CN102068694A - Method for producing triple inactivated vaccine for newcastle disease, infectious bronchitis and infectious bursal disease - Google Patents
Method for producing triple inactivated vaccine for newcastle disease, infectious bronchitis and infectious bursal disease Download PDFInfo
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Abstract
The invention relates to a method for producing a triple inactivated vaccine for newcastle disease, infectious bronchitis and infectious bursal disease. In the method, the inactivated vaccine is prepared by adopting a newcastle disease virus La Sota strain, an infectious bronchitis virus M41 strain, and an Escherichia coli genetic engineering bacteria E. coil BL21/pET28a-VP2 strain which expresses an infectious bursal disease virus VP2 protein serving as production bacteria toxic species, a super concentration process and a high-quality adjuvant. After immunizing animals, antibodies are produced rapidly; the potency is high; the protection period is long; and the outbreak and the spreading of epidemic diseases are reduced.
Description
Technical field
The present invention is the preparation method of a kind of newcastle that is used to prevent chicken, infectious bronchitis, three kinds of eqpidemic disease oil emulsion vaccines of infectious bursal disease, belongs to the veterinary biologics field.
Background technology
(newcastle disease ND) has very high sickness rate and case fatality rate to newcastle disease, and all over the world have generation more, and in case outburst will bring crushing blow to poultry breeding industry, be a kind of main infectious disease of harm aviculture.OIE classifies it as category-A eqpidemic disease.(Yin Zhen, Liu Jinghua, 1997. animal virologies. Beijing. Science Press)
Infectious bronchitis of chicken (Infectious Bronchitis IB) infects and can cause chickling, future development of broilers slow, laying hen lay eggs reduce and egg product matter under degradation.OIE classifies it as category-B eqpidemic disease.(Yin Zhen, Liu Jinghua, 1997. animal virologies. Beijing. Science Press)
(Infectious bursal disease virus is a kind of special disease that is caused by the infectious bursal disease virus in the birnavirus section IBD) to infectious bursal disease, damages young chicken bursa.Feature is diarrhoea, depletion, and remarkable inflammation and increase take place earlier fabricius bursa, succeeded by atrophy, and future trouble chicken death.Primary disease often causes that also the chicken immune of recovering suppresses except that causing some chickling death, cause the immunity inoculation failure, increases the susceptibility of chickling to numerous diseases such as newcastle diseases.(Yin Zhen, Liu Jinghua, 1997. animal virologies. Beijing. Science Press)
The commercial prod kind of listing is a lot of now both at home and abroad, but all be very single traditional vaccine, repeatedly use especially inactivated vaccine of vaccine, not only increase chicken house man power and material burden, the injection of repeatedly grabbing chicken simultaneously stress, also can influence fertility performance, cause the chicken group that the susceptibility of disease is increased.The continuous variation of strain in recent years in addition, though the vaccine of the feasible multiple choices of releasing, but still the situation that epidemic situation develops appears losing control of.So force we have the poison that looses in the past, antibody titer is high, the immunoprotection phase long, influenced on the basis of oil emulsion inactivated vaccine of advantage such as little by maternal antibody develops the product that renewal better is more suitable for preventing these three kinds of present situation epidemic diseases.
Summary of the invention
The objective of the invention is to prepare three kinds of antigens of newcastle disease, infectious bronchitis, infectious bursal disease, and carry out composite newcastle disease, infectious bronchitis, three kinds of eqpidemic disease oil emulsion vaccines of infectious bursal disease made rationally, can effectively prevent the generation of newcastle disease, infectious bronchitis, infectious bursal disease, also can significantly reduce simultaneously people's labor intensity, reduce aviculture raiser's risk, and guaranteed public health.
The main related content of this trigeminy vaccine technology and the technological means that is adopted:
(1) the new strain of selection-breeding will pass through tests such as serology, molecular biology and carries out a large amount of clinical pathological material of disease Analysis and Identification work, selects the height of tiring, immunogenicity at last well and can resist the comparatively ideal seedling strain of a strain that the popular poison of infectious bronchitis of chicken is attacked.
(2) composite in conjunction with genetic engineering subunit vaccine on the basis of traditional totivirus inactivated vaccine, and guarantee that each single virus has the effect the same with single Seedling at connection Miao Zhongjun.
(3) the Millipore thickener that utilizes U.S.'s import concentrates antigen through superpower, and antibody produces fast, the height of tiring, and the protection period is long, the generation of reduction eqpidemic disease and spreading.(manufacturer of thickener, U.S. Mi Libo.Model is CUP50).
(4) technical scheme mainly may further comprise the steps:
1) producing with the bacterium kind is that the proteic bacillus coli gene engineering bacteria of IBDV VP2 is expressed in La Sota strain newcastle disease virus, M41 strain infectious bronchitis virus and E.coli BL21/pET28a-VP2 strain;
2), gather in the crops viral liquid after concentrate and inactivation technology is prepared into inactivation of viruses liquid respectively according to a conventional method with Embryo Gallus domesticus breeding La Sota strain newcastle disease virus and M41 strain infectious bronchitis virus; With the proteic bacillus coli gene engineering bacteria of the LB fluid medium culture expression IBDV VP2 E.coli BL21/pET28a-VP2 strain that adds kanamycin (the present invention or abbreviate " genetic engineering bacterium " as), be prepared into the infectious bursal disease virus VP 2 albumen of expression through broken bacterium, ammonium sulfate extraction and sterilization process;
3) prepare the deactivation Adjuvanted vaccines according to a conventional method after three kinds of inactivation antigens mixing with above preparation.
Detailed description of the present invention
1. production of vaccine is with the source of bacterium kind
(1) newcastle disease virus La Sota strain and effect inspection are all purchased No. 8 China Veterinery Drug Inspection Office in the ZhongGuanCun south Street, Haidian District, BeiJing City with the strong malicious Beijing strain of newcastle disease (CVCC AV1611 strain);
(2) No. 8 China Veterinery Drug Inspection Office in the ZhongGuanCun south Street, Haidian District, BeiJing City are purchased in avian infectious bronchitis virus M41 strain;
(3) express the proteic bacillus coli gene engineering bacteria of IBDV VP2 E.coli BL21/pET28a-VP2 strain (Chinese typical culture collection center (CCTCC) keeping, the bacterial strain deposit number is M204038, the Rong Jun of Changjiang University etc. provides the optimization of Rong Jun .IBDVVP2 gene recombination plasmid Pbv220 expression condition." animal medicine progress " the 4th phase in 2007; Rong Jun. infectious bursal disease recombinant subunit vaccine immune efficacy and safety testing. " Zhejiang agricultural sciences " the 6th phase in 2007); No. 8 China Veterinery Drug Inspection Office in the ZhongGuanCun south Street, Haidian District, BeiJing City are purchased in infectious bursal disease virus BC6-85 strain (CVCC AV7 strain);
2. produce with kind of the preparation of poison:
Newcastle disease virus La Sota strain is produced and is prepared with seed culture of viruses: seed culture of viruses is done suitably dilution (as 10 with sterile saline
-4Or 10
-5), inoculation 10 age in days SPF Embryo Gallus domesticus in the allantoic cavity, every embryo 0.1ml.Death in 72～120 hours of choosing inoculation back and the tangible Embryo Gallus domesticus of lesion are gathered in the crops Embryo Gallus domesticus liquid (allantoic fluid and amniotic fluid) respectively, are loaded in the sterilization container.To check Embryo Gallus domesticus liquid aseptic, that 1% chicken red blood cell agglutination titer is not less than 1: 512 (micromethod) to mix, quantitatively be sub-packed in the aseptic ampoule freezing preservation.Indicate harvest date, seed culture of viruses generation etc.
Avian infectious bronchitis virus M41 strain is produced and is prepared with seed culture of viruses: seed culture of viruses is done suitably dilution (as 10 with sterile saline
-2～10
-3), inoculation 10 age in days SPF Embryo Gallus domesticus in the allantoic cavity, every embryo 0.1ml.The dead Embryo Gallus domesticus and the fluid of chick embryo (allantoic fluid and amniotic fluid) of survival in 48 hours are loaded in the sterile chamber in the choosing inoculation back 30～48 hours.To check aseptic, viral level 〉=10
6.0EID
50/ 0.1ml, the negative blastochyle of 1% chicken red blood cell agglutination test is mixed, quantitatively be sub-packed in the aseptic ampoule freezing preservation.Indicate harvest date, seed culture of viruses generation etc.
Express the proteic production strain preparation of chicken infectivity bursa of Fabricius virus VP 2: first order seed breeding and evaluation are inoculated in each strain freeze-drying lactobacillus respectively in the LB fluid medium that adds kanamycin, cultivated 24 hours for 35～36 ℃, streak inoculation is cultivated on the LB solid medium that adds kanamycin then, choosing 10 of standard compliant colonies typicals is mixed in a small amount of LB culture fluid, be inoculated in some of LB agar slants, put 35～36 ℃ of cultivations 20～24 hours, as first order seed.2～8 ℃ of preservations, be no more than 14; On culture medium, go down to posterity, should be no more than for 4 generations.The secondary seed breeding is got first order seed and is inoculated in the LB culture fluid that adds kanamycin, cultivates 20～24 hours for 35～36 ℃.Put 2～8 ℃ of preservations, should be no more than 3.
3. seedling is with the preparation of viral liquid:
(1) preparation of Newcastle disease venom
Inoculation is got to produce and is used seed culture of viruses, does suitably dilution (as 10 with sterile saline
-4Or 10
-5), inoculation 10～11 age in days susceptible Embryo Gallus domesticus, every embryo 0.1ml, sealing pin hole in inoculation back is put 36～37 ℃ and is continued to hatch, and needn't turn over embryo.
After hatching and observing egg inoculation, every day is according to embryo 1 time, and dead Embryo Gallus domesticus before 60 hours is discarded.After this, the photograph embryo was 1 time in per 4～6 hours, and dead embryo takes out at any time, and to 96 hours, no matter whether death, all takes out, and air chamber upwards places 2～8 ℃ of coolings 12～24 hours.
Results are taken out refrigerative Embryo Gallus domesticus, results chick embryo allantoic liquid (receiving dead germ behind the embryo of accepting orders for repairs or processing earlier).Draw blastochyle and be put in the 50000ml sterilization container, the red cell agglutination valency is measured in sampling.Agglutination titer is lower than 1: 256, and (micromethod) person should discard.Carry out steriling test (can not transplant) by existing " Chinese veterinary drug allusion quotation " simultaneously, answer asepsis growth.2～8 ℃ of preservations, should be no more than 5 before the blastochyle deactivation of results.
The concentrated blastochyle that will gather in the crops is under 2～8 ℃ of conditions, concentrate with the ultrafiltration and concentration machine, at least concentrate 4 times, the red cell agglutination valency is measured in sampling, agglutination titer stops to concentrate when being not less than 1: 2048 (micromethod), by People's Republic of China's veterinary drug allusion quotation (Chinese veterinary drug allusion quotation committee. three ones of in 2005 versions of People's Republic of China's veterinary drug allusion quotation. Chinese agriculture publishing house, 2005, the present invention is designated hereinafter simply as " Chinese veterinary drug allusion quotation ") regulation method carry out steriling test (can not transplant), answer asepsis growth, and keep sample and survey malicious valency fully.Blastochyle after concentrating is carried out deactivation immediately.
Deactivation imports the Newcastle disease venom in the deactivation jar, is metered into 10% formalin, opens blender and stirs, and it is fully mixed, and the ultimate density of formaldehyde is 0.1%.Import in another deactivation jar after adding formalin, fail to contact inactivator to avoid near jar mouthful adherent virus.Take out 16 hours (reach 37 ℃ with temperature in the jar and pick up counting, open the blender continuous stirring) backs of 37 ℃ of deactivations, puts 2～8 ℃ of preservations, should be no more than 1 month.
(2) preparation of avian infectious bronchitis virus liquid
Inoculation is got to produce and is used seed culture of viruses, does suitably dilution (as 10 with sterile saline
-2Or 10
-3), inoculation 10～11 age in days susceptible Embryo Gallus domesticus, every embryo 0.1ml, sealing pin hole in inoculation back is put 36～37 ℃ and is continued to hatch, and needn't turn over embryo.
Hatch and observe after the egg inoculation 24 hours photograph embryos 1 time, discard dead germ.After this photograph embryo was 1 time in per 4 hours, and dead Embryo Gallus domesticus takes out at any time, and until 48 hours, no matter whether death, all takes out, and air chamber is upwards upright, places 2～8 ℃ to cool off 12～24 hours.
Results are taken out refrigerative Embryo Gallus domesticus, results chick embryo allantoic liquid (receiving dead germ behind the embryo of accepting orders for repairs or processing earlier).Draw blastochyle and be put in the 50000ml sterilization container, the sampling Detection viral level answers 〉=10
6.0EID
50/ 0.1ml.Carry out steriling test (can not transplant) by existing " Chinese veterinary drug allusion quotation " simultaneously, answer asepsis growth.2～8 ℃ of preservations, should be no more than 5 before the blastochyle deactivation of results.
Concentrate will results blastochyle under 2～8 ℃ of conditions, concentrate with the ultrafiltration and concentration machine, concentrate 4 times at least, carry out steriling test (can not transplant) by " Chinese veterinary drug allusion quotation " simultaneously, answer asepsis growth, and keep sample and survey malicious valency fully.Blastochyle after concentrating is carried out deactivation immediately.
Deactivation imports infectious bronchitis virus liquid in the deactivation jar, is metered into 10% formalin, opens blender and stirs, and it is fully mixed, and the ultimate density of formaldehyde is 0.1%.Import in another deactivation jar after adding formalin, fail to contact inactivator to avoid near jar mouthful adherent virus.Take out 16 hours (reach 37 ℃ with temperature in the jar and pick up counting, open the blender continuous stirring) backs of 37 ℃ of deactivations, puts 2～8 ℃ of preservations, should be no more than 1 month.
(3) the proteic preparation of chicken infectivity bursa of Fabricius virus VP 2
Bacterium liquid is cultivated and to be used the culture tank aerobic culture, by volume pack into 70% culture medium (the LB fluid medium that adds kanamycin) and Oleum Arachidis hypogaeae semen defoamer.The sterilization back is by 2%～4% inoculation secondary seed bacterium liquid of cultivating base unit weight, and 37 ℃ of cultivations treat that the OD600 value of bacterium liquid reaches 0.6～1.0, add the 0.2mol/L alpha-lactose, make final concentration reach 0.02mol/L, continue to cultivate 5～8h again.Beginning is ventilation in a small amount, increases tolerance gradually.
After broken bacterium is cultivated and finishes, centrifugal collection thalline.The thalline of collecting cleans 2 times with PBS liquid, the thalline of collecting is added an amount of PBS liquid carry out resuspendedly, uses ultrasonic disruption under 4 ℃.Bacterium liquid after the fragmentation, 3000r/min, centrifugal 30min collects supernatant.Behind ammonium sulfate precipitation, collect the VP2 protein liquid and carry out deactivation immediately.
Sterilization is with the formalin that adds 10% in the supernatant of collecting in proportion, the unlatching blender stirs, it is fully mixed, the ultimate density of formalin is 0.2%, then blastochyle is imported in another deactivation jar, 37 ℃ of deactivations 12 hours (reaching 37 ℃ with the pot liquid temperature picks up counting) are with deactivation remaining escherichia coli and toxin.2～8 ℃ of preservations are no more than 7; Preserve below-15 ℃, be no more than 60.
4. the preparation of inactivated vaccine:
Carry out vaccine production through after the assay was approved semi-finished product antigen.
94 parts of high-quality injection white oils are got in oil phase preparation, 2 parts of aluminium stearate, in oil phase preparation jar mix homogeneously and be heated to 80 ℃ after, Jia Siben-80 6 part again, when temperature reaches 125 ℃, kept 30 minutes, the cooling back is standby.
Water prepares the Newcastle disease venom, infectious bronchitis virus liquid, the chicken infectivity bursa of Fabricius virus VP 2 albumen mixed in equal amounts that are up to the standards, contains to make 0.1ml aqueous phase newcastle disease virus content be not less than 10
8.0EID
50, M41 strain infectious bronchitis virus content is not less than 10
6.0EID
50Aqueous phase infectious bursal disease virus VP 2 albumen is tired and was not less than 1: 16 by tiring to calculate with the fine jade expansion of infections chicken cloacal bursa virus standard positive serum.Get 4 parts of tween 80s after the sterilization, add in the Agitation Tank, add 96 parts of hybrid antigen liquid simultaneously, shake well starts stirring motor and stirred 20～30 minutes, and tween 80 is dissolved fully.
Emulsifying is got oil phase and is put into emulsion tank for 3 parts, starts motor, and slow rotation stirs, add 1 part of water simultaneously slowly after, stirred 30～40 minutes with 4000r/min again, adding 1% thimerosal solution before stopping stirring, making its ultimate density is 0.01%.After the emulsifying, get vaccine 10ml and add in the centrifuge tube, with 3000r/min centrifugal 15 minutes, the pipe end water of separating out should≤0.5ml.
The technology difference with the prior art that the present invention adopts is:
1. express the proteic bacillus coli gene engineering bacteria of chicken infectivity bursa of Fabricius virus VP 2 E.coli BL21/pET28a-VP2 strain (producing the engineering strain that gene engineering subunit vaccine of chicken's infectious bursal disease is used) and be the strain of cloning VP2 gene at the popular IBDV highly virulent strain of China with molecular biological method, be loaded on the expression plasmid carrier pET28a after gene order suitably modified, make behind the transformed into escherichia coli BL21.This recombinant bacterial strain can be expressed the VP2 albumen of infectious bursal disease virus, extract the VP2 protein Preparation and become vaccine, good immune effect, simple in production process operation, production cost is low, purity is high, safe in utilization, different with seedling process in the past, and combine the composite of genetic engineering subunit vaccine on the basis with traditional totivirus inactivated vaccine, guarantee that simultaneously each single virus has the effect the same with single Seedling at connection Miao Zhongjun.
2. concentration technology of the present invention is to utilize the Millipore thickener of U.S.'s import to concentrate; though producers more both domestic and external all adopt this type of concentration technique at present; but we have strengthened the antigen cycles of concentration in the concentration technology link of this product; through the antigen after superpower the concentrating; antibody produces fast behind the preparation vaccine; the height of tiring, the protection period is long, has reduced the generation of eqpidemic disease and spreads.
Positive effect of the present invention
The present invention relates to the production method of a kind of newcastle disease, infectious bronchitis, infectious bursal disease triple inactivated vaccine.The present invention adopts Avian pneumo-encephalitis virus La Sota strain, infectious bronchitis virus M 41 strains and expresses the proteic bacillus coli gene engineering bacteria of chicken infectivity bursa of Fabricius virus VP 2 E.coli BL21/pET28a-VP2 strain as producing the bacterium kind; superpower concentration technology and high-quality adjuvant are prepared into inactivated vaccine; antibody produces fast behind the immune animal; the height of tiring; protection period is long, has reduced the generation of eqpidemic disease and spreads.
Description of drawings:
Fig. 1 process chart of the present invention
A1 fumigation: be meant that disinfectant adopts stifling method that the couveuse of cultivating Embryo Gallus domesticus is carried out thorough disinfection.
A3 NDV, IBV inoculated into chick embryo: refer to that Embryo Gallus domesticus is inoculated newcastle disease when hatching to 10～11 ages in days, infectious bronchitis is bred in Embryo Gallus domesticus.
A4 receives poison: in the period of cultivating regulation, the Embryo Gallus domesticus liquid virus of breeding in the Embryo Gallus domesticus is gathered in the crops.
A5 virus concentrates: the toxic Embryo Gallus domesticus liquid that will gather in the crops adopts U.S. Millipore ultrafiltration and concentration machine to carry out antigenic concentrating under 2～8 ℃ of conditions, makes it improve antigenic content, and simultaneously antigen has been done further purification.
A6 surveys malicious valency: survey its antigenic blood clotting valency.
B5 fragmentation: refer to that the centrifugal back of the VP2 albumen of infectious bursa of Fabricius collects thalline, add an amount of PBS liquid and carry out resuspendedly, use ultrasonic disruption down, its endobacillary albumen is fully discharged at 4 ℃.
The C deactivation: all use an amount of formalin to carry out deactivation the antigen liquid of A group and B group, antigen active and extracellular toxin are fallen in deactivation.
The D inspection of semifinished product: be meant and carry out deactivation check, the detection of VP2 protein content, steriling test, endotoxin check, make its each index qualified when semi-finished product, just can carry out next step.
E psma ligand ratio: the psma ligand ratio that refers to the water of 3 groups of newcastle diseases, infectious bronchitis, infectious bursa of Fabricius is 1: 1: 1.
F emulsifying: refer to mix fully according to certain matching method, promptly become the vaccine state of finished product with containing antigenic water and oil phase.
Preferred forms:
Embodiment 1
(1) antigen preparation and the inspection of semifinished product:
1. the preparation of Newcastle disease venom
(1) inoculation is got to produce and is used seed culture of viruses, does suitably dilution (as 10 with sterile saline
-4Or 10
-5), inoculation 10～11 age in days susceptible Embryo Gallus domesticus, every embryo 0.1ml, sealing pin hole in inoculation back is put 36～37 ℃ and is continued to hatch, and needn't turn over embryo.
(2) hatch and observe egg inoculation after, every day is according to embryo 1 time, and dead Embryo Gallus domesticus before 60 hours is discarded.After this, the photograph embryo was 1 time in per 4～6 hours, and dead embryo takes out at any time, and to 96 hours, no matter whether death, all takes out, and air chamber upwards places 2～8 ℃ of coolings 12～24 hours.
(3) results are taken out refrigerative Embryo Gallus domesticus, results chick embryo allantoic liquid (receiving dead germ behind the embryo of accepting orders for repairs or processing earlier).Draw blastochyle and be put in the 50000ml sterilization container, the red cell agglutination valency is measured in sampling.Agglutination titer is lower than 1: 256, and (micromethod) person should discard.Carry out steriling test (can not transplant) by existing " Chinese veterinary drug allusion quotation ", answer asepsis growth.2～8 ℃ of preservations, should be no more than 5 before the blastochyle deactivation of results.
(4) the concentrated blastochyle that will gather in the crops is under 2～8 ℃ of conditions, concentrate with the ultrafiltration and concentration machine, at least concentrate 4 times, the red cell agglutination valency is measured in sampling, agglutination titer stops to concentrate when being not less than 1: 2048 (micromethod), carry out steriling test (can not transplant) by " Chinese veterinary drug allusion quotation ", answer asepsis growth, and keep sample and survey malicious valency fully.Blastochyle after concentrating is carried out deactivation immediately.
(5) deactivation imports the Newcastle disease venom in the deactivation jar, is metered into 10% formalin, opens blender and stirs, and it is fully mixed, and the ultimate density of formaldehyde is 0.1%.Import in another deactivation jar after adding formalin, fail to contact inactivator to avoid near jar mouthful adherent virus.Take out 16 hours (reach 37 ℃ with temperature in the jar and pick up counting, open the blender continuous stirring) backs of 37 ℃ of deactivations, puts 2～8 ℃ of preservations, should be no more than 1 month.
2. the preparation of avian infectious bronchitis virus liquid
(1) inoculation is got to produce and is used seed culture of viruses, does suitably dilution (as 10 with sterile saline
-2Or 10
-3), inoculation 10～11 age in days susceptible Embryo Gallus domesticus, every embryo 0.1ml, sealing pin hole in inoculation back is put 36～37 ℃ and is continued to hatch, and needn't turn over embryo.
(2) hatch and observe after the egg inoculation 24 hours photograph embryos 1 time, discard dead germ.After this photograph embryo was 1 time in per 4 hours, and dead Embryo Gallus domesticus takes out at any time, and until 48 hours, no matter whether death, all takes out, and air chamber is upwards upright, places 2～8 ℃ to cool off 12～24 hours.
(3) results are taken out refrigerative Embryo Gallus domesticus, results chick embryo allantoic liquid (receiving dead germ behind the embryo of accepting orders for repairs or processing earlier).Draw blastochyle and be put in the 50000ml sterilization container, the sampling Detection viral level answers 〉=10
6.0EID
50/ 0.1ml.Carry out steriling test (can not transplant) by existing " Chinese veterinary drug allusion quotation ", answer asepsis growth.2～8 ℃ of preservations, should be no more than 5 before the blastochyle deactivation of results.
(4) concentrate will results blastochyle under 2～8 ℃ of conditions, concentrate with the ultrafiltration and concentration machine, concentrate 4 times at least, carry out steriling test (can not transplant) by " Chinese veterinary drug allusion quotation ", answer asepsis growth, and keep sample and survey malicious valency fully.Blastochyle after concentrating is carried out deactivation immediately.
(5) deactivation imports infectious bronchitis virus liquid in the deactivation jar, is metered into 10% formalin, opens blender and stirs, and it is fully mixed, and the ultimate density of formaldehyde is 0.1%.Import in another deactivation jar after adding formalin, fail to contact inactivator to avoid near jar mouthful adherent virus.Take out 16 hours (reach 37 ℃ with temperature in the jar and pick up counting, open the blender continuous stirring) backs of 37 ℃ of deactivations, puts 2～8 ℃ of preservations, should be no more than 1 month.
3. the proteic preparation of chicken infectivity bursa of Fabricius virus VP 2
(1) bacterium liquid is cultivated and to be used the culture tank aerobic culture, by volume pack into 70% culture medium (the LB fluid medium that adds kanamycin) and Oleum Arachidis hypogaeae semen defoamer.The sterilization back is by 2%～4% inoculation secondary seed bacterium liquid of cultivating base unit weight, and 37 ℃ of cultivations treat that the OD600 value of bacterium liquid reaches 0.6～1.0, add the 0.2mol/L alpha-lactose, make final concentration reach 0.02mol/L, continue to cultivate 5～8h again.Beginning is ventilation in a small amount, increases tolerance gradually.
(2) after broken bacterium is cultivated and finishes, centrifugal collection thalline.The thalline of collecting cleans 2 times with PBS liquid, the thalline of collecting is added an amount of PBS liquid carry out resuspendedly, uses ultrasonic disruption under 4 ℃.Bacterium liquid after the fragmentation, 3000r/min, centrifugal 30min collects supernatant.Behind ammonium sulfate precipitation, collect the VP2 protein liquid and carry out deactivation immediately.
(3) sterilization is with the formalin that adds 10% in the supernatant of collecting in proportion, the unlatching blender stirs, it is fully mixed, the ultimate density of formalin is 0.2%, then blastochyle is imported in another deactivation jar, 37 ℃ of deactivations 12 hours (reaching 37 ℃ with the pot liquid temperature picks up counting) are with deactivation remaining escherichia coli and toxin.2～8 ℃ of preservations are no more than 7; Preserve below-15 ℃, be no more than 60.
4. the inspection of semifinished product
(1) Newcastle disease venom
The blastochyle of taking out before the deactivation after 1) viral level is measured and will be concentrated is carried out 10 times of serial dilutions, gets 10
-7, 10
-8, 10
-93 dilution factors, inoculation 10～11 age in days SPF Embryo Gallus domesticus are 5 in each allantoic cavity, every embryo 0.1ml puts 36～37 ℃ and continues to hatch, and shine embryo 2 every day, observed 5, no matter dead germ, the embryo of living all should be measured the red cell agglutination valency, and agglutination titer is not less than 1: 128 (micromethod) person, is judged to infection, calculate EID50, every 0.1ml viral level 〉=10
8.7EID
50Blastochyle, can be used for seedling.
2) the red cell agglutination valency is measured to get and is concentrated the preceding blastochyle of back deactivation, measures by existing " Chinese veterinary drug allusion quotation ", and its agglutination titer is not less than 1: 2048 (micromethod) person, can be used for seedling.
3) 6 of 10 age in days SPF Embryo Gallus domesticus are got in the deactivation check, inoculation inactivation of viruses liquid in the allantoic cavity, and each 0.2ml puts 36～37 ℃ and continues to hatch, and shine embryo 2 every day, observed 5, and the non-special death of Embryo Gallus domesticus should be no more than 1.All blastochyles are measured the red cell agglutination valency respectively, coagulation all should not occur, blastochyle is gathered in the crops a blind passage generation again, when still not having agglutination titer, think that deactivation is complete.
4) the steriling test blastochyle of getting deactivation is tested by existing " Chinese veterinary drug allusion quotation ", answers asepsis growth.
(2) infectious bronchitis virus liquid
The blastochyle of taking out before the deactivation after 1) viral level is measured and will be concentrated is carried out 10 times of serial dilutions, gets 10
-5, 10
-6With 10
-73 dilution factors are inoculated 5 of 10 age in days SPF Embryo Gallus domesticus in the allantoic cavity respectively, every embryo 0.1ml, and shine embryo 2 every day, observed 6.Collect Embryo Gallus domesticus after 6 days, no matter dead germ, the embryo of living (inoculate in back 24 hours except the dead person) are all weighed and are observed the Embryo Gallus domesticus pathological changes, its fetus has dehydration, crispaturas, grows little specificity lesion persons such as (comparison of inoculation fetal weight are according to more than few 2 grams of the lightest fetal weight of embryo), is judged to infection, calculates EID
50Every 0.1ml viral level 〉=10
6.7EID
50, can be used for seedling.
2) steriling test is got the viral liquid of deactivation, tests by existing " Chinese veterinary drug allusion quotation ", answers asepsis growth.
3) 6 of 10 age in days SPF Embryo Gallus domesticus are got in the deactivation check, inoculation inactivation of viruses liquid in the allantoic cavity, and each 0.2ml puts 36～37 ℃ and continues to hatch, and shine embryo 2 every day, observed 5, and the non-specific death of Embryo Gallus domesticus should be no more than 1.All Embryo Gallus domesticus are tested, should dehydration all not occur, crispatura, grow phenomenons such as little.Blastochyle is gathered in the crops a blind passage generation again, dehydration still do not occur, when rolling up, growing phenomenon such as little, think that deactivation is complete.
(3) chicken infectivity bursa of Fabricius virus VP 2 albumen
1) the VP2 content detection is according to a conventional method carried out agar gel diffusion test with the VP2 protein liquid to chicken infectivity bursa of Fabricius virus positive serum (available from China Veterinery Drug Inspection Office) with seedling, the titre of expression product VP2 (AGP tires) was not less than 1: 64 in the supernatant, can be used for seedling.
2) steriling test is undertaken by " Chinese veterinary drug allusion quotation ", answers asepsis growth.
3) the VP2 protein liquid after the endotoxin detection will be sterilized carries out endotoxin by " note " method and detects, endotoxin content is not higher than 1000EU/ml person, and (sodium chloride injection with endotoxin＜0.125EU/ml dilutes 1000 times with VP2 albumen, tachypleus amebocyte lysate box detects negative), can be used for seedling.
(2) vaccine production:
(1) 94 parts of high-quality injection white oils are got in oil phase preparation, 2 parts of aluminium stearate, in oil phase preparation jar mix homogeneously and be heated to 80 ℃ after, Jia Siben-80 6 part again, when temperature reaches 125 ℃, kept 30 minutes, the cooling back is standby.
(2) the water preparation makes 0.1ml aqueous phase newcastle disease virus content be not less than 10 the Newcastle disease venom, infectious bronchitis virus liquid, the chicken infectivity bursa of Fabricius virus VP 2 albumen mixed in equal amounts that are up to the standards
8.0EID
50, avian infectious bronchitis virus content is not less than 10
6.0EID
50Aqueous phase infectious bursal disease virus VP 2 albumen is tired and is not less than 1: 16 by tiring with the fine jade expansion of infections chicken cloacal bursa virus standard positive serum.Get 4 parts of tween 80s after the sterilization, add in the Agitation Tank, add 96 parts of hybrid antigen liquid simultaneously, shake well starts stirring motor and stirred 20～30 minutes, and tween 80 is dissolved fully.
(3) emulsifying is got oil phase and is put into emulsion tank for 3 parts, starts motor, and slow rotation stirs, add 1 part of water simultaneously slowly after, stirred 30～40 minutes with 4000r/min again, adding 1% thimerosal solution before stopping stirring, making its ultimate density is 0.01%.After the emulsifying, get vaccine 10ml and add in the centrifuge tube, with 3000r/min centrifugal 15 minutes, the pipe end water of separating out should≤0.5ml.
(4) the quantitative packing of packing seals.
Embodiment 2
Product inspection
1. character
Appearance milky white Emulsion.
The dosage form water-in-oil type.Get a cleaning suction pipe, draw a small amount of vaccine and splash in the cold water, except that the 1st, all should indiffusion.
Stability is drawn vaccine 10ml and is added in the centrifuge tube, with 3000r/min centrifugal 15 minutes, the pipe end water of separating out should≤0.5ml.
Viscosity is drawn about 25 ℃ vaccine 1ml with 1ml suction pipe (the end opening internal diameter is 1.2mm, and internal diameter suitable for reading is 2.7mm), makes its vertical outflow naturally, and record flows out the required time of 0.4ml, should be in 6 seconds.
2. steriling test is undertaken by existing " Chinese veterinary drug allusion quotation ", answers asepsis growth.
3. safety verification is with 10 of 7～14 age in days SPF chickens, and every muscle or cervical region subcutaneous injection vaccine 1ml observed 14, any part and the systemic adverse reactions that are caused by vaccine should not occur.
4. efficacy test
(1) newcastle disease part efficacy test adopts serological method to test, and when the result is against regulation, can adopt immune counteracting toxic substances method to test.
1) serological method is with 15 of 30～60 age in days SPF chickens, 10 each subcutaneous or intramuscular injection vaccine 20 μ l, 5 not immune comparing in addition.Inoculation back 21～28 days, every chicken is taken a blood sample respectively, and separation of serum carries out the HI antibody titer by existing " Chinese veterinary drug allusion quotation " and measures.The geometrical mean of immune group HI antibody titer should be not less than 1: 16 (micromethod), and the geometrical mean of not immune matched group HI antibody titer should not be higher than 1: 4 (micromethod).
2) immune counteracting toxic substances method is with 15 of 30～60 age in days SPF chickens, 10 each subcutaneous or intramuscular injection vaccine 20 μ l, 5 not immune comparing in addition.Inoculation back 21～28 days, (the CVCC AV1611 strain) 10 of every strong poison of each intramuscular injection newcastle disease virus Beijing strain of chicken
5.0ELD
50, observed 14.Matched group should be all dead, and immune group should be protected at least 7.
(2) infectious bronchitis of chicken is partly used 25 of 14～42 age in days SPF chickens, and wherein 20 each eye dripping, collunarium inoculative infection bronchitis live vaccine (H120 strain) 1 plumage part (0.05ml) are not inoculated in contrast for 5 in addition.Inoculation back 21～28 days, blood sampling respectively, separation of serum, immune chicken is distinguished intramuscular inoculation triple inactivated vaccine 0.3ml more simultaneously, and two exempt from the back took a blood sample separation of serum respectively with immune chicken and contrast chicken on 21st～28 again.Twice serum is surveyed the HI antibody titer respectively.It is high more than 4 times that the geometrical mean that immune group two is exempted from serum HI antibody titer should be exempted from serum than head, and the geometrical mean of not immune matched group serum HI antibody titer should not be higher than 1: 8 (micromethod).
(3) the following method of infectious bursal disease part efficacy test is appointed and is selected one.
1) serological method is got 20 of 21～28 age in days SPF chickens, wherein 10 each muscle or cervical region subcutaneous injection vaccine 0.2ml, other 10 in contrast not immune, support with group feeding.Inoculation back 21～28 days, every chicken is taken a blood sample respectively, and separation of serum is done the agar immunodiffusion.The immunity chicken should have at least 8 fine jades to expand to tire to be not less than 1: 8, and the contrast chicken answers total negative.
2) immune counteracting toxic substances method is got 20 of 21～28 age in days SPF chickens, wherein 10 each muscle or cervical region subcutaneous injection vaccine 0.2ml, other 10 in contrast not immune, with isolated rearing under the condition.Inoculation back 21～28 days, all immune chickens and contrast chicken, the strong malicious BC6-85 strain venom 0.1ml of infectious bursal disease (real toxic amount 〉=100 BID) of 100 times of dilutions of every eye dripping approach inoculation.Behind the counteracting toxic substances, observe the clinical manifestation of chicken every day, record morbidity and dead chicken number to 72～96 hours, are slaughtered the survival chicken, by only analysing, observe pathological changes such as fabricius bursa.The immunity chicken is should at least 8 normal, the fabricius bursa pathological changes do not occur; The contrast chicken should at least 8 morbidities, tangible fabricius bursa pathological changes (, fabricius bursa enlargement hemorrhage as chest muscle or lower limb flesh strip or atrophy, jaundice, in more than one pathological changes such as gel-shaped secretions are arranged) occurs.
5. formaldehyde, the antiseptic mercurials determination of residual amount are measured by existing " Chinese veterinary drug allusion quotation ", meet " regulation of veterinary biologics general rule ".
Note:
Gel method is checked bacterial endotoxin
The quick detection kit of Pyrosate endotoxin
The quick detection kit of Pyrosate gel method endotoxin is mainly used in the endotoxic fast detecting of finished products such as water, lysate, dialysis solution as unique in the world quick detectable of gel method endotoxin.
[composition] sample detection pipe (SPL): inside pipe wall has tachypleus amebocyte lysate
Sample positive detection control tube (PPC): inside pipe wall has endotoxin
Suction pipe
The quick detection kit of [specificity] Pyrosate gel method endotoxin only produces specificity, the false positive results that causes except (1,3)-β-D-glucans side by side to endotoxin.
The sensitivity of [sensitivity] this test kit is 1EU/ml.
The endotoxin that [effect and purposes] is used for finished products such as water, lysate, dialysis solution detects.
[storage] room temperature preservation, effect duration is 120 months.
[specification] 10 times/box.
Formula is stated in operation:
(1) the used utensil of preheating in 37 ℃ of incubators.
(2) with test sample with sodium chloride injection (endotoxin＜0.125EU/ml) suitably be diluted to different multiples, to be measured.
(3) test sample that 0.5ml is diluted different multiples carefully adds sample detection pipe (SPL), and mixing gently.
(4) test sample of drawing 0.25ml carefully with pyrogen-free suction pipe from the sample detection pipe adds sample positive detection pipe (PPC), mixes gently 60 seconds, gets final product.
(5) sample detection pipe behind the complete mixing and sample positive detection pipe are positioned over 37 ℃ of cultivation 30min.
(6) be inverted sample detection pipe and sample positive detection pipe, observation has or not coagulation.
(7) declaring test when the complete coagulation of positive control detector tube (PPC) sets up.
(8) if the sample detection pipe has agglutination, endotoxic content 〉=1.0EU/ml in this dilution test sample is described; If sample detection Guan Zhongwu agglutination illustrates endotoxin content＜1.0EU/ml in this dilution test sample.Calculate endotoxin content in the former test sample according to dilution factor.
Claims (3)
1. a newcastle disease, infectious bronchitis, infectious bursal disease triple inactivated vaccine is characterized in that the infectious bursal disease virus VP 2 albumen and the vaccine adjuvant of La Sota strain newcastle disease virus, M41 strain infectious bronchitis virus and E.coli BL21/pET28a-VP2 strain bacillus coli gene engineering bacterium expression that this vaccine main component is deactivation.
2. the production method of a newcastle disease, infectious bronchitis, infectious bursal disease triple inactivated vaccine is characterized in that:
(1) producing with the bacterium kind is La Sota strain newcastle disease virus, M41 strain infectious bronchitis virus and the proteic bacillus coli gene engineering bacteria of expression chicken infectivity bursa of Fabricius virus VP 2 E.coli BL21/pET28a-VP2 strain;
(2), gather in the crops viral liquid after concentrate and inactivation technology is prepared into inactivation of viruses liquid respectively according to a conventional method with Embryo Gallus domesticus breeding La Sota strain newcastle disease virus and M41 strain infectious bronchitis virus; With the proteic bacillus coli gene engineering bacteria of the LB fluid medium culture expression chicken infectivity bursa of Fabricius virus VP 2 E.coli BL21/pET28a-VP2 strain that adds kanamycin, be prepared into the infectious bursal disease virus VP 2 albumen of expression through broken bacterium, ammonium sulfate extraction and sterilization process;
(3) prepare the deactivation Adjuvanted vaccines according to a conventional method after three kinds of inactivation antigens mixing with above preparation.
3. as the production method of a kind of newcastle disease, M41 strain infectious bronchitis, infectious bursal disease triple inactivated vaccine as described in the claim 2, it is characterized in that three kinds of inactivation antigen mixed in equal amounts, contain and make 0.1ml aqueous phase newcastle disease virus content be not less than 10
8.0EID
50, M41 strain infectious bronchitis virus content is not less than 10
6.0EID
50Aqueous phase infectious bursal disease virus VP 2 albumen is tired and is not less than 1: 16 by tiring with the fine jade expansion of infections chicken cloacal bursa virus standard positive serum.
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