CN108070571A - A kind of virulent duck enteritis virus and its application - Google Patents
A kind of virulent duck enteritis virus and its application Download PDFInfo
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Abstract
The present invention provides a kind of chicken embryo adaptability virulent duck enteritis virus, and deposit number is CCTCC No.V201761.The chicken embryo adaptability virulent duck enteritis virus of the present invention is used to prepare vaccine.The present invention filters out the good virulent duck enteritis virus of immunogenicity from advantage prevalence strain, through being passed in SPF chicken embryos, it is stable to reach obtain after 58 generations one plant, the good chicken embryo of immunogenicity adapts to vaccine and prepares strain, inoculated into chick embryo harvests dead embryo idiosome and blastochyle, virus liquid is collected after ground, freeze thawing, after formalin inactivation, vaccine is made in oiling adjuvant mixing and emulsifying.This vaccine can prevent the Duck Hepatitis Virus as caused by New Type Duck Hepatitis Virus, have many advantages, such as that efficient, security is good, protective rate is high.
Description
Technical field
The invention belongs to vaccine preparation technology fields, and in particular to a kind of virulent duck enteritis virus and its application.
Background technology
Duck virus hepatitis (duck viral hepatitis, DVH) is by virulent duck enteritis virus (duck
Hepatitis virus, DHV) a kind of acute, height using liver swelling and bleeding as main pathological change of duckling is caused to cause
Dead property, contagious disease.The nervous symptoms such as clinical manifestation spasm, twitch and opisthotonos, using liver swelling and bleeding as spy
Venereal disease is levied to become.The duckling of 1~3 week old is susceptible, and morbidity is serious, propagates rapidly, and duckling is interior dead usually when 1 after there is symptom is small
It dies, the death rate 90~95% after the infection of 10 age in days ducklings has become one of principal disease for seriously endangering duck culturing industry.
Duck hepatitis virus (DHV) serotype DHV has 4 independent serotypes, 1 type, 2 types, 3 types and new (prepattern).
Model book just wait (Fan Shucai, Li Hong, Yuan lead separation identification China Preventive Veterinary Medicine report of the New Type Duck Hepatitis Virus such as treasure, 2009,
10) prove to exist simultaneously 1 type and new DHV prevalences in China at present by testing.Serum neutralization test shows 1 type and new
Between lack cross-protection.In the DHV of 4 serotype, 1 type and new DHV prevalences are most extensive, and virulence was most strong, to 3 weeks
Duckling lethality in age can be more than 80%；Only in Britain, 3 types only have been reported that 2 type DHV in the U.S.；The two is in sporadic
Prevalence, lethality are low (about 20%).
Since market SPF duck embryos source is relatively difficult, so existing duck hepatitis inactivated vaccine uses duck at present
Embryo manufactures, and duck embryos are it is difficult to ensure that the duck virus hepatitis neutralizing antibody of duck embryos is negative and pure property, therefore the present invention has carried out newly
The experiment that type duck virus hepatitis is passed in SPF chicken embryos.
The content of the invention
The purpose of the present invention is preparing the duck virus hepatitis antigen of new embryo-culture viruses, new oil emulsion inactivation is made
Vaccine with this vaccine immunity animal, can prevent Duck Hepatitis Virus caused by New Type Duck Hepatitis Virus, this vaccine has potency
The advantages that height, protective rate are high, security is good.
Present invention firstly provides a kind of chicken embryo adaptability virulent duck enteritis virus, are by wild duck virus hepatitis disease
What poison obtained after being passed in chicken embryo can stablize the virus being proliferated in chicken embryo；
The chicken embryo adaptability virulent duck enteritis virus, one kind for YBHXN plants of virulent duck enteritis virus, in
On November 23rd, 2017 send the China typical culture collection center for being deposited in Wuhan University, deposit number CCTCC
No.V201761。
The chicken embryo adaptability virulent duck enteritis virus of the present invention is used to prepare vaccine；
Another aspect of the present invention provides a kind of vaccine, wherein viral hepatitis of the antigen used for inactivation
YBHXN plants；
The inactivation is inactivated using formalin；
Vaccine provided by the present invention, comprising vaccine adjuvant, wherein adjuvant is white oil for animals, aluminum stearate and Si Ben 80；
The quality parts ratio of white oil for animals, aluminum stearate and Si Ben 80 in above-mentioned adjuvant:47：1：3；
The present invention filters out the good virulent duck enteritis virus of immunogenicity from advantage prevalence strain, through in SPF chickens
Embryo passes on, and it is stable to reach one plant obtained after 58 generations, and immunogenicity good chicken embryo adapts to vaccine and prepares strain, inoculated into chick embryo,
The dead embryo idiosome of harvest and blastochyle collect virus liquid after ground, freeze thawing, after formalin inactivation, oiling adjuvant mixing and emulsifying
Vaccine is made.This vaccine can prevent the Duck Hepatitis Virus as caused by New Type Duck Hepatitis Virus, have efficiently, security it is good,
The advantages that protective rate is high.
Description of the drawings
Fig. 1：N-DHV primer amplification histograms.
Specific embodiment
On the basis of 1 type duck virus hepatitis embryo-culture viruses research early period, applicant leads to new duck liver poison hepatitis
The continuous passage in chicken embryo is crossed, has investigated new embryo-culture viruses, the multiplication that virus can be stablized in chicken embryo.With this strain
It prepares vaccine and carries out immune kind of duck, filial generation can obtain good protection.Result of study shows the new embryo-culture viruses of development
Inactivated vaccine potency is high, and security is good, and protective rate is high, overcomes that duck embryos source is unstable and duck embryos maternal antibody is to virus multiplication
Influence, and the experiment substantially reduced and the cost of production have established heavily fortified point for the popularization and use of duck virus hepatitis vaccine
Real basis.
The present invention is described in detail with reference to embodiment.
Embodiment 1：The screening of chicken embryo adaptability virulent duck enteritis virus
The good virulent duck enteritis virus of immunogenicity is filtered out from advantage prevalence strain, through continuous in SPF chicken embryos
Passage, reaches that obtained during 40 generation one plant is stable, and the good chicken embryo of immunogenicity adapts to vaccine and prepares strain, after reach 58 generations poison
Strain shows good stability, ELD50106.50~106.78Between.
1st, the freeze thawing 3 times of YBHXN plants of seed culture of viruses, 4000r/min centrifugation 30min are taken supernatant to make 100 times of dilutions by viral passages,
Allantoic cavity is inoculated with 9 10 pieces of age in days SPF chicken embryos, per embryo 0.2ml, 37 DEG C of incubations, 2 times a day according to embryo inspection.48~120 is small after inoculation
When interior death chicken embryo, put respectively 2~8 DEG C place 4~12 it is small when after, collect allantoic fluid, amniotic fluid and idiosome, idiosome go to turn around and
Four limbs.After above-mentioned tissue is homogenized, continue to pass in the same way, record chicken embryo death situation, and measure viral level.Knot
Fruit refers to table 1, and for virus when SPF embryos reached for 40 generation, SPF chicken embryos 8/10~10/10 are dead, and disease has been carried out to the virus from 40 generations
The measure of malicious content, the results showed that, ELD50106.50~106.78Between, strain passage is stablized.
Table 1：YBHX plants of E1~E58 pass on table for seed culture of viruses SPF chicken embryos
New embryo-culture viruses YBHXN plants of seedling immunogene virulent duck enteritis virus, by the easy public journey of nation's biology in Qingdao
Co., Ltd's separation, identification, keeping and supply.New YBHXN plants is 2008 from the liver that Shandong Province duck dies of illness duck
The poison obtained after separation after reaching for 58 generations in chicken embryo.More than strain is delivered on November 23rd, 2017 to Wuhan University
China typical culture collection center, deposit number be CCTCC No.V201761.
The new embryo-culture viruses YBHXN plants of VP1 protein gene sequences analysis of 2 virulent duck enteritis virus
N-DHV AP-04114 plants of (DQ812093) genom sequence of 2.1 design of primers and synthesis South Korea, selects VP1
Gene conserved regions, using gene runner Software for Design 2 to specific primer, by Shanghai, bioengineering Co., Ltd synthesizes.
N-DHV:
Upstream:CAGATGGCCGCCAATGATCA
Downstream:GTCTCTGACATTTCGAAATTGG
2.2RNA extraction by the YBHX strain virus before passage and passage the 5th, 10,15,20,25,30,35,40,45,50,
YBHXN strain virus after 55 and 58 generations extracts total serum IgE according to RNA extracts kits specification respectively.
50 DEG C of 30min of 2.3RT-PCR amplification N-DHV VP1PCR reaction conditions；94 DEG C of pre-degeneration 2min；94 DEG C of denaturation
30s, 54 DEG C of annealing 30s, 72 DEG C of extension 60s, 30 Xun Huans；72 DEG C of extension 5min；4 DEG C reaction was completed.PCR product is given birth to by Shanghai
Object Engineering Co., Ltd is sequenced.
2.4 sequence analysis application DNAMan and MEGA4.0 softwares analyze the sequencing result of acquisition, by sequence with
The amino acid sequence that the DHV reference virus strain logged in GenBank derives carries out sequence alignment.
2.5RT-PCR testing results carry out RT-PCR amplifications using the primer of South Korea N-DHV plants of VP1 gene, as a result show
Show, the YBHX strain virus before passage and the YBHXN strains after the 5th, 10,15,20,25,30,35,40,45,50,55 and 58 generations of passage
Virus amplifies the purpose band (Fig. 1) of size about 760bp.
2.6VP1 gene sequencings carry out sequence using DNAMan softwares to the VP1 genes of the virus of the different generations of amplification
The difference of the amino acid sequence of derivation is compared in row analysis.The results show that the virus is after chicken embryo was uploaded to for 40 generations, amino acid
The variation that sequence occurs, by
GDSNQLGDDEPVCFLNFETANVPIQGESHTLVKHLFGRQWLVRTVQHTSEVQELDLPVPDQGHASLLRF
FAYFSGEVILTIVNNGTTPCMVAHSYTMDNLTSEYAVTAMGGILIPANSAKNINIPFYSVTPLRPTRPMPASQGGGL
TFGRLYIWTQSGSVSVFMGLHKPALFFPLPAPTYTTHTLLNKIETMNLHDQSDQPDCHLCKICRKMKKWFRNHRPFR
FCLRLKTLAFELHLEIE
Become
GDSNQLGDDEPVCFLNFETANVPIQGESHTLVKHLFGRQWLVRTVQHTSEVQELDLPVPDQGHASLLRF
FAYFSGEVILTIVNNGTTPCMVAHSYTMDNLTSEYAVTAMGGILIPANSAKNINIPFYSVTPLRPTRPMPASQGGGL
TFGRLYIWTHSGSVSVFMGLHKPALFFPLPAPTYTTHTWLNKIETMNLHDQSDQPDCHLCKICRKMKKWFRNHRPFR
FCLRLKTLAFELHLEIE
That is 156 bit aminos become H from Q, and 185 become W from L；Show that the YBHX strains that the present invention screens have adapted to chicken embryo
Passage.
The preparation of 2 vaccine of embodiment
(1) prepared by vaccine (immunogene)：
Production is made 100 times of dilutions for YBHXN plants by the preparation of 1 seedling virus liquid with seed culture of viruses, and allantoic cavity is inoculated with 9 ages in days respectively
100 pieces of 100 pieces of SPF chicken embryos and the nonimmune chicken embryo of 9 ages in days, per embryo 0.2ml, 36~37 DEG C of incubations, 2 times a day according to embryo inspection.It connects
Kind after 48~120 it is small when interior death chicken embryo, put respectively 2~8 DEG C placements 4~12 it is small when after, collect allantoic fluid, amniotic fluid and embryo
Body, idiosome goes to turn around and four limbs.After blastochyle is homogenized, freeze thawing 3 times, 4000r/min centrifugation 30min take supernatant to be mixed in sterile
In container, 2~8 DEG C of preservations are put.(referring to table 2).
Table 2：The preparation of venom
| Strain name | Embryo kind | It is inoculated with embryo number (piece) | Embryo age in days | Harvest blastochyle (ml) |
| YBHXN plants | SPF chicken embryos | 100 | 9 | 1500 |
| YBHXN plants | Nonimmune chicken embryo | 100 | 9 | 1400 |
2 inactivations respectively import SPF embryos and the New Type Duck Hepatitis Virus liquid of nonimmune chicken embryo in inactivation tank, metered
10% (volume ratio) formalin, is sufficiently mixed it, and the ultimate density of formalin is 0.2%.It is imported after adding formalin
In another inactivation tank, fail to contact inactivator to avoid the virus that tank mouth nearby adheres to.(with temperature in tank when 37 DEG C of inactivations 16 are small
Reach 37 DEG C of beginning timing) it takes out afterwards, put 2~8 DEG C of preservations.
The 3 vaccine inspection of semifinished product
(1) steriling test takes the SPF chicken embryos of inactivation and nonimmune chicken embryo liquid respectively, by existing《Chinese veterinary pharmacopoeia》Annex
It carries out, asepsis growth.
(2) viral level measures
1) 10 times of virus liquid sterile saline work after concentration is serially diluted by new SPF embryos virus liquid, takes 10-4、
10-5、10-6、10-74 dilution factors, difference allantoic cavity is inoculated with 95 pieces of age in days SPF chicken embryos, per embryo 0.2ml.Set inoculation physiology simultaneously
5 pieces of saline control, per embryo 0.2ml.It puts 36~37 DEG C to continue to be incubated, often sunshine embryo 2 times, when observation 168 is small.With chicken embryo death simultaneously
There is chorioallantoic membrane oedema to thicken, the change of the systemic bleedings venereal disease such as head, neck, back is judged to infect, ELD50, it is 106.9。
2) 10 times of virus liquid sterile saline work after concentration is serially diluted by new nonimmune embryo virus liquid, is taken
10-4、10-5、10-6、10-74 dilution factors, difference allantoic cavity inoculation nonimmune 5 pieces of the embryo of 9 ages in days, per embryo 0.2ml.Set life simultaneously
Saline control is managed, per embryo 0.2ml.It puts 36~37 DEG C to continue to be incubated, often sunshine embryo 2 times, when observation 168 is small.With chicken embryo death simultaneously
There is chorioallantoic membrane oedema to thicken, the change of the systemic bleedings venereal disease such as head, neck, back is judged to infect, ELD50, it is 106.9。
(3) inactivation is examined is inoculated with 9 age in days SPF chicken embryos by the virus liquid allantoic cavity after SPF embryos and the inactivation of nonimmune embryo respectively
Each 10 pieces with the nonimmune chicken embryo of 9 ages in days, per embryo 0.2ml, put 36~37 DEG C and continue to be incubated, be observed continuously 168 it is small when, SPF chicken embryos
With nonimmune chicken embryo without death.
The preparation of 4 inactivated vaccines：Vaccine, which is carried out, by semi-finished product antigen after the assay was approved prepares (each liquid in preparation below
Body ingredient is counted by volume, referring to table 2, table 3).
(1) oil phase preparation takes 94 parts of white oil for animals, and 2 parts of aluminum stearate is placed in oil phase preparation tank after being heated to 80 DEG C,
6 parts of Jia Siben -80 again until when temperature reaches 115 DEG C, maintain 30min, spare after cooling.
(2) water, which mutually prepares to inactivate, examines qualified YBHXN strain virus liquid to mix, and detects and contains per in 0.2ml water phases
YBHXN strain virus contents, 106.3ELD50.4 parts of Tween-80 after sterilizing is taken, is added in Agitation Tank, while adds hybrid antigen liquid
It 96 parts, starts stirring motor and stirs 20~30min, be completely dissolved Tween-80.
(3) emulsification takes 1.5 parts of oil phase to be put in high-speed shearing machine, starts the stirring of motor slow rotation, while adds in slowly
1.5 parts of water phase with 12000r/min, emulsifies 6 minutes.After emulsification, 10ml is taken, is centrifuged 15 minutes with 3000r/min, tube bottom is anhydrous
Mutually it is precipitated.
(4) quantitative separating is dispensed, is sealed.
Table 3SPF chicken embryos vaccine emulsifies and packing
4 nonimmune chicken embryo vaccine of table emulsifies and packing
Embodiment 3
New duck virus hepatitis inactivated vaccine prepared by SPF chicken embryos and nonimmune chicken embryo is distinguished in vaccine product inspection
Carry out the product inspection of vaccine.
1 character
(1) appearance is milky emulsion.
(2) dosage form is water-in-oil type, takes a cleaning suction pipe, draws a small amount of vaccine drop in cold water, does not expand in oil droplet shape
It dissipates.
(3) stability is drawn vaccine 10ml and is added in centrifuge tube respectively, is centrifuged 15 minutes with 3000r/min, the equal nothing of tube bottom
Water is mutually precipitated.
(4) viscosity presses current edition respectively《Chinese veterinary pharmacopoeia》Annex carries out, result 61.6cp, 58.3cp.
2 loading quantity inspections press current edition respectively《Chinese veterinary pharmacopoeia》Annex carries out, and complies with standard.
3 steriling tests press current edition respectively《Chinese veterinary pharmacopoeia》Annex carries out, asepsis growth.
4 safety verifications are divided into 3 groups, the 1st group of 10 chickens, every neck is subcutaneously injected with 21~35 age in days SPF chickens 25
New duck virus hepatitis inactivated vaccine 2.0ml prepared by SPF chicken embryos, the 2nd group of 10 chickens, every neck are subcutaneously injected nonimmune
New duck virus hepatitis inactivated vaccine 2.0ml prepared by chicken embryo, the 3rd group of 5 chickens are not immune to be compared.At identical conditions
Raising, is observed continuously 14, test chicken and control chicken is all strong lives, and do not occur it is any caused by vaccine locally and systemically
Reaction.
5 residual formaldehydes measure presses current edition respectively《Chinese veterinary pharmacopoeia》Annex is measured, and residual formaldehyde is respectively
0.09%th, 0.07%.
6 efficacy tests are divided into 3 groups, SPF chickens are subcutaneously injected in the 1st group of 10 chickens, every neck with 24 age in days SPF chickens 25
Nonimmune chicken embryo is subcutaneously injected in new duck virus hepatitis inactivated vaccine 1.0ml prepared by embryo, the 2nd group of 10 chickens, every neck
The new duck virus hepatitis inactivated vaccine 1.0ml prepared, the 3rd group of 5 chickens are not immune to be compared.21 days after exempting from, blood sampling separation
Serum measures new duck hepatitis neutralize antibody titers respectively.
5 vaccine potency inspection result of table
It 3/10 is 1 that new duck virus hepatitis inactivated vaccine prepared by SPF chicken embryos, which is immunized neutralize antibody titers after chicken to be,:
64,6/10 be 1:128,1/10 be 1:256；During new duck virus hepatitis inactivated vaccine prepared by nonimmune chicken embryo is immunized after chicken
It 2/10 is 1 to be with antibody titer:64,5/10 be 1:128,3/10 be 1:256,5 chickens of control group are feminine gender.(referring to table 5).
The above results show that vaccine prepared by the present invention has good immune effect.
Claims (7)
1. a kind of virulent duck enteritis virus, which is characterized in that the deposit number of the virulent duck enteritis virus is CCTCC
No.V201761。
2. application of the virulent duck enteritis virus described in claim 1 in vaccine is prepared.
3. a kind of vaccine, which is characterized in that the antigen used in the vaccine includes the duck of inactivation described in claim 1
Viral hepatitis.
4. vaccine as claimed in claim 3, which is characterized in that the inactivation is inactivated using formalin.
5. vaccine as claimed in claim 3, which is characterized in that the vaccine includes vaccine adjuvant.
6. vaccine as claimed in claim 5, which is characterized in that the vaccine adjuvant is white oil for animals, aluminum stearate and department
This 80.
7. vaccine as claimed in claim 5, which is characterized in that white oil for animals, aluminum stearate and department in the vaccine adjuvant
This 80 quality parts ratio:47：1：3.
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