.7805, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is court of BeijingNo. 3, No. 1, North Star West Road institute of sun district. According to the embodiment of the present invention, described NDV is from the normal immune ewcastle disease of processThe morbidity chicken group of inactivated vaccine separates and obtains, and its malicious valency HA tires higher than 1:256, therefore, and described newcastle disease virus strain virulenceHeight, has good immunogenicity, can be used as inactivated vaccine and produces strain and inspection seed culture of viruses.
In a second aspect of the present invention, the present invention proposes above-mentioned NDV preparing in ewcastle disease inactivated vaccinePurposes, in one embodiment of the invention, according to NDV cross immunity, protection test proves, described NDVThere is good cross immunity protective effect. Therefore utilize this NDV to produce ewcastle disease inactivated vaccine, can effectively carryThe immunogenicity of high ewcastle disease inactivated vaccine, thus attacking of the strong poison of NDV and other Virulent Newcastle Disease Virus strain can be resistedHit.
In third aspect present invention, the present invention proposes a kind of ewcastle disease inactivated vaccine, according to the embodiment of the present invention, this is newCity epidemic disease inactivated vaccine comprises the NDV of deactivation. Thereby effectively improve the immunogenicity of ewcastle disease inactivated vaccine, furtherImprove ewcastle disease inactivated vaccine security and immunogenicity.
In fourth aspect present invention, the present invention proposes a kind of method of preparing ewcastle disease inactivated vaccine, according to the present inventionEmbodiment, the method comprises: above-mentioned NDV is carried out to deactivation, to obtain the NDV through deactivation; UtilizeThe described NDV through deactivation is prepared water; And described water and oil phase are carried out to mixing and emulsifying, to obtain instituteState ewcastle disease inactivated vaccine. Thus, can effectively improve the immunogenicity of ewcastle disease inactivated vaccine, thereby further improve new cityThe security of epidemic disease inactivated vaccine and immunogenicity.
According to one embodiment of the invention, the method for preparing ewcastle disease inactivated vaccine of the above embodiment of the present invention also comprisesFollowing additional technical feature:
According to the embodiment of the present invention, before described NDV is carried out to deactivation, comprise according to the following step and obtainingNDV nutrient solution: described NDV is inoculated in nonimmune chicken embryo, and cultivated at 35.5～37.5 DEG C,Collect the chick embryo allantoic liquid obtaining; Separate described chick embryo allantoic liquid, to obtain described NDV nutrient solution.
According to one embodiment of the invention, described NDV is carried out to deactivation and comprise: at 37 DEG C, utilize final concentrationBe that 0.2% formaldehyde carries out inactivation treatment 16～36 hours to NDV nutrient solution; Contain deactivation Newcastle Disease to obtainThe solution of poison. According to one embodiment of the invention, the described water of preparing comprises: by the solution that contains deactivation NDV with tellTemperature-80 is mixed according to the volume ratio of 96:4, to obtain described water, wherein, the final concentration of described Tween-80 is4%。
According to one embodiment of the invention, the described oil phase of preparing comprises: by injection white oil MARCOL52, Si Ben-80 withAnd aluminum stearate mixes according to the mass ratio of 94:6:2, to obtain described oil phase. Thus, improve ewcastle disease inactivated vaccineImmunogenicity, thereby further improve the security of ewcastle disease inactivated vaccine and immunogenicity.
According to one embodiment of the invention, described water is mixed and is comprised with oil phase: by described water and oil phase according to bodyLong-pending than carrying out mixing and emulsifying stirring for 2:3～2:4, before stirring stops, adding final concentration is that 0.01% thimerosal is to obtainDescribed ewcastle disease inactivated vaccine. Can make thus can prevent thus corruption, extend the storage life of vaccine, improve ewcastle disease and go outThe stability of live vaccine and immunogenicity.
Additional aspect of the present invention and advantage in the following description part provide, and part will become from the following descriptionObtain obviously, or recognize by practice of the present invention.
Brief description of the drawings
Above-mentioned and/or additional aspect of the present invention and advantage from conjunction with below accompanying drawing to becoming the description of embodimentObviously and easily understand, wherein:
Fig. 1 is preparation method's Technology Roadmap of ewcastle disease inactivated vaccine according to an embodiment of the invention.
Detailed description of the invention
The following discovery of the present invention based on inventor completes: inventor has separated a strain NDV, this diseasePoison is being used as and preparing ewcastle disease inactivated vaccine after deactivation, for preventing and resist NDV and other new cityThe attack of epidemic disease velogen strain.
In a first aspect of the present invention, the present invention proposes a kind of NDV, according to the embodiment of the present invention, this new cityEpidemic disease poison deposit number CGMCC:7805, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, groundLocation is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City. According to the embodiment of the present invention, NDV is from the normal new city of processThe morbidity chicken group of epidemic disease inactivated vaccine immunity separates and obtains, and its HA that tires, higher than 1:256, therefore, separates the NDV obtainingStrain virulence is high, has good immunogenicity, can be used as inactivated vaccine and produces strain and inspection seed culture of viruses, a reality according to the present inventionExecute example, the protection test of NDV cross immunity proves, this NDV has good cross immunity protective effect. CauseThis can utilize this NDV to produce ewcastle disease inactivated vaccine, effectively improves the immunogenicity of ewcastle disease inactivated vaccine, fromAnd further improve the security of ewcastle disease inactivated vaccine and immunogenicity.
NDV of the present invention has good specificity and immunogenicity, the erythrocytic work of chick embryo allantoic liquid aggegationWith not suppressed, can not be suppressed by anti-H5, H7, H9 subtype avian influenza virus positive serum by anti-EDS76 positive serum, and energySuppressed by ewcastle disease positive serum. Immunized chicks can produce the special HI antibody of ewcastle disease. Neutralize with ewcastle disease specific corrosioning anteserumRear inoculated into chick embryo, chicken embryo is all not dead, and unneutralized contrast inoculation embryo is all dead, and this further illustrates strain of the present inventionSpecificity.
In a second aspect of the present invention, the present invention proposes above-mentioned NDV preparing in ewcastle disease inactivated vaccinePurposes, in one embodiment of the invention, according to NDV cross immunity, protection test proves, this NDV hasWell cross immunity protective effect. Therefore utilize this NDV to produce ewcastle disease inactivated vaccine, can effectively improve newThe immunogenicity of city epidemic disease inactivated vaccine, thus ewcastle disease inactivated vaccine immunogenicity and immune effect further improved.
In a third aspect of the present invention, the present invention proposes a kind of ewcastle disease inactivated vaccine, according to the embodiment of the present invention, shouldEwcastle disease inactivated vaccine comprises the NDV of deactivation. According to the embodiment of the present invention, the immunity of this ewcastle disease inactivated vaccine is heldRenew at least and can maintain 4 months. According to the embodiment of the present invention, this ewcastle disease inactivated vaccine can be preserved 12 at 2～8 DEG CMonth, therefore this vaccine has good stability. Ewcastle disease inactivated vaccine of the present invention, immunogenicity is good, and the immunity cycle is long, rawProduct cost is low, and technique is reasonable, has extremely wide application.
According to the embodiment of the present invention, above-mentioned ewcastle disease inactivated vaccine is inactivated, and the inactivator kind that can adopt alsoBe not particularly limited, as long as can effectively destroy the nucleic acid structure of NDV and make protein denaturation, but not brokenThe antigenicity of bad NDV and blood clotting. According to one embodiment of the invention, the inactivator adopting is formaldehyde, and formaldehyde canEffectively to destroy the DNA structure of NDV, and then reach the object of deactivation NDV. According to the embodiment of the present invention,The inactivator formaldehyde final concentration adopting is also not particularly limited, as long as effective deactivation NDV. According to thisInvent an embodiment, the final concentration of the formaldehyde adopting is 0.2%.
In a fourth aspect of the present invention, the present invention proposes a kind of method of preparing ewcastle disease inactivated vaccine, according to thisBright embodiment, the method comprises carries out deactivation to above-mentioned NDV, to obtain the NDV through deactivation; UtilizeNDV through deactivation is prepared water; And water and oil phase are carried out to mixing and emulsifying, to obtain ewcastle disease deactivationVaccine. Thus, effectively improve the immunogenicity of ewcastle disease inactivated vaccine, thereby further improve ewcastle disease inactivated vaccine immunogeneProperty and immune effect.
According to one embodiment of present invention, before NDV is carried out to deactivation, comprise according to the following step and obtainingObtain NDV nutrient solution: NDV is inoculated in nonimmune chicken embryo, and cultivates at 35.5～37.5 DEG C,Collect the chick embryo allantoic liquid obtaining, to obtain NDV nutrient solution. According to the embodiment of the present invention, nonimmune chicken embryo isInstar chicken embryo on the 9th～11, can effectively cultivate NDV. According to one embodiment of the invention, at NDVBefore being inoculated in nonimmune chicken embryo, first NDV is carried out rarely, after inoculation, at 35.5～37.5 DEG C, cultivate 72 littleTime, discard dead chicken embryo in 24 hours, collect 24～72 hours dead chicken embryos. Thereby be conducive to obtain the chick embryo allantois that malicious valency is highLiquid, and then improve the immune protective efficiency of preparing ewcastle disease inactivated vaccine.
According to one embodiment of the invention, NDV is carried out the condition of deactivation and is not particularly limited, according to thisThe specific embodiment of invention, can adopt formaldehyde to carry out deactivation to NDV. The inactivator formaldehyde final concentration adopting alsoBe not particularly limited, as long as effective deactivation NDV. According to a particular embodiment of the invention, the first adoptingThe final concentration of aldehyde is 0.1%～0.4%. According to the embodiment of the present invention, the time of inactivator deactivation and temperature are also not particularly limited,As long as can effectively destroy the nucleic acid structure of NDV. According to a particular embodiment of the invention, utilize final concentration to be0.1%～0.4% formaldehyde carries out inactivation treatment 16～36 hours to NDV nutrient solution, thus can be by NDVFully deactivation.
According to one embodiment of present invention, in the above-mentioned method of preparing ewcastle disease inactivated vaccine, can utilize following sideLegal system is for water: the solution that contains deactivation NDV is mixed with Tween-80.
What the solution that according to a particular embodiment of the invention, contains deactivation NDV and Tween-80 mixed joinsCompare and be not particularly limited, according to concrete example of the present invention, can mix according to the volume ratio of 96:4. On adoptingState proportioning and concentration is prepared water.
According to one embodiment of present invention, in the above-mentioned method of preparing ewcastle disease inactivated vaccine, can utilize following sideLegal system is for oil phase: injection white oil MARCOL52, Si Ben-80 and aluminum stearate are mixed, to obtain oil phase. According toSpecific embodiments of the invention, the quality that injection white oil MARCOL52, Si Ben-80 and aluminum stearate can adopt is 94:6:2, can improve the immunogenicity of ewcastle disease inactivated vaccine thus, thereby further improves ewcastle disease inactivated vaccine immunogenicityAnd immune protective effect.
According to one embodiment of present invention, by water and oil phase mixing and emulsifying, can be according to water and oil phase volume ratioFor 2:3～2:4 carries out mixing and emulsifying stirring, be that 0.01% thimerosal is to obtain new city stopping adding final concentration before stirringEpidemic disease inactivated vaccine. Can prevent thus corruption, improve stability and the immunogenicity of ewcastle disease inactivated vaccine.
Below with reference to specific embodiment, present invention is described, it should be noted that, these embodiment describeProperty, and do not limit the present invention in any way.
Separation and the qualification of embodiment 1 NDV
1, virus separates
Gather morbidity live-bird sample and comprise tracheae or cloaca swab etc., collected specimens is placed on to the pH value that contains antibioticIn 7.0～7.4 isotonic phosphate buffer liquid (PBS), pathological material of disease is ground, or be fully spread in PBS, sample liquid is through 1500r/The centrifugal 10min of min, gets supernatant 0.22 μ m bacterial filter and filters, by the aseptic viral sample liquid of handling well through fine hair allantoisChamber inoculation 9～11 age in days SPF chicken embryos, inoculate 10 pieces altogether, and 0.1mL/ piece, puts 37 DEG C and hatch, and every 8h, according to an embryo, discards in 24hNon-specific dead embryo, collects the not dead embryo allantoic liquid of living of the dead embryo of 24～72h and 72h, places-20 DEG C of Cryopreservations for subsequent use.
According to the method described above, from the pathological material of disease of sick chicken, be separated to the virus of blood clotting, HA evidence NDVCan aggegation chicken red blood cell, this viral blood clotting performance of HI evidence is suppressed, but by NDV (NDV) antiserum simultaneouslyCan not be suppressed by anti-AIVH5, H7, H9 hypotype positive serum. The results are shown in Table 2.
The first separating resulting of table 2 pathological material of disease
NDV of the present invention has good specificity and immunogenicity, the erythrocytic work of chick embryo allantoic liquid aggegationWith not suppressed, can not be suppressed by anti-H5, H7, H9 subtype avian influenza virus positive serum by anti-EDS76 positive serum, and energySuppressed by ewcastle disease positive serum. Immunized chicks can produce the special HI antibody of ewcastle disease. Neutralize with ewcastle disease specific corrosioning anteserumRear inoculated into chick embryo, chicken embryo is all not dead, and unneutralized contrast inoculation embryo is all dead, and this further illustrates strain of the present inventionSpecificity.
2, qualification
The chick embryo allantoic liquid of separation is suppressed to be accredited as NDV, called after BJ strain through blood clotting and blood clotting. This poisonStrain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 26th, 2013 and (is called for shortCGMCC) preservation, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Classification And Nomenclature is newcastle disease virus (BJ strain), protectsTibetan number is CGMCCNo.7805.
The Evaluation of Immunogenicity of embodiment 2 newcastle disease viruses
With 20 of 1～2 monthly age SPF chickens, the ewcastle disease that wherein every subcutaneous vaccination of 10 chickens is made into by NDVInactivated vaccine 20 μ L, another 10 subcutaneous vaccination equivalent physiological saline compare, and isolated rearing is respectively taken a blood sample and is separated blood for 21 days afterwardsDo HI test with antigen clearly. The results are shown in Table 3. Result shows that this virus has good immunogenicity.
The immunogenicity of table 3 NDV
Embodiment 3 newcastle disease virus vaccine preparations
Preparation technology's flow chart below with reference to Fig. 1 Newcastle Disease Virus Vaccine is prepared Newcastle Disease Virus Vaccine.
1, the preparation of vaccine antigen liquid and malicious valency are measured
The seed disease of working venom, with after normal saline dilution, is inoculated 10 age in days SPF chicken embryos, and 37 DEG C are cultured to 72 hours(discarding non-specific dead chicken embryo in 24h), after 4 DEG C of refrigeration 24h, the aseptic allantoic fluid of collecting, measures malicious valency: 107.0～108.5ELD50, HA tires in 1:215～512. It is for subsequent use that virus liquid is put-20 DEG C of preservations.
2, antigen liquid deactivation
Formalin is added in virus liquid, and final concentration is 0.1%～0.3%, puts the interior jolting effect of 37 DEG C of incubators 16 hours,Prepare antigen liquid through the conduct after the assay was approved of inoculation 9`11 age in days SPF chicken embryo.
3, oil phase adjuvant preparation
Mix in the preparation of 94:6:2 ratio with the special white oil of injection, Si Ben-80 and aluminum stearate, through high-temperature sterilization standbyWith.
4, water preparation
A certain amount of Tween-80, after high-temperature sterilization, is joined in inactivation antigen liquid, and Tween-80 final concentration is 4%,Be water through fully dissolving to mix.
5, vaccine preparation
A certain proportion of oil phase and water (ratio is 2:3) in vertical colloid mill machine after premix, then are noted mixtureEnter to high-pressure homogenization pump internal emulsification, make water-in-oil emulsion, every milliliter contains virus quantity and is more than or equal to 105.5ELD50。
6, safety testing
Get 10 of SPF chickens in 2 week age, 3 times of immunizing dose inactivated vaccines of every chicken neck hypodermic injection. Establish control group chicken simultaneously10,14d continuously, it is bad whether observation occurs that the inactivated vaccine of being prepared by NDV is inoculated any part or the whole body that causeReaction. Result proves that all inoculation chickens all do not have limping symptom to occur, also without other any clinical pathology symptom or pathological change,Without any part or general reaction.
Embodiment 4 NDV inactivated vaccine immune protective tests
Get 50 of 28 age in days SPF chickens, be divided into 5 groups, 10 every group, first and second group is as attacking malicious Protection, and first group isImmune group, second group is control group. Immune group inoculation NDV inactivated vaccine, every intramuscular injection single dose vaccine0.5ml, after 28 days, does challenge test with ewcastle disease standard velogen strain (CVCCAV1611). Remaining is control group, only attacks poisonNot vaccine inoculation. Attack toxic agent amount and be 105.0ELD50. After attacking poison, observe animal dead situation.
Known (result of the test is in table 4) by experiment, immune group has 90% animal to be protected, and animal does not go outExisting ewcastle disease clinical symptoms; And control group 100% death. Result shows that NDV inactivated vaccine can make chicken avoid this poisonThe attack of strain, shows that this strain has good immune protective effect.
The 3rd group and the 4th group be as viral separation test, and the 3rd group does not connect as a control group as immune group, the 4th groupKind. Immune group inoculation NDV inactivated vaccine, every intramuscular injection single dose vaccine 0.5ml, after 28 days, uses ewcastle disease markAccurate velogen strain (CVCCAV1611) does challenge test. Attack toxic agent amount and be 105.0ELD50. After attacking poison, within 3～7 days, gather cotton swab,Inoculate instar chicken embryo on the 10th and separate this virus.
Known (result of the test is in table 4) by experiment, cotton swab virus separating resulting is: 10 of immune group are separated to virusQuantity be 0, it is 10 that 10 of control groups are separated to viral quantity. Cotton swab result shows NDV inactivated vaccine energyEnough make chicken avoid the attack of this strain, show that this strain has good immune protective effect.
Table 4SPF animal is attacked poison and viral separating experiment result
The 5th group as antibody detection: with blood sampling afterwards in 14,21,28 and 35 days after an intramuscular injection of 0.5ml, separation of serumMeasure its HI numerical value to NDV standard strain (CVCCAV1611). After 14 days, most of chickens have shown different journeysThe HI antibody of degree. After 21 days, all immune chickens all produce the HI antibody of anti-ewcastle disease, and significantly rise during compared with 14 days, and HI is anti-Body titre peaked in the time of 35 days, on average can reach 10log2.
Embodiment 5 Immunization cross-protection tests
Get commercialization ewcastle disease inactivated vaccine A strain and the inactivated vaccine of using the NDV of the embodiment of the present invention 1 to makeCarry out cross immunity protection test, the immune protection performance of checking inactivated vaccine.
Get 50 of 28 age in days SPF chickens, be divided into 6 groups, the 1st group, the 2nd group, the 4th group and the 5th group is all 10, the 3rd group and the 6thGroup is all 5, the 1st group and the 2nd group of muscle and every 0.5ml/ of subcutaneous vaccination A strain inactivated vaccine only, the 4th group and the 5th group of muscleWith subcutaneous vaccination NDV (Beijing Strain) inactivated vaccine 0.5ml/ only, the 3rd group and the 6th group of muscle and subcutaneous vaccination are asepticPBS0.5ml/ only. In immunity latter 28 days, attack poison 10 with NDV Beijing Strain for the 1st, 3,4 groups5.5ELD50/ only, the 2nd, 5,6Group is attacked poison 10 with ewcastle disease standard velogen strain F48E85.5EID50/ only; After attacking poison, observe the chicken public sentiment condition every day, observe two weeks, andWithin the 5th day, gather oral cavity and cloacal swabs, carry out that virus separates etc. Attack poison latter the 14th day, cut open all test chickens of inspection, in observationPopular name for change etc.
Result of the test shows (result of the test is in table 5): attack after poison, obvious clinical symptoms all appear in all groups; And it is cottonSwab virus separating resulting shows, the 1st group has 2 parts of cotton swabs can be separated to NDV, the 3rd group and the 6th of inoculation PBSGroup all can be separated to virus. The antibody that NDV A strain produces can be resisted the attack of standard Virulent Newcastle Disease Virus strain, andCan only produce 80% immune protective efficiency to the attack of NDV Beijing Strain. And NDV (Beijing Strain) inactivated vaccineCan, for chicken provides good protection, can resist the attack of ewcastle disease standard velogen strain and NDV Beijing Strain, enterOne step illustrates that NDV inactivated vaccine of the present invention has good cross-protection, can resist this Virus andThe attack of other Virulent Newcastle Disease Virus strains.
Table 5 is attacked malicious cross protection experiment with the SPF chicken immune of A strain commodity inactivated vaccine
The preparation of embodiment 6 NDV antigens
NDV is inoculated to 10 age in days SPF chicken embryos, discard 24h dead germ, the allantoic fluid of results 24h-72h dead germ, willAllantoic fluid carries out deactivation, centrifugal after, add proper adjuvant to make the stable antigen of NDV, this antigen is using and is laying inTime there is not loose malicious danger, reduced the harm to environment and human body because use live virus antigen, this product has veryGood stability, under 2～8 DEG C of conditions, preserves 12 months above these product blood clotting valencys constant, can be used for the HI of NDVDetect (the results are shown in Table 6).
Antigen storage stability result of the test prepared by table 6 NDV
In the description of this description, reference term " embodiment ", " some embodiment ", " example ", " specifically showExample " or the description of " some examples " etc. mean the specific features, structure, material or the spy that describe in conjunction with this embodiment or examplePoint is contained at least one embodiment of the present invention or example. In this manual, to the schematic statement of above-mentioned term notNecessarily refer to identical embodiment or example. And specific features, structure, material or the feature of description can be anyOne or more embodiment or example in suitable mode combination.
Although illustrated and described embodiments of the invention above, be understandable that, above-described embodiment is exampleProperty, can not be interpreted as limitation of the present invention, those of ordinary skill in the art is not departing from principle of the present invention and aimSituation under can change above-described embodiment within the scope of the invention, amendment, replacement and modification.
Claims (10)
1. a NDV, it is preserved in Chinese microorganism strain preservation conservator with preserving number CGMCCNo.7805Can common micro-organisms center.
2. NDV claimed in claim 1 is in the purposes of preparing in ewcastle disease inactivated vaccine.
3. an ewcastle disease inactivated vaccine, is characterized in that, comprises:
The NDV claimed in claim 1 of deactivation.
4. ewcastle disease inactivated vaccine according to claim 3, is characterized in that, described NDV is by formalin-inactivated.
5. a method of preparing ewcastle disease inactivated vaccine, is characterized in that, comprising:
NDV claimed in claim 1 is carried out to deactivation, to obtain the NDV through deactivation;
Utilize the described NDV through deactivation to prepare water; And
Described water and oil phase are carried out to mixing and emulsifying, to obtain described ewcastle disease inactivated vaccine.
6. method according to claim 5, is characterized in that, before described NDV is carried out to deactivation, comprisesObtain NDV nutrient solution according to the following step:
Described NDV is inoculated in nonimmune chicken embryo, and cultivates at 35.5～37.5 DEG C;
Collect the chick embryo allantoic liquid obtaining, to obtain described NDV.
7. method according to claim 5, is characterized in that, described NDV is carried out to deactivation and comprise:
At 37 DEG C, to utilize final concentration be 0.1%～0.3% formaldehyde to NDV nutrient solution carry out inactivation treatment 16～36 hours.
8. method according to claim 5, is characterized in that, the described water of preparing comprises:
Mix according to the volume ratio of 96:4 with Tween-80 containing deactivation NDV chicken blastochyle, to obtain instituteState water,
Wherein, the final concentration of described Tween-80 is 4%.
9. method according to claim 5, is characterized in that, the described oil phase of preparing comprises:
Injection white oil MARCOL52, Si Ben-80 and aluminum stearate are mixed according to the mass ratio of 94:6:2, so thatTo described oil phase.
10. method according to claim 5, is characterized in that, described water is mixed and comprised with oil phase:
Be that 2:3～2:4 carries out mixing and emulsifying stirring by described water and oil phase according to volume ratio, before termination is stirred, add dense eventuallyDegree is 0.01% thimerosal, to obtain described ewcastle disease inactivated vaccine.
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| CN101474401B (en) * | 2008-12-11 | 2011-08-31 | 瑞普（保定）生物药业有限公司 | Method for producing concentrated inactivate vaccine for newcastle disease |
| CN102579339A (en) * | 2012-03-12 | 2012-07-18 | 天津瑞普高科生物药业有限公司 | Oil emulsion vaccine for broilers and preparation method thereof |
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