CN102973930A - Chicken subunit bivalent vaccine, as well as preparation and application thereof - Google Patents
Chicken subunit bivalent vaccine, as well as preparation and application thereof Download PDFInfo
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Abstract
The invention relates to preparation and application of a newcastle disease-infectious bursal disease VP2 subunit bivalent vaccine. The preparation comprises the following steps of performing egg inoculation on a newcastle disease Clone30 strain to obtain blastochyle, and inactivating; mixing with chicken infectious bursal disease VP2 protein liquid of bacillus coli in a certain proportion; adding mineral oil adjuvant, and emulsifying to obtain bivalent vaccine; immunizing a 21-day SPF (specific pathogen free)chick with the bivalent vaccine; performing toxicity attack on newcastle disease virulent strain isolated in Beijing and infectious bursal disease virulent BC6-85 respectively after 21 days of immunization, results indicate that light bursa fabricius symptom appears on a chick in 20 chicken in the immunized group, and the rest chicken does not have abnormality; and 10/10 chicken are attacked and dead in the newcastle disease control group, and 8/10 chicken in the infectious bursal disease virus attack control group are attacked.
Description
Technical field
The invention belongs to bird vaccine and make the field, be specifically related to a kind of chicken subunit bigeminy vaccine and preparation and application, i.e. a kind of newcastle disease and infectious bursal disease bigeminy vaccine.
Background technology
Newcastle disease (newcastle disease, ND) is the birds acute infectious disease that is caused by newcastle disease virus (NDV), and spread speed is fast, has brought huge threat for for a long time China's aviculture.Although chicken Newcastle disease live vaccine is effective, cheap, easy to use, but its duration of immunity is short, immunity is subject to maternal antibody and disturbs, and cause easily the respiratory tract side effect, interference experiment chamber diagnosis virus is separated, therefore, since 1980, used the newcastle disease inactivated vaccine to become gradually China's chicken house and prevented and treated one of conventional method of newcastle disease.
Infectious bursal disease (Infectious Bursal Disease, IBD) claim again infectious bursal disease, is a kind of acute, the social disease that is caused by infectious bursa of Fabricius virus.Be badly damaged as feature take fabricius bursa inflammation, necrosis, atrophy and fabricius bursa endolymph cell, can cause the immunodeficiency disease of chicken, disturb the immune effect of various vaccines.This disease sickness rate is high, is one of most important disease of present aviculture.And, owing to the appearance of virulent, traditional vaccine immunity failure occurs repeatedly, therefore in recent years, be necessary to screen the not iso-allele of infections chicken cloacal bursa virus, and provide a new approach by the method for genetic engineering subunit vaccine for the infectious bursal disease prevention.
In order effectively to control ND and IBD, reduce inoculation times, be convenient to production application, therefore, be necessary to provide a kind of newcastle disease and infectious bursal disease bigeminy vaccine to come the requirement of satisfying the market.
Summary of the invention
The objective of the invention is to prepare and a kind ofly can prevent simultaneously the particularly vaccine that infects of virulent of newcastle disease and infectious bursal disease, infectious bursal disease wherein partly is the subunit vaccine that adopts the major antigen protective protein VP2 albumen of escherichia coli expression to make.
Bigeminy vaccine of the present invention is comprised of antigen and vaccine adjuvant, and described antigen is that newcastle disease virus and sequence are the infectious bursal disease VP2 albumen of SEQ ID NO:1.
The preparation method of above-mentioned bigeminy vaccine is with behind the Avian pneumo-encephalitis virus inoculated into chick embryo, and the results blastochyle adds formalin-inactivated; Then with the Avian pneumo-encephalitis virus blastochyle with after Infectious bursal disease virus VP2 mixes, add that vaccine adjuvant emulsifying makes.
Above-mentioned Avian pneumo-encephalitis virus is Avian pneumo-encephalitis virus Clone30.
The bigeminy vaccine of the present invention's preparation can prevent newcastle disease and infectious bursal disease simultaneously, and immune effect is similar with infectious bursal disease list Seedling effect to the single Seedling of newcastle disease, has reached the purpose that a pin two is prevented.
The specific embodiment
The major structural protein VP2 albumen of IBDV is main host protective antigen; the present invention is with the increased VP2 gene of IBD virulent of PCR method; be cloned on the pET-28a carrier; transform BL21 (DE3); VP2 albumen has obtained expression after IPTG induces, will express the fragmentation of thalline high-pressure homogenization after the centrifuging and taking supernatant be the VP2 protein solution.
10 age in days SPF embryos are inoculated in Avian pneumo-encephalitis virus Clone30 strain, choose the embryo results blastochyle of cultivating 72 ~ 120 hours after inoculating, and the ultrafiltration and concentration method is concentrated with virus liquid, and the adding final concentration is 0.1% formaldehyde.
The VP2 protein liquid is mixed rear emulsifying join Seedling with the Newcastle Disease venom.
Below in conjunction with specific embodiment newcastle disease of the present invention-infectious bursal disease virus VP 2 subunit bigeminy vaccine is described in detail.
One, the preparation of Newcastle Disease venom
With Avian pneumo-encephalitis virus Clone30 strain (available from China Veterinery Drug Inspection Office), do suitable dilution with sterile saline, through allantoic cavity inoculation 9 ~ 10 age in days susceptible Embryo Gallus domesticus, every embryo 0.1ml, sealing pin hole after the inoculation, straight 36 ~ 37 ℃ are continued to hatch.Dead Embryo Gallus domesticus before discarding 48 hours, after this 4 ~ 8 hours photograph eggs once, dead embryo takes out at any time, until all took out in 96 hours, puts 2 ~ 8 ℃ of coolings 12 ~ 24 hours.Results chick embryo allantoic liquid and amniotic fluid discard the blood clotting valency and are lower than 1:128, carry out steriling test.
Concentrating of Newcastle Disease venom
The virus liquid of results is removed large granular impurity with secondary pre-filtering post, then with viral ultrafiltration concentration systems the Newcastle Disease venom is concentrated into 1/3 of original volume.
Deactivation
Virus liquid after concentrated is placed in the deactivation tank, and the adding final concentration is 0.1% formaldehyde, with adding with stirring, it is fully mixed.37 ℃ of deactivations 16 hours.
Two, the screening of VP2 gene
With the Primer5.0 software design the full gene primer of pair for amplification chicken infectivity bursa of Fabricius virus VP 2; and at forward primer 5 ' end adding protectiveness base and BamH I restriction enzyme site; add protectiveness base and Xho I restriction enzyme site at 5 of downstream primer ' end, primer sequence is:
Primer?1：5′-GCG?TCG?ACA?TGA?CAA?ACC?TGC?AAG?ATC?-?3′
Primer?2：5′-CCC?TCG?AGT?TAT?CCT?TAT?GGC?CCG?GAT?T?-3′
Get the egg inoculation chorioallantoic membrane suspension supernatant that 200 μ l infect with the pathological material of disease of the doubtful IBDV of clinical separation, press the described method of RNAisoReagent description and extract viral RNA, carry out the synthetic cDNA of reverse transcription by reverse transcription test kit operation instructions, carry out pcr amplification, amplification system is (50 μ l): 10 * PCR Buffer, 5 μ l, dNTP (10mM each) 1 μ l, each 1 μ l of upstream and downstream primer (10mM), cDNA 3 μ l, 25mM MgCl2 4 μ l, Taq enzyme (5U/ μ l) 1 μ l, ddH2O 34 μ l; PCR response parameter: 95 ℃ of 5min, 95 ℃ of 1min, 58 ℃ of 2min, 72 ℃ of 2min, 35 circulations, 72 ℃ of 10min.Obtain a fragment about 1300bp, cut glue and reclaim this fragment, be connected with the pMD-18T carrier.PCR reclaims fragment 6 μ l, T carrier 2 μ l (after diluting 10 times), and T4 dna ligase 1 μ l, T4 buffer 2.5 μ l, water: 13.5 μ l amount to 25 μ l.16 ℃ of connections of spending the night, get 20 μ l and connect product transformed competence colibacillus e. coli jm109, selecting 10 white colonies cultivates, extract plasmid identification correct choose 3 order-checkings, the sequencing result of 3 plasmids is all consistent as a result, the nucleotide fragments that proof obtains does not produce mistake in the process of amplification, its nucleotides sequence is classified SEQ ID NO:2 as, and the aminoacid sequence of translation is SEQ ID NO:1.
This nucleotides sequence is listed in the upper Blast comparison of NCBI, has tens bases that point mutation has occured, show that this strain is the new epidemic isolates of a strain.This is because VP2 albumen is the main host protective antigen, often undergos mutation and causes antigenicity generation difference.
The expression of VP2 recombiant protein
To identify that a correct above-mentioned plasmid reclaims genetic fragment with BamH I and Xho I double digestion, be connected with the pET-28a carrier with these two enzyme action, the transformed competence colibacillus e. coli jm109, extract the correct rear BL21(DE3 that transforms receptor of plasmid identification), picking list bacterium colony, switching 5mlLB tubule culture medium, 37 ℃ of shaken cultivation 3 ~ 4 hours to OD600 be 0.6 o'clock, the adding final concentration is that the IPTG of 0.8mM induced 4 ~ 5 hours, and the SDS-PAGE electrophoresis result turns out to be secreting, expressing.
It is resuspended with 6 times of thalline weight in wet base PBS to express bacterium, the broken antibacterial of high-pressure homogenization, and the centrifuging and taking supernatant is done the agar immunodiffusion, and as a result fine jade expansion is tired and is not less than 1:64.
The purification of VP2 albumen
Use ni-sepharose purification, the imidazoles eluting of 200mM, eluent PBS dialysed overnight.The gained dialysis solution is the albumen of purification, can be used for seedling.
The preparation of VP2 subunit vaccine
Get 94 parts of injection white oils, Si Ben-80 6 part, add 2 parts of heating of aluminium stearate after mixing and dissolves, 116 ℃ of sterilizations 30 minutes are as the seedling oil phase; Then 4 parts of tween 80s that add sterilization add 96 parts of the VP2 recombiant protein fermented extracted liquid of above-mentioned preparation, and shake well dissolves fully as the seedling water tween 80; Get 3 parts of oil phases and be put in the colloid mill, start the motor slow rotation and stir, slowly add simultaneously water l part, stirred 2～5 minutes with l0000r/min after adding again, add 1% thimerosal solution before stopping stirring, making its ultimate density is 0.01%.
The immune effect of VP2 subunit vaccine:
20 SPF chickens are divided into two groups at random, every group 10, wherein one group when 21 age in days with 0.3ml/ dosage subcutaneous injection VP2 subunit vaccine only, immunity after 21 days together with every of not immune matched group through the strong malicious BC6-85 strain venom of eye dripping approach inoculation 100BID infectious bursal disease, observe clinical manifestation and the death condition of chicken behind the counteracting toxic substances every day, record morbidity and dead chicken number, cut open inspection and observe the pathological changes such as dead chicken bursa, slaughtered the survival chicken in 72～96 hours, by only analysing, observe the pathological changes such as fabricius bursa.8 chicken morbidities of the not immune matched group of result, and the immune group chicken is without morbidity.The VP2 subunit vaccine of the above results proof the present invention preparation has good immunogenicity.
Three, the preparation of bigeminy vaccine
Add 2 parts of aluminium stearate after getting 94 parts of injection white oils, Si Ben-80 6 part mixing, heating is dissolved, and sterilizes 30 minutes as the seedling oil phase for 116 ℃; 4 parts of the tween 80s of adding sterilization; The deactivation Newcastle Disease venom of above-mentioned preparation and VP2 recombiant protein fermented extracted liquid are according to the ratio mix homogeneously of 1:2; Get 96 parts of mixed uniform antigen liquids as the seedling water; Get 3 parts of oil phases and be put in the colloid mill, start the motor slow rotation and stir, slowly add simultaneously water l part, stirred 2～5 minutes with l0000r/min again after adding, add 1% thimerosal solution before stopping stirring, making its ultimate density is 0.01%, finishes the preparation of bigeminy vaccine.
The result of use of bigeminy vaccine
40 21 age in days SPF chickens are divided into 4 groups at random, every group 10, two groups of subcutaneous injections immunity bigeminy vaccine 0.3ml wherein, rear 21 days of immunity, get one group of immunity chicken together with not immune contrast newcastle Beijing Strain counteracting toxic substances, another organizes immune chicken together with the not immune contrast of another group chicken infectivity bursa of Fabricius virus counteracting toxic substances, and 20 chickens of immune group have one slight fabricius bursa symptom to occur as a result, and all the other are all without unusual; And newcastle counteracting toxic substances matched group 10/10 Mortality, 8/10 morbidity of chicken infectivity bursa of Fabricius virus counteracting toxic substances matched group.
Claims (4)
1. a bigeminy vaccine is comprised of antigen and vaccine adjuvant, and described antigen is that newcastle disease virus and sequence are the infectious bursal disease VP2 albumen of SEQ ID NO:1.
2. bigeminy vaccine as claimed in claim 1 is characterized in that described newcastle disease virus is newcastle disease virus Clone30.
3. the preparation method of bigeminy vaccine claimed in claim 1, be with this vaccine with the Avian pneumo-encephalitis virus inoculated into chick embryo after, the results blastochyle adds formalin-inactivated; Then with the Avian pneumo-encephalitis virus blastochyle with after Infectious bursal disease virus VP2 mixes, add that vaccine adjuvant emulsifying makes.
4. the application of bigeminy vaccine claimed in claim 1 in prevention newcastle disease and infectious bursal disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069489A (en) * | 2014-07-09 | 2014-10-01 | 江苏省农业科学院 | Newcastle disease and infectious bursal bivalent inactivated vaccine and preparation method thereof |
| CN112501139A (en) * | 2020-12-11 | 2021-03-16 | 新乡学院 | Recombinant Newcastle disease virus strain and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688486A (en) * | 2012-06-18 | 2012-09-26 | 青岛易邦生物工程有限公司 | Production method of triple inactivated vaccine for newcastle disease, infectious bursal disease and viral arthritis |
-
2012
- 2012-12-03 CN CN201210506662.7A patent/CN102973930B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688486A (en) * | 2012-06-18 | 2012-09-26 | 青岛易邦生物工程有限公司 | Production method of triple inactivated vaccine for newcastle disease, infectious bursal disease and viral arthritis |
Non-Patent Citations (2)
| Title |
|---|
| SUN-HEE CHO ET.AL.: "Characterization of a Recombinant Newcastle Disease Virus Vaccine Strain", 《CLINICAL AND VACCINE IMMUNOLOGY》 * |
| 程太平等: "新城疫和传染性法氏囊病二联疫苗的免疫效力研究", 《湖北农业科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069489A (en) * | 2014-07-09 | 2014-10-01 | 江苏省农业科学院 | Newcastle disease and infectious bursal bivalent inactivated vaccine and preparation method thereof |
| CN104069489B (en) * | 2014-07-09 | 2016-04-20 | 江苏省农业科学院 | Newcastle disease and infectious bursa of Fabricius bivalent inactivated vaccine and preparation method thereof |
| CN112501139A (en) * | 2020-12-11 | 2021-03-16 | 新乡学院 | Recombinant Newcastle disease virus strain and preparation method and application thereof |
| CN112501139B (en) * | 2020-12-11 | 2023-03-21 | 新乡学院 | Recombinant Newcastle disease virus strain and preparation method and application thereof |
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