CN102397539A - Duckling flavivirus disease inactivated vaccine and preparation method thereof - Google Patents
Duckling flavivirus disease inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a duckling flavivirus disease inactivated vaccine and a preparation method thereof. The duckling flavivirus disease inactivated vaccine contains an inactivated duckling flavivirus strain and an adjuvant. The preparation method comprises the following steps of: culturing seed viruses for producing duckling flavivirus disease strains on a large scale to collect a virus liquid; inactivating the collected virus liquid, adding into the adjuvant, and emulsifying to obtain the duckling flavivirus disease inactivated vaccine. The invention further discloses a duckling flavivirus JM strain which is collected in the China Center for Type Culture Collection on October 14th, 2011 with the collection number of CCTCCV201131. The ''duckling flavivirus disease'' inactivated vaccine has the advantages of high safety, no untoward effect on ducklings, good immune effect, capability of remarkably enhancing the resistance of laying ducks to duckling flaviviruses, clinical application to prevention of the ''duckling flavivirus disease'' and remarkable economic and social benefits.
Description
Technical field
The invention belongs to the animal vaccine field, relate to a kind of duckling virosis inactivated vaccine and preparation method thereof.
Background technology
" duckling virosis " (temporarily name) has another name called " duck hemorrhagic oophoritis ", " laying ducks jaundice viral disease ", " duck hyperpyrexia egg drop syndrome " and " egg drop syndrome that novel banzi virus BYD causes " is a kind of new duck disease of finding first on Chinese Zhejiang, Fujian, Shandong, Guangdong and other places in 2010, mainly betides laying ducks, also betides kind of a goose, meat-type duck.When this disease took place laying ducks, clinical manifestation was the limbs astasia, difficulty in walking, and feed intake descends, and egg production reduces suddenly, and it is very big to subtract the egg amplitude, subtracts egg in 5 days and surpasses 90% perhaps total crop failure, and the sick duck of part is death in a few days, and mortality rate is 5%～10%.Cut open the inspection pathological changes and be that ovary takes place is hemorrhage, atrophy, follicular rupture or atrophy etc.Before this, do not see similar eqpidemic disease both at home and abroad.Rattan pool in 2010 is vast, Cao Zhenzhen, Wan Chun and etc., this disease pathogen of proof belongs to flaviviridae Flavivirus duckling virus such as Su Jing in 2011 is good (
Flaviviridae, Flavivirus, Duck flavivirus).
Hu Qilin in 2011 etc. are separated to 1 strain duckling virus JM strain from the laying ducks of Guangdong Province's generation " duckling virosis ", JM is identified, have measured JM strain part physics and chemistry and biological characteristics, and have carried out animal recurrence test etc.
Up to now, do not see the particularly report of inactivated vaccine research of domestic and international relevant " duckling virosis " vaccine.Therefore, development a kind of " duckling virosis " safely and effectively vaccine is significant to the development of the foster duck industry of China.
Summary of the invention
The object of the present invention is to provide a kind of duckling virosis inactivated vaccine, this inactivated vaccine safety is good, and immune effect is reliable.
Another object of the present invention is to provide the method for preparing of above-mentioned vaccine.
The technical scheme that the present invention adopted is following:
A kind of duckling virosis inactivated vaccine, contain through the duckling of deactivation virus (
Flaviviridae, Flavivirus, Duck flavivirus) strain and adjuvant.
Preferably, said adjuvant is the French SEPPIC Montanide ISA of company 71 VG adjuvants.
Preferably, said duckling virus stain is duckling virus JM strain, delivers Chinese typical culture collection center preservation on October 14th, 2011, and deposit number is CCTCC V201131.
The method for preparing of above-mentioned " duckling virosis " inactivated vaccine comprises the steps:
1) production of duckling virus stain is carried out large-scale culture with kind of a poison, gather in the crops viral liquid;
2) the viral liquid deactivation that will gather in the crops;
3) the viral liquid after the deactivation is added in the adjuvant, promptly get after the emulsifying.
Preferably; The concrete operations of step 1) are: with the production of duckling virus stain with kind of poisons allantoic cavity inoculation 10 ~ 12 age in days sheldrake embryos or instar chicken embryo on the 8th～11 or 10 ~ 13 age in days Muscovy duck embryos; 37 ℃ of cultivations; Collect 48 ~ 120 hours dead embryos in inoculation back, place 4 ~ 8 ℃ of coolings 4 ~ 24 hours, the toxic allantoic fluid of aseptic results.
Preferably, concrete operations step 2) are: the toxic allantoic fluid that will gather in the crops added 0.2% ~ 0.3% of formaldehyde to total amount, in 37 ℃ of deactivations 20 ~ 28 hours.
Preferably, the concrete operations of step 3) are: the viral liquid after 20 ~ 40 parts by volume deactivations is added in the adjuvant of 60 ~ 80 parts by volume emulsifying in the high-speed homogenization machine.
Duckling virus JM strain is delivered Chinese typical culture collection center preservation on October 14th, 2011, and deposit number is CCTCC V201131.
Beneficial effect of the present invention is:
Inactivated vaccine safety of the present invention is good, respectively with 1.5ml/ only and 2.5ml/ do not see obvious adverse reaction after only inoculating laying ducks; Good immune effect; To laying ducks with this vaccine 1.5ml/ only back 14 days/12 days of immunity attack with strong poison, the result, the counteracting toxic substances matched group began to lay eggs behind the counteracting toxic substances and obviously descends on the 2nd day; Began in the 8th day to stop to lay eggs or being reduced to 20%; Behind the counteracting toxic substances in 17 days average laying rate be 17.8%/20.3%, and behind the vaccine immunity group counteracting toxic substances of the present invention in 17 days average laying rate be 67.03%/67.2%～71.7%, protective rate is 60%/59%～65%; Behind the white-oil adjuvant vaccine control group counteracting toxic substances in 17 days average laying rate be 29.3%/33.1%; Protective rate is 14%/16.4%, shows that vaccine immunity duck egg production of the present invention is significantly increased than not immune counteracting toxic substances matched group and white-oil adjuvant vaccine control group duck, proves that this vaccine can protect the attack of the strong poison of laying ducks opposing; Can be used for preventing clinically " duckling virosis ", have great economic and social benefit.
Description of drawings
Fig. 1 is the electromicroscopic photograph (100,000 *) of flaviviridae Flavivirus duckling virus Guangdong strain (JM);
(swimming lane 1-6 is the clinical pathological material of disease of doubtful ARV to Fig. 2, swimming lane 7 is ddH for the PCR product agarose gel electrophoresis figure of JM strain
2O, swimming lane 8,9,14,15 and 17 is respectively: JMDE3, JMDE4, JMDE1, JMDE2 and JMDE5; Swimming lane 10-13 is respectively NDV, ARV, IBDV and MDRV; Swimming lane 16 is DNA molecular weight Marker)
Fig. 3 is the RT-PCR amplification of 7 fragment genes of duckling virus JM pnca gene group ORF;
(a is the normal control group to Fig. 4, and b is an infected group for the inspection photo is cutd open in sheldrake infection JM strain after the 5th day.）。
The specific embodiment
Below in conjunction with embodiment the present invention is further described, but is not limited thereto.
Embodiment 1
One, production of vaccine is used strain
1, viral source, preservation situation
This production of vaccine is the Guangdong strain (JM) of flaviviridae Flavivirus duckling virus with kind of a strain; Be the inventor in 2011 from the tissues such as laying ducks ovary of Guangdong Province's Jiangmen city generation egg drop reduction with the sheldrake embryo through allantoic cavity inoculation separation and get; Deliver Chinese typical culture collection center preservation on October 14th, 2011, deposit number is CCTCC V201131.Return methods such as test, morphology of virus electron microscopic observation and identify through PCR, virus.
, the Strain characteristic
(1) form of duckling virus JM strain
Get toxic allantoic fluid JMDE4 in 4 ℃, behind the centrifugal 20min of 3000r/min, get supernatant; In 4 ℃, the centrifugal 40min of 5000r/min gets supernatant again; Supernatant is in 4 ℃, and 40, the centrifugal 120min of 000r/min gets deposition, uses a small amount of 0.01M, and pH7.0PBS is resuspended, and this resuspended liquid is preliminary purification virus.Preliminary purification virus is with 2.5
%The phosphotungstic acid negative staining is observed under the JE-1200EX transmission electron microscope, and the result can see about diameter 50nm, circular virion (as shown in Figure 1).
(2) molecular biology identification of duckling virus JM strain
According to reports such as Su Jingliang
(Jingliang Su; Shuang Li; Xudong Hu, et al. Duck Egg-Drop Syndrome Caused BYDVirus, aNew Tembusu-RelatedFlavivirus. PLoS ONE 6 (3): e18106.); Synthesized a pair of duckling virus special primer TV-3 (f), TV-3 (r), the expection amplified production is 401bp.The viral RNA extracting; Get toxic allantoic fluid JMDE3, JMDE4, JMDE1, JMDE2 and JMDE5; Simultaneously with NDV etc. as negative control, carry out extracting with Takara MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 extraction agent box by operation instructions.
Reverse transcription (cDNA is synthetic): get 21ul viral RNA extract product, add Random 6mers 2ul, add dNTPs 4ul, mixing, 65 ℃; 10min, ice bath 2～3min adds DTT 4ul, 5 * M-MLV buffer 8ul again in above-mentioned reverse transcription pipe; Add M-MLV (Invitrogen Company products), mixing, 30 ℃; 10min, 37 ℃ again, 60min.
The pcr amplification of pcr amplification primer TV-3 (f), TV-3 (r) is undertaken by following: reaction volume 25 μ l, DEPC water 26ul, ExTaq buffer 5ul; DNTPs 3ul, primer TV-3 (f) 3ul, TV-3 (r) 3ul; ExTaq 0.25ul; Be divided into 2 parts behind the mixing, every part 20 ul adds the 5ulcDNA template again in reaction system.The PCR reaction condition is: 94 ℃ of 5min; 94 ℃ of 30sec, 52 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations; At last, 72 ℃ are extended 10min.1% agarose gel electrophoresis with containing Goldenview (day damp genome company product) is analyzed the PCR product.Negative control and water are not all seen any band as a result, have only the JM strain dna fragmentation (as shown in Figure 2) of expection size to occur.
A is with reference to the duckling virus BYD-1 strain (Su Jingliang etc. that announced among the GenBank; Whole genome sequence ditto); On the basis of carrying out Flavivirus multisequencing compare of analysis, utilize in the banzi virus genome than conservative region, Using P rimer Premier 5.0 software designs 7 pairs of primers; Be respectively applied for the duckling viral gene fragment (primer sequence is seen table 1) of the about 1200-1900 bp length of amplification, all primers are synthetic by Invitrogen.
Primer location is with reference to duckling virus BYD-1 strain (NCBI serial number: JF312912)
The extraction of B JM strain the 4th generation allantoic fluid poison (JMDE4) RNA
TRIZOL LS Reagent RNA extracts the test kit operation instructions to carry out.In 1.5 mL microcentrifugal tubes, add 250 μ L JMDE4 allantoic fluids and 750 μ L TRIzol, abundant mixing, room temperature is placed 10 min; Add 200 μ L chloroforms, 15 sec that commove, after room temperature leaves standstill 5 min, 4 ℃ of 12 000 centrifugal 15 min of rpm; Get supernatant in new sterilization 1.5 mL microcentrifugal tubes, add the equal-volume isopropyl alcohol, abundant mixing, room temperature is placed 10 min, 4 ℃ of 12 000 centrifugal 10 min of rpm; Abandoning supernatant, deposition is with 70% ethanol, 1000 μ L, and mixing washs 1 time gently, 4 ℃ of 12 000 centrifugal 10 min of rpm; Supernatant discarded, air-dry; With the distilled water dissolution precipitation of 10 μ L through the no RNA enzyme of DEPC processing.
The pcr amplification of each genetic fragment of C JM strain
Use the RT-PCR two-step method to carry out, press the operation of the ReverTra Ace of TOYOBO company reverse transcription description.
The mensuration and the sequence comparing analysis of each gene fragment order of D JM strain
The bacterium liquid sample (4 clones) that will contain recombiant plasmid send Guangzhou handsome order-checking.Application of DNA Star 5.07 analysis software package are spliced arrangement to the sequence of the JM strain that order-checking obtains, and (Genbank serial number: JN811559): the genome ORF total length of JM strain is 10278bp to the complete genome sequence of acquisition JM strain.From ncbi database, search for and download the gene order of the representative strain of 41 strain Flavivirus; Use Clustalx and respectively gene is carried out the multisequencing comparison; On this basis; Use the Phylogenetic Analysis that the MEGA4.0 analysis software carries out gene, adopt the ortho position commonly used method (Neighbor-Joining) of joining to make up cladogram, and assess the reliability of cladogram with the Bootstrap value.Analyze the similarity between strain, variation situation and genetic evolution relation.Submit to NCBI BLAST SERVER to carry out the homology search of sequence the complete genome sequence of JM strain simultaneously, result for retrieval is as shown in table 2.JM strain ORF sequence B LAST result shows, the sequence similarity of isolating light yellow viral JS804 was the highest from Jiangsu with 2010 in the JM strain, reached 99%, and the difference of 55 bases is only arranged, and secondly the sequence of the viral BYD-1 of isolating duckling also reached 99% from Hebei with 2010; Represent the genetic evolution tree (figure slightly) of strain and JM strain duckling viral genome total length to find out from 41 strain Flavivirus; 2 strain ducklings virus JM and BYD-1 strain and new isolating light yellow viral JS804 strain (Huang Xinmei in 2010; Li Yin; Zhao Dongmin etc. the separation of novel light yellow viral JS804 strain and evaluation. Jiangsu agricultural journal; 2011,2:354～360) with Flavivirus in the sibship of tembusu virus nearest, this 4 strain poison has constituted little branch on the cladogram with bagaza virus.6 strain sequences on this subbranch are carried out the sequence similarity analysis with DNAstar software; Result's (seeing table 2) shows: the similarity of JM strain and JS804 strain and BYD-1 strain is very high; Be respectively 99.5%, 99.4%, promptly separate in the period of China 2011 from the duckling virus JM in Guangdong strain with separate from the light yellow virus in Jiangsu with separate between the duckling virus genome sequence in Hebei similarity all up to more than 99%.
(3) pathogenicity of JM strain
Healthy sheldrake field is divided and is bought 94,98 ages in days for 2 times and close on out and produce 50 and 60 of sheldrakes from Sanshui District, Foshan City; Adaptability is raised after 120 days and is divided into groups under laboratory condition; Normal saline matched group and respectively 13 of test group are established in test for the first time, respectively through leg muscle injecting normal saline and JMDE4 allantoic fluid (ELD
50Be 10
-4.5), 2mL/ only tests for the second time and establishes 19 of normal saline matched groups, 16 of test group, isolated rearing.Observe clinical symptoms, 5 d, 7 d and 10 d after inoculation get 3,2 ducks from matched group and infected group respectively and compel to kill simultaneously, cut open inspection observation pathological changes.As a result, 2 tests are all found, do not see after the normal saline winding kind obviously unusually, and visible the searching for food of JMDE4 inoculation group beginning in the 4th day slightly reduce, draw green to know just, and egg production obviously descends, and experimental period does not see that duck is only dead.5 d, 7 d and 10 d cut open inspection after infection, and all visible infected group follicle is hemorrhage, and contrast duck normal (see Fig. 4, a is the normal control group, and b is an infected group).
(4) stability of JM strain
With JM strain the 7th generation allantoic fluid poison (JMDE7, ELD
50Be 10
-6.0) on the duck embryo of healthy sheldrake field, go down to posterity 5 times continuously, every generation is pressed the method for Yin Zhen etc. [Yin Zhen, Liu Zhenhua chief editor, animal virology, second edition, 329-330, Science Press, Beijing] and is measured ELD
50, the ELD of JMDE8, JMDE9, JMDE10, JMDE11 and JMDE12 as a result
50Be respectively: 10
-6.25, 10
-6.5, 10
-6.75, 10
-6.5With 10
-6. 75, show that JM strain heritability is stable.
(5) specificity of JM strain
Two, duckling virosis inactivated vaccine and preparation method thereof
Experiment material: duckling virus Guangdong strain JM strain; By Guangdong Provincial Academy Of Agricultural Sciences Veterinary Institute fowl diseases research department in 2011 from Guangdong Province Jiangmen city separate and identify; Deliver Chinese typical culture collection center preservation on October 14th, 2011, deposit number is CCTCC V201131.9～11 age in days sheldrake embryos are available from the healthy sheldrake field of planting of Guangzhou Fanyu District.94 age in days health are laid eggs sheldrake available from Sanshui District, Fushan City, Guangdong Province health sheldrake field of laying eggs; Buy back the back in the Guangdong Provincial Academy Of Agricultural Sciences Veterinary Institute healthy animal drylot feeding support to 127 ages in days (at this moment; Laying rate is 22/67 (32.8%)), be used for safety and immuning effect test.Analytical pure formaldehyde is by Guangzhou Chemical Reagent Factory production.Montanide ISA 71 VG adjuvants are French SEPPIC Company products, available from Shanghai representative office of France match BIC Corp.
The preparation process of inactivated vaccine is following:
1, seed culture of viruses breeding
With JM strain the 7th generation allantoic fluid poison (code name JMDE7, ELD
50Be 10
-6.0/ ml) inoculate 11 age in days sheldrake embryos, 0.2mL/ embryo, 37 ℃ of cultivations through allantoic cavity; Shine embryo 2 every day, discards the 24h dead embryo, and dead afterwards duck embryo is put 4 ℃ of refrigerators; Aseptic results dead germ allantoic fluid (to call toxic allantoic fluid in the following text) in 24h, mark is JMDE8, puts-80 ℃ of refrigerators and preserves subsequent use.
Get JMDE8 allantoic fluid poison and measure ELD by the same method with the sheldrake embryo
50, result, the ELD of JMDE8
50Be 10
-6.25/ ml, subsequent use with seed culture of viruses as basic seed with production.
, virus breeding
Method by above-mentioned " seed culture of viruses breeding " is inoculated 11 age in days sheldrake embryos with producing with seed culture of viruses, and results allantoic fluid (code name JMDE9) is also measured malicious valency, and malicious valency is 10
6.5ELD
50/ ml is greater than 10
5.5ELD
50/ ml, it is qualified to be judged to, and is used to prepare vaccine.
, inactivation of virus and check
JMDE9 allantoic fluid poison low-speed centrifugal is removed post precipitation, adds 0.3% formaldehyde, in 37 ℃ of deactivation 24h, during jolting for several times.The deactivation rearmounted 4 ℃ of refrigerators that finish are preserved subsequent use.
Get the good 5 piece of 11 age in days sheldrake embryo of toxic allantoic fluid inoculation of deactivation, the 0.2mL/ embryo, 37 ℃ of cultivations, shine embryo 2 every day, observed 7 days, and 5 pieces of embryos all become alive as a result, show that inactivation of virus is thorough.
, seedling
It is an amount of to get the good toxic allantoic fluid of deactivation, is in the ratio adding Montanide ISA 71 VG adjuvants of 30:70 in viral liquid and adjuvant, 8000r/min emulsifying 2 minutes, and at a distance from 3 minutes, the same again emulsifying 1 time promptly obtained " duckling virosis " of the present invention vaccine.4 ℃ of refrigerators of the vaccine for preparing are preserved subsequent use.
Embodiment 2
Duckling virosis inactivated vaccine and preparation method thereof
Step by embodiment 1 prepares the good toxic allantoic fluid of deactivation; It is an amount of to get the good toxic allantoic fluid of deactivation; In viral liquid and adjuvant is that 8000r/min emulsifying 2 minutes was at a distance from 3 minutes in the ratio adding Montanide ISA 71 VG adjuvants of 20:80; The same again emulsifying 1 time promptly obtains " duckling virosis " of the present invention vaccine.4 ℃ of refrigerators of the vaccine for preparing are preserved subsequent use.
Duckling virosis inactivated vaccine safety testing
2 tests have been carried out altogether.It is 0801 that the vaccine lot number is used in test for the first time, get 31 the 127 healthy laying ducks of age in days and be divided into 3 groups at random, and first group 13, be the blank group, through the inboard intramuscular inoculation normal saline of shank, 1.5ml/ is only; Second group 10, be the 1.5ml test group, through the inboard intramuscular inoculation embodiment of shank 1 obtained vaccine, 1.5ml/ is only; The 3rd group 8, be the 2.5ml test group, through the inboard intramuscular inoculation embodiment of shank 1 obtained vaccine, 2.5ml/, isolated rearing was observed 15 days.As a result, matched group, 1.5ml/ only and 2.5ml/ dose groups have 1,1 and 1 duck to occur walking lamely the same day respectively, walked lamely in second day and to recover, laying rate and matched group are close, do not see untoward reaction, show that this inactivated vaccine safety is good.
It is 0802 that the vaccine lot number is used in test for the second time, get 26 the 135 healthy sheldrakes of age in days and be divided into 2 groups at random, and first group 20, be the blank group, through the inboard intramuscular inoculation normal saline of shank, 2.5ml/ is only; Second group 6, be test group, through the inboard intramuscular inoculation embodiment of shank 2 obtained vaccines, 2.5ml/, isolated rearing was observed 15 days.As a result, matched group and test group have 2 and 1 duck appearance on same day limping respectively, the recovery of walking lamely in second day, and laying rate and matched group are close, do not see untoward reaction, show that this inactivated vaccine safety is good.
Duckling virosis inactivated vaccine potency test
2 tests have been carried out altogether.It is 0801 that the vaccine lot number is used in test for the first time, get 57 the 127 healthy sheldrakes of age in days and be divided into 4 groups at random, and first group 13, be the blank group, through the inboard intramuscular inoculation normal saline of shank, 1.5ml/ is only; Second group 16, be the counteracting toxic substances matched group, through the inboard intramuscular inoculation normal saline of shank, 1.5ml/ is only; The 3rd group 14, be test group (being labeled as the D4-P group) that through the vaccine of inboard intramuscular inoculation embodiment 1 preparation of shank, 1.5ml/ only; The 4th group 14 is that white-oil adjuvant vaccine control group (has only adjuvant with different with the vaccine of the present invention's preparation; All the other compositions are all with identical with the vaccine of the present invention's preparation; Be labeled as the D4-line of oils), through the inboard muscle vaccination of shank, 1.5ml/ is only; Each organizes isolated rearing, observes each group spirit, kinestate every day, searches for food, situation such as feces.After the immunity inoculation 14 days, except that first group, all the other each groups were all carried out counteracting toxic substances with JMDE5: every duck is through the inboard intramuscular inoculation 2.0 * 10 of shank
5.25ELD
50JMDE5; Isolated rearing; Observe every day, situation such as each group of record is laid eggs quantity, spirit, kinestate, search for food, feces, simultaneously, 5 d, 7 d and 10 d behind counteracting toxic substances; All respectively get 2,3 and 2 urgent killing of ducks from blank group, counteracting toxic substances matched group and immune group respectively, cut open inspection and observe pathological changes; Each group of statistics is from day that begins to laying eggs day to recover of counteracting toxic substances average laying rate of totally 17 days; Calculate immune group protective rate, protective rate (%)={ [the average laying rate of 1-counteracting toxic substances matched group]-[the average laying rate of 1-immune group] } ÷ [the average laying rate of 1-counteracting toxic substances matched group] * 100% by following formula.As a result, the counteracting toxic substances matched group began to lay eggs behind the counteracting toxic substances and obviously descends in second day, and the 8th day begins to stop to lay eggs, and average laying rate is 17.8% in 17 days, and the test group protective rate is 60%, and white-oil adjuvant vaccine control group protective rate is 14% (seeing table 4).
It is 0802 that the vaccine lot number is used in test for the second time, get 78 the 135 healthy sheldrakes of age in days and be divided into 5 groups at random, and first group 20, be the blank group, through the inboard intramuscular inoculation normal saline of shank, 1.5ml/ is only; Second group 16, be the counteracting toxic substances matched group, through the inboard intramuscular inoculation normal saline of shank, 1.5ml/ is only; The 3rd group 13, for testing 1 group (being labeled as the D4-P1 group), through the vaccine of inboard intramuscular inoculation embodiment 1 preparation of shank, 1.0ml/ only; The 4th group 13, for testing 2 groups (being labeled as the D4-P2 group), through the vaccine of inboard intramuscular inoculation embodiment 1 preparation of shank, 1.5ml/ only; The 5th group is that white-oil adjuvant vaccine control group (has only adjuvant with different with the vaccine of the present invention's preparation; All the other compositions are all with identical with the vaccine of the present invention's preparation; Be labeled as the D4-line of oils), through the inboard muscle vaccination of shank, 1.5ml/ is only; Except that the counteracting toxic substances time is the 12d of immunity back, all the other are all with test for the first time.As a result, the counteracting toxic substances matched group began to lay eggs behind the counteracting toxic substances and obviously descends on the 2nd day, and average laying rate is 20.3% in 17 days, and D4-P1 group protective rate is 65%, and D4-P2 group protective rate is 59%, and white-oil adjuvant vaccine control group protective rate is 16% (seeing table 5).
More than experiment shows that " duckling virosis " of the present invention inactivated vaccine safety is good, can only not produce any untoward reaction to duck; Good immune effect can significantly improve the resistance of laying ducks to duckling virus, can be used for preventing clinically " duckling virosis ", has great economic and social benefit.
Above embodiment is merely and introduces preferred case of the present invention; In practical operation; Deactivation strain in the duckling virosis inactivated vaccine of the present invention through the duckling virus JM of deactivation strain, also can be selected other duckling virus stain for use, like BYD-1 strain, JS804 strain etc. except available; Used adjuvant is Montanide ISA 71 VG, all can obtain " duckling virosis " inactivated vaccine of good immune effect.
In the method for preparing of duckling virosis inactivated vaccine; Except being inoculated into and carrying out the large-scale breeding in the sheldrake embryo with planting poison; Also can be inoculated in Embryo Gallus domesticus and Muscovy duck embryo and sheldrake embryo, Embryo Gallus domesticus and the Muscovy duck embryo fibroblast planting poison; Carry out large-scale culture, obtain toxic allantoic fluid or cell culture poison.
Above embodiment is merely and introduces preferred case of the present invention, and to those skilled in the art, any conspicuous variation and the improvement in the scope that does not deviate from spirit of the present invention, carried out all should be regarded as a part of the present invention.
< 110>Guangdong Provincial Academy Of Agricultural Sciences Veterinary Institute
< 120>duckling virosis inactivated vaccine and preparation method thereof
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Claims (8)
1. a duckling virosis inactivated vaccine contains duckling virus (Flaviviridae, Flavivirus, Duck flavivirus) strain and the adjuvant through deactivation.
2. duckling virosis inactivated vaccine according to claim 1 is characterized in that, said adjuvant is the French SEPPIC Montanide ISA of company 71 VG adjuvants.
3. duckling virosis inactivated vaccine according to claim 1 is characterized in that, said duckling virus stain is duckling virus JM strain, delivers Chinese typical culture collection center preservation on October 14th, 2011, and deposit number is CCTCC V201131.
4. the method for preparing of each described duckling virosis inactivated vaccine of claim 1 ~ 3 comprises the steps:
1) production of duckling virus stain is carried out large-scale culture with kind of a poison, gather in the crops viral liquid;
2) the viral liquid deactivation that will gather in the crops;
3) the viral liquid after the deactivation is added in the adjuvant, promptly get after the emulsifying.
5. the method for preparing of duckling virosis inactivated vaccine according to claim 4; It is characterized in that; The concrete operations of step 1) are: with kind of poisons allantoic cavity inoculation 10 ~ 12 age in days sheldrake embryos or instar chicken embryo on the 8th～11 or 10 ~ 13 age in days Muscovy duck embryos, 48 ~ 120 hours dead embryos in inoculation back are collected in 37 ℃ of cultivations with the production of duckling virus stain; Place 4 ~ 8 ℃ of coolings 4 ~ 24 hours, the toxic allantoic fluid of aseptic results.
6. the method for preparing of duckling virosis inactivated vaccine according to claim 4 is characterized in that step 2) concrete operations be: the toxic allantoic fluid that will gather in the crops added 0.2% ~ 0.3% of formaldehyde to total amount, in 37 ℃ of deactivations 20 ~ 28 hours.
7. the method for preparing of duckling virosis inactivated vaccine according to claim 4 is characterized in that, the concrete operations of step 3) are: the viral liquid after 20 ~ 40 parts by volume deactivations is added in the adjuvant of 60 ~ 80 parts by volume emulsifying in the high-speed homogenization machine.
8. duckling virus JM strain is delivered Chinese typical culture collection center preservation on October 14th, 2011, and deposit number is CCTCC V201131.
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| CN102925592B (en) * | 2012-11-28 | 2013-12-25 | 湖北省农业科学院畜牧兽医研究所 | Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection |
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