CN104069487A - Preparation method of identifiable inactivated vaccine for animals - Google Patents
Preparation method of identifiable inactivated vaccine for animals Download PDFInfo
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- CN104069487A CN104069487A CN201410225569.8A CN201410225569A CN104069487A CN 104069487 A CN104069487 A CN 104069487A CN 201410225569 A CN201410225569 A CN 201410225569A CN 104069487 A CN104069487 A CN 104069487A
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Abstract
The invention discloses a preparation method of identifiable inactivated vaccine for animals, and relates to the technical field of animal vaccines, in a preparation process, a gene engineering method, a natural subculture method or a chemical synthesis method is adopted to reduce one or multiple segments of genes from antigenic substance to generate new properties, and the antigenic substance with new properties is prepared into the identifiable inactivated vaccine. The preparation method disclosed by the invention has the beneficial effects that after the vaccine is inoculated to an animal, an antibody can be verified by detecting the properties, by verifying whether the antibody is generated by the non-treated antigen (namely, wild virus) or by treated antigen (namely, vaccine virus), the antibodies generated by the vaccine and the wild virus can be distinguished to diagnose whether the animal is infected with wild virus strain, so as to eliminate and remove animals with virus to eliminate diseases, thereby being beneficial to eliminate diseases in farms.
Description
Technical field
The present invention relates to animal vaccine technical field, be specifically related to the preparation method that can differentiate inactivated vaccine for a kind of animal.
Background technology
Animal diseases are more and more now, and vaccine has become prevention and controlled the main tool of disease, and attenuated live vaccine occurs and controls outbreak of communicable diseases and played very important effect in infection prevention disease.But, using attenuated live vaccine to produce the safety problem by bacterium kind, is that Long term Animal is being used attenuated live vaccine may occur that vaccine strain is residual, and gene recombinaton, genovariation, virulence are returned the phenomenons such as strong, cause disease again popular, be unfavorable for purification and the removing of infectious disease.
The play vital effect of animal vaccine to prevention Animal diseases, domestic and international many infectious diseases are all prevented and are controlled by immunity inoculation vaccine, but carry out disease purification and remove needing to differentiate vaccine especially identifiable inactivated vaccine.
Summary of the invention
One of object of the present invention is in order to make up the deficiencies in the prior art, provides a kind of and can effectively eliminate and remove with malicious animal, realizes the preparation method that can differentiate inactivated vaccine for animal that disease purifies.
The technical scheme that the present invention realizes its object employing is:
The preparation method that can differentiate inactivated vaccine for a kind of animal, described preparation process is for adopting gene engineering method, natural propagating method or chemical synthesis process, cause antigenic substance to reduce one or more snippets gene, and then produce new characteristic, the more described antigenic substance with new features is prepared into and can differentiates inactivated vaccine.
As preferred technical scheme: described antigenic substance is antibacterial, virus, parasite, mycoplasma or chlamydia.
As preferred technical scheme: described virus is Pseudorabies virus, swine fever virus, pig circular ring virus, foot and mouth disease virus, reproductive and respiratory syndrome poison, epidemic diarrhea virus, bird flu virus, egg drop syndrome virus, duck plague virus or DHV.
As preferred technical scheme: described antigenic substance can be to have immunogenic protein or synthetic peptide.
As preferred technical scheme: described gene engineering method is by one section of antigenic substance or multistage gene knockout or importing, by expressing the new characteristic of rear generation, and keep the good cultural character of antigen and immunogenicity constant.
As preferred technical scheme: described natural method, for being cultivated and caused antigenic substance to lack one or more snippets gene by continuous passage in cell, embryo, animal body, produces new characteristic after expressing, and keep antigen well cultivation property and immunogenicity constant.
As preferred technical scheme: describedly differentiate that inactivated vaccine can be produced into emulsion inactivated vaccine by emulsifying process, or make lyophilizing inactivated vaccine by lyophilization.
As preferred technical scheme: described Emulsion inactivated vaccine is for containing oil adjuvant killed vaccine.
As preferred technical scheme: describedly comprise oil-in-water type, water-in-oil type, W/O/W type containing oil adjuvant killed vaccine, wherein oil comprises mineral oil or vegetable oil.
As preferred technical scheme: described Emulsion inactivated vaccine is hydrophilic dosage form inactivated vaccine, described hydrophilic dosage form inactivated vaccine adopts solvent to comprise aluminium hydroxide gel, aluminum phosphate colloid, propolis, high molecular polymer, resin.
The final stage of infectious disease prevention and control is purification and removings of disease; this stage needs to use inactivated vaccine; especially with the identifiable inactivated vaccine of feature; after this type of vaccination animal, not only can produce strong protection; and can differentiate antibody by detecting; can whether infect wild poison by Differential Diagnosis animal, thereby can remove and eliminate and be with malicious animal, reach the object that animal population disease purifies.Though the needed bacterium/seed culture of viruses of inactivated vaccine is bacterium/seed culture of viruses that virulence is strong, owing to preparing vaccine process by the thorough deactivation of bacterium kind, therefore, in use there will not be gene recombinaton, genovariation, virulence to return the phenomenons such as strong.In the situation that not affecting Virus culture characteristic, immunogenicity, immune efficacy, by gene engineering method and natural propagating method, production of vaccine can be differentiated with characteristic by bacterium kind or protein, synthetic peptide etc., be produced into again and can differentiate inactivated vaccine, can produce the antibody that can differentiate that thing is corresponding.Animal immune is inoculated after such vaccine, and detection can be differentiated antibody, can distinguish the antibody that vaccine and wild poison produce, and diagnoses out infection to cross the animal of wild poison, thereby eliminates and malicious animal is with in removing, is conducive to the purification of plant's disease.
Production of vaccine is identical with conventional method with bacterium (poison) strain cultural method, just by screening and optimizing, go out the best condition of culture of differentiating rear vaccine bacterium (poison) strain, then according to the quality standard of vaccine, the stock solution of cultivating is diluted to required antigenic content, carry out deactivation, screen suitable immunological adjuvant, according to best proportion, be mixed with inactivated vaccine.Certainly safety verification and efficacy test will meet the requirements.
The beneficial effect that the present invention has:
Animal immune is inoculated after such vaccine, by detected characteristics, can differentiate antibody, differentiate that this antibody is that after being produced or processed by the antigen before processing (i.e. wild poison), antigen (vaccine virus) produces, can distinguish the antibody that vaccine and wild poison produce, whether diagnosis animal infected street strain, thereby malicious animal is with in superseded and removing, realize disease and purify, be conducive to the purification of plant's disease.
The specific embodiment
Below in conjunction with embodiment, the invention will be further described, but protection scope of the present invention is not only confined to embodiment.
Embodiment 1
PRV (Pseudorabies virus) can be differentiated the preparation of inactivated vaccine:
The separation of the new epidemic isolates of pseudorabies: gather the tissues such as brain and lung, kidney from the piglet sterile working of pig farm Mortality, after mixing, in the mortar of sterilizing, shred, according to said method processed the pathological material of disease of 3 piglets, with DMEM, made 1:5 and be doubly diluted to suspension ,-70 ℃ of multigelations 3 times.Through 3000r/min centrifugal 30 minutes, get supernatant after 0.22 μ m filtering with microporous membrane, add appropriate penicillin and streptomycin, be inoculated in the culture bottle of the BHK-21 cell that grows up to monolayer, be placed in 37 ℃ of calorstat absorption 1 hour, then add DMEM maintenance medium to cultivate and within 72 hours, receive poison.Purification, be accredited as Pseudorabies virus.
GE knocks out: transform first 2 hours and on LB flat board, be coated with in x-gal (40 μ g/ml) and 37 ℃ of incubators of IPTG (20 μ g/ml) and be inverted plate, make X-gal and IPTG fully suck in agar, from-70 ℃ of refrigerators, take out 2 pipe competent cells, after thawing, immediately pipe is transferred in ice bath, place after 10 minutes, in ice bath, plasmid to be transformed (volume <10 μ l will be added in pipe, <50ng), rotation mixes gently, on dry ice, place 30 minutes, then proceed to rapidly in 42 ℃ of water-baths thermal shock 90 seconds, proceed to rapidly again in ice bath cooling 2 minutes, the LB fluid medium that adds 400 μ l37 ℃ preheatings, 37 ℃ of shaking tables slowly shake (250 revs/min) incubation 45 minutes, on the LB flat board that coating contains Amp (50ug/m1) respectively, incubated overnight in 37 ℃ of incubators.Recombiant plasmid pBsA is transformed to DH5, and the single bacterium colony of picking, by LB fluid medium propagation, extracts plasmid, and BamHI enzyme action, reclaims the fragment that size is about 6.6kb.This PRV6.6kbDNA fragment is connected in pUC18BamHI site, obtains recombiant plasmid pUc6.6, continue, with this PRV6.6kb DNA fragmentation of Kpnl enzyme action, to reclaim PRV4.lkb fragment, as vaccine strain simultaneously.
This can be differentiated to the cells such as vaccine strain inoculation PK15, BHK, VERO, ST are cultivated, 24～48 hours results virus-culturing fluids, then in culture fluid, add 0.1%～0.5% inactivator, shake up, put 2～8 ℃ 48 hours.Check after complete inactivation of viruses liquid, virus is also mixed with suitable adjuvant or freeze drying protectant, subpackage carries out emulsifying or lyophilizing, is prepared into semi-finished product, and then subpackage, is finished product after the assay was approved, puts 2～8 ℃ of preservations.
Embodiment 2
Swine fever can be differentiated the preparation of inactivated vaccine:
The vaccine of prevention swine fever is C strain live vaccine at present, and immune effect is good, but can not distinguish with street strain.On the basis of C strain, raq gene manually knocks out, and makes it lack 54 aminoacid, JiB/C district 693～746.After knocking out, do not affect cultivation and immunogenicity and the effect of this strain, can resist the attack of the strong poison of swine fever.
This can be differentiated to swine Fever Vaccine strain inoculation ST cell, bovine testicle cell etc. are cultivated, 36～48 hours results virus-culturing fluids, then in culture fluid, add 0.1%～0.5% inactivator, shake up, put 2～8 ℃ 48 hours.Check after complete inactivation of viruses liquid; virus liquid after deactivation and freeze drying protectant are mixed according to volume ratio 1:1; divide and install in peace glass bottle (2ml); according to suitable freeze-drying curve; be lyophilized into powdery, be prepared into semi-finished product, according to quality standard, do steriling test, exogenous virus check, safety examination and efficacy test; be finished product after the assay was approved, put 2～8 ℃ of preservations.
Embodiment 3
Streptococcus can be differentiated the preparation of inactivated vaccine:
Upstream and downstream homology arm P1 and P2 and the full length sequence thereof of srtA gene have increased, utilize responsive to temperature type " suicide " plasmid pSET4s to build recombiant plasmid pSET4s-P1-P2, and this plasmid electricity is transformed in wild strain SS2 (SC21), by antibiotic and temperature dual, screen, obtain srtA gene delection bacterial strain, deletion mutation bacterial strain can genetic stability, does not affect cultural character, still possesses good immunogenicity.
The cultivation of vaccine, deactivation, join Seedling, identical with conventional vaccine manufacture method.
Vaccine of the present invention is checked through following: steriling test, exogenous virus check, safety examination, efficacy test be qualified can use all.
Finally it should be noted that: above embodiment is only in order to illustrate the present invention and unrestricted technical scheme described in the invention; Therefore, although this description has been described in detail the present invention with reference to each above-mentioned embodiment,, those of ordinary skill in the art should be appreciated that still and can modify or be equal to replacement the present invention; And all do not depart from technical scheme and the improvement thereof of the spirit and scope of the present invention, it all should be encompassed in claim scope of the present invention.
Claims (9)
1. the preparation method that can differentiate inactivated vaccine for an animal, it is characterized in that, described preparation process is for adopting gene engineering method, natural propagating method or chemical synthesis process, cause antigenic substance to reduce one or more snippets gene, and then produce new characteristic, the more described antigenic substance with new features is prepared into and can differentiates inactivated vaccine.
2. the preparation method that can differentiate inactivated vaccine for animal according to claim 1, is characterized in that, described antigenic substance is antibacterial, virus, parasite, mycoplasma, chlamydia, has immunogenic protein or synthetic peptide.
3. animal use according to claim 2 can be differentiated the preparation method of inactivated vaccine, it is characterized in that, described virus is Pseudorabies virus, swine fever virus, pig circular ring virus, foot and mouth disease virus, reproductive and respiratory syndrome poison, epidemic diarrhea virus, bird flu virus, egg drop syndrome virus, duck plague virus or DHV.
4. animal use according to claim 1 can be differentiated the preparation method of inactivated vaccine, it is characterized in that, described gene engineering method is by one section of antigenic substance or multistage gene knockout or importing, by expressing the new characteristic of rear generation, and keep the good cultural character of antigen and immunogenicity constant.
5. animal use according to claim 1 can be differentiated the preparation method of inactivated vaccine, it is characterized in that, described natural method is for being cultivated and caused antigenic substance to lack one or more snippets gene by continuous passage in cell, embryo, animal body, after expressing, produce new characteristic, and keep antigen well cultivation property and immunogenicity constant.
6. the preparation method that can differentiate inactivated vaccine for animal according to claim 1, is characterized in that, describedly differentiates that inactivated vaccine can be produced into emulsion inactivated vaccine by emulsifying process, or makes lyophilizing inactivated vaccine by lyophilization.
7. the preparation method that can differentiate inactivated vaccine for animal according to claim 6, is characterized in that, described Emulsion inactivated vaccine is for containing oil adjuvant killed vaccine.
8. the preparation method that can differentiate inactivated vaccine for animal according to claim 8, is characterized in that, the described oil adjuvant killed vaccine that contains comprises oil-in-water type, water-in-oil type, W/O/W type, and wherein oil comprises mineral oil or vegetable oil.
9. animal use according to claim 7 can be differentiated the preparation method of inactivated vaccine, it is characterized in that, described Emulsion inactivated vaccine is hydrophilic dosage form inactivated vaccine, and described hydrophilic dosage form inactivated vaccine adopts solvent to comprise aluminium hydroxide gel, aluminum phosphate colloid, propolis, high molecular polymer, resin.
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| CN104928261A (en) * | 2015-07-03 | 2015-09-23 | 江苏省农业科学院 | Pseudo-rabies virus LA-A strain and establishing method and application thereof |
| CN105010235A (en) * | 2015-08-14 | 2015-11-04 | 湖南新南方养殖服务有限公司 | Method for cleaning pig farm to prevent diseases |
| CN105462840A (en) * | 2015-12-10 | 2016-04-06 | 浙江卫信生物药业有限公司 | Method for preparing living freeze-dried body of mycoplasma orale |
| CN113238048A (en) * | 2021-05-11 | 2021-08-10 | 上海真测生物科技有限公司 | Diagnostic marker and application thereof in distinguishing new coronavirus infection and new coronavirus inactivated vaccination |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104928261A (en) * | 2015-07-03 | 2015-09-23 | 江苏省农业科学院 | Pseudo-rabies virus LA-A strain and establishing method and application thereof |
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| CN105010235A (en) * | 2015-08-14 | 2015-11-04 | 湖南新南方养殖服务有限公司 | Method for cleaning pig farm to prevent diseases |
| CN105462840A (en) * | 2015-12-10 | 2016-04-06 | 浙江卫信生物药业有限公司 | Method for preparing living freeze-dried body of mycoplasma orale |
| CN113238048A (en) * | 2021-05-11 | 2021-08-10 | 上海真测生物科技有限公司 | Diagnostic marker and application thereof in distinguishing new coronavirus infection and new coronavirus inactivated vaccination |
| CN113238048B (en) * | 2021-05-11 | 2024-03-15 | 抗码(苏州)生物科技有限公司 | Diagnostic markers and their use in differentiating between new coronavirus infection and new coronavirus inactivated vaccination |
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