CN105154455A - Swine fever virus gene recombinant adenovirus lyophilized vaccine and preparation method thereof - Google Patents
Swine fever virus gene recombinant adenovirus lyophilized vaccine and preparation method thereof Download PDFInfo
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- CN105154455A CN105154455A CN201510677195.8A CN201510677195A CN105154455A CN 105154455 A CN105154455 A CN 105154455A CN 201510677195 A CN201510677195 A CN 201510677195A CN 105154455 A CN105154455 A CN 105154455A
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Abstract
The invention aims to provide a swine fever virus gene recombinant adenovirus lyophilized vaccine. The vaccine antigen is prepared by the following steps: artificially screening gene segments which are beneficial to preparing the vaccine according to different characteristics of swine fever virus natural protective antigens, modifying and connecting to obtain a new antigen gene segment; and carrying out homologous recombination on the new antigen gene segment to the human 5-type replication-defective adenovirus vector. The vaccine antigen has favorable immunization effect, and ensures the safety of the vaccine due to the characteristic of replication defect. The heat-resistant protective agent can maximally reduce the damage of various physicochemical factors to the virus activity in the product subpackaging and lyophilization process, and reduce the virus titer loss before and after lyophilization. The heat-resistant protective agent live vaccine has high antiaging degree; the virus loss rate does not exceed 0.2 titer after the vaccine is stored at 37 DEG C for 10 days; and after the vaccine is stored at 2-8 DEG C for 36 months, the vaccine titer still does not change obviously, thereby effectively prolonging the storage life of the vaccine.
Description
Technical field
The invention belongs to virus vaccines preparing technical field, be specifically related to a kind of Pestivirus suis gene recombinant adenovirus freeze dried vaccine and preparation method thereof.
Background technology
Swine fever (CSF) is the high degree in contact sexually transmitted disease of the boar caused by Pestivirus suis (Classicalswinefevervir μ s, CSFV), and mortality ratio is high, causes huge financial loss to world's pig industry.Swine fever is classified as domestic animal category-A transmissible disease by OIE (OIE), and China is also classified as a class animal epidemic.Clinically occur bleeding profusely a little for feature to delay high fever, skin and mucous membrane.Practical experience for many years proves, vaccine inoculation is the Main Means of prevention and corntrol animal epidemic.The sixties in 20th century, China develops fever virus lapinized Chinese Strain (also known as the C strain) vaccine obtained through induced mutations and serve decisive role in CSFV prevention and control, effectively controls swine fever popular on a large scale in China and even world wide.Swine fever C strain attenuated vaccine is current domestic and international widely used vaccine, and its security and immune efficacy are by social recognition.But it disturbs by maternal antibody, cause there is report during the phenomenon of immuning failure, the antibody that the antibody inoculating the generation of this attenuated vaccine cannot produce with wild virus infection is distinguished mutually, also bring huge challenge to the formulation of immunity and national swine fever prevention and control purification, govern China live pig and the Trade Development of pork product in nonimmune countries and regions.
Summary of the invention
The object of this invention is to provide a kind of Pestivirus suis gene recombinant adenovirus freeze dried vaccine and preparation method thereof, i.e. a kind of heat resistant type Pestivirus suis gene recombinant adenovirus freeze dried vaccine.
The present invention first provide a kind of can for the preparation of the gene fragment of Pestivirus suis gene recombinant adenovirus, its nucleotides sequence is classified as SEQIDNO:1; The aminoacid sequence made new advances of encoding is SEQIDNO:2, makes its antigenicity better.
The present invention also provides a kind of Pestivirus suis gene recombinant adenovirus, is above-mentioned gene fragment homologous recombination to be prepared to the genomic multiple clone site of people 5 type replication-defective adenoviral.
Pestivirus suis gene recombinant adenovirus prepared by the present invention is for the preparation of vaccine;
The present invention provides a kind of freeze dried vaccine on the other hand, is add vaccine heat resisting protective to prepare as antigen with above-mentioned Pestivirus suis gene recombinant adenovirus;
Vaccine protectant composed as follows used: 2.5 ~ 3.5%NZ-amine, 0.2 ~ 0.4% monopotassium glutamate salt, 15 ~ 25% sucrose, 1.5 ~ 2.5% lactoalbumin hydrolysates, 4 ~ 6% gelatin, 2 ~ 8% glycerine; Surplus is water.
Also add penicillin, Streptomycin sulphate and anti-mycoplasma drug in above-mentioned protective material, its final concentration is respectively penicillin 100I Μ/ml, Streptomycin sulphate 80 μ g/ml, and anti-mycoplasma drug is 5 μ g/ml.
Above-mentioned heat-resisting lyophilized protecting agent, its preparation method is as follows:
1) first take NZ-amine, monopotassium glutamate salt, sucrose and lactoalbumin hydrolysate, after dissolving, filtered liquid is made in filtration sterilization; Millipore filtration aperture used is 0.1 μm;
2) taking gelatin joins in 70-80 DEG C of water for injection, stir until dissolve completely, add 2-8% glycerine together autoclaving make gelatin solution; It is 25% that the concentration of gelatin is preferably W/V; Autoclave conditions is 116 DEG C, 20min;
3) by step 1) filtered liquid prepared and step 2) gelatin solution 4:1 mix after, make lyophilized vaccine.
The present invention's vaccine antigen used is the different qualities according to Pestivirus suis natural protection antigen, manually the gene fragment being conducive to preparing vaccine carried out screening and transform the antigen gene fragment connecting and make new advances, homologous recombination is prepared from people 5 type replication-defective adenoviral vector, this vaccine antigen good immune effect, meanwhile, the characteristic of its replication defective ensure that the safety of vaccine; The present invention's heat resisting protective used can reduce goods various chemical factors in packing and freeze-drying process to greatest extent, to the damage of virus activity, to be made the loss of tiring of virus before and after freeze-drying less, is no more than 1 titre; The ageing-resistant degree of heat resisting protective living vaccine of the present invention is high; the rate of loss of preserving 10 days virus under 37 DEG C of conditions is no more than 0.2 titre; preserve after 36 months under 2 ~ 8 DEG C of conditions, still there is not obvious change in vaccine valence, thus effectively extends the storage life of vaccine.Heat-resisting lyophilized protecting agent of the present invention not only resistance toheat good, can the activity of available protecting virus, but also there is system component and collocation method is simple, cost is low, can realize the advantages such as production in enormous quantities, reduce production cost.
Embodiment
Applicant is on the Research foundation of live vaccines of hog cholera, the infective molecule cloning that utilized human adenovirus type 5 genome as framework construction, insert in its genomic multiple clone site district filter out the gene antigen Predominance Area (totally 585 amino acid) of CSFV.The recombinant virus obtained passed for 60 generations continuously on cell; there is not any sudden change or loss in the exogenous genetic fragment inserted; and by Immunofluorescence test to foreign gene stably express; and the protectiveness provided and spleen drench seedling indifference; and there is adenovirus characteristic gene; can distinguish mutually with vaccine virus and wild virus infection, the differential diagnostic method simultaneously distinguishing CSFV and spleen pouring seedling (comprising wild poison) is set up.This vaccine is once come into operation, not only can distinguish vaccine immunity animal and wild virus infection animal by antibody authentication technique, greatly promote purification or the elimination of CSFV, the survival rate of pig can be improved, increase the breeding stock of pig and the amount of delivering for sale, the supply of livestock product relevant on Pork Market and stable price are had great importance.
Freeze-dried live vaccine is one of main vaccine of prevention livestock and poultry transmissible disease, and lyophilized vaccine mainly sucrose, skim-milk and sucrose, the simple proportioning of gelatin that intradermal vaccine enterprise of state is conventional, prescription is simple, and protectiveness is poor.If preserved under 2 ~ 8 DEG C of conditions, preservation period only has short 3 ~ 6 months, approximately needs to preserve under-20 DEG C of conditions.This just requires that vaccine carries out as far as possible under cryogenic in preservation, transport and use procedure, otherwise tiring of vaccine will be had a greatly reduced quality.In production of vaccine, transport and use procedure, if the cold chain system control of any one link is improper, hot environment will cause vaccine potency to decline, and finally causes the popular of epidemic disease because of immuning failure.Lyophilized vaccine may affect biological products quality, tire and stability; heat resisting protective has immunocompetence and parmacodynamics-less activity; it not only has the active effect of protection product biological; and there is the effect of vehicle and antioxidant; directly be associated with the titre of vaccine and stability; the stability of vaccine can be maintained when freeze-drying is preserved; guarantee that goods are under 2 ~ 8 DEG C of conditions; preservation period is 36 months; tire without obviously declining, even if also more than ten days can be preserved under 37 DEG C of conditions.Thus solve conventional vaccine in long-distance transport, long-term to preserve and in use procedure, thermo-labile, that storing temp is low, preservation period is short and harsh freezing conditions causes the difficult problems such as mass energy waste.But the lyophilized vaccine used at present is used for the preservation effect of Pestivirus suis gene recombinant adenovirus vaccine and bad.
In vaccine freeze-drying protective agent preparation field, the selection of protective material component and the preservation effect of proportioning to vaccine have material impact.Preserve for a long time to enable Pestivirus suis gene recombinant adenovirus vaccine of the present invention; applicant has carried out long-term research to the component of lyophilized vaccine of the present invention; and the matched proportion density optimized to each other, thus make that there is between each component maximum composite effect.
For the component that lyophilized vaccine of the present invention uses, its character is as follows:
1.NZ amine (NZ-AmineAS), is also called the enzymically hydrolyse thing of sieve albumen;
2. monopotassium glutamate salt (Monopotassium L-Glutamate): be a kind of tensio-active agent, the tension force at interface can be reduced, freeze with dehydration in can reduce freezing and deformation of dewatering caused by ice, water interfacial tension, in reconstitution process, wetting agent effect can be played to active ingredient again.
3. lactoalbumin hydrolysate is also known as hydrolyzing lactoalbumin, is the product of protease hydrolysis condensed whey protein or opalescin, containing enough necessary amino acid.
4. sucrose has osmosis, and harmful microorganism can be suppressed to grow, and extends product preservation term, has well water-soluble.Microbial survival rate, forms homogeneous suspension, plays moisture mitigation, can prevent active ingredient sex change.
Be described below for lyophilized vaccine component of the present invention, NZ-amine, monopotassium glutamate salt and the equal available from Sigma of gelatin, lactoalbumin hydrolysate is purchased from GIBCO, and sucrose is purchased from Merck KGaA company.
Pestivirus suis gene recombinant adenovirus of the present invention is that laboratory utilizes people 5 type replication-defective adenoviral vector to connect the gene constructed new generation vaccine strain of Pestivirus suis.
Below freeze dried vaccine of the present invention is described in detail.
Embodiment 1: the preparation of Pestivirus suis gene recombinant adenovirus
By analyzing the genome sequence of the strong poison (CSFV) of swine fever crossdrift system, obtain and have antigenic nucleotide fragments, its nucleotides sequence is classified as SEQIDNO:1, and the aminoacid sequence of coding is SEQIDNO:2.The gene clone of screening is entered PET32a (+) polyclone restriction enzyme site, transform DH5a intestinal bacteria, be coated with ammonia benzyl resistant panel, picking mono-clonal, order-checking qualification.Select the positive bacterium colony that order-checking is correct, picking mono-clonal, order-checking qualification.
By goal gene directed cloning in shuttle vector pAdTrack-CMV, adopt " two-step conversion method " at endotoxin test method, construct the recombinant adenovirus metastasis transplanting physique grain pAdEasy-E02 carrying goal gene, after PacI linearization for enzyme restriction, Human embryonic's nephrocyte (HEK293), successfully packs out the recombinant adenovirus of Pestivirus suis goal gene.
Embodiment 2: the preparation of lyophilized vaccine
The formula table (point A liquid and B liquid two portions, wherein the content concn of component is quality concentration of volume percent g/ml) of each embodiment of table 1 heat-resisting lyophilized protecting agent of the present invention
1) A liquid preparation: take NZ-amine, monopotassium glutamate salt, sucrose, lactoalbumin hydrolysate respectively, above composition is dissolved in order in 3500ml water for injection, shake up after abundant dissolving, add water for injection to 4000ml, shake up, degerming with 0.1 μm of membrane filtration, save backup in 37 DEG C of greenhouses.
2) B liquid preparation: get gelatin, after fully dissolving with the water for injection of 800ml70-80 DEG C, glycerol adding also shakes up after adding water for injection to 1000ml, 121 DEG C, 15 pounds autoclavings 20 minutes, are kept at after sterilizing in 37 DEG C of greenhouses and save backup.
3) A liquid and B liquid are carried out proportioning, mix and be heat-resisting lyophilized protecting agent.
Embodiment 3: preparation Pestivirus suis recombinant adenovirus freeze dried vaccine
The preparation of 1 recombinant adenovirus venom
The recovery of 1.1HEK293 cell, cultivate and take out HEK293 cell cryopreservation tube rapidly from liquid nitrogen container, put rapidly fast melt in 37 DEG C of water-baths.Cell in each cryopreservation tube is transferred to and fills 5ml cell growth medium (DMEM90%; New-born calf serum 10%, adjust pH is to 7.2-7.4.) centrifuge tube in, with 1000r/min centrifugal 5 minutes, discard supernatant liquor.With 10ml cell growth medium suspension cell, put 37 DEG C, containing the adherent static gas wave refrigerator of 5%CO2 incubator.After within 24 ~ 48 hours, forming good individual layer, discard growth media, wash cell face 2 times with PBS, with cell dissociation buffer (0.25% pancreatin-0.02%EDTA), cell dissociation is got off, in time there is the gap of needle point size in cell face, cell dissociation buffer is abandoned in suction, pats cell bottle, and cell evenly comes off along bottle wall, now draw cell growth medium with suction pipe, blow and beat cell dispersion gently, make cell dispersal be individual cells, by the cell suspension of dispersion by 1; The ratio of 2 ~ 1:3 carries out enlarged culturing.
The breeding of 1.2 viruses: when cell covers with the 80-90% of bottle wall, is not less than 10 by every 1ml viral level
7.0after recombinant adenovirus seed culture of viruses serum-free DMEM prepared by the embodiment 1 of IF Μ dilutes 50 times, be inoculated on HEK293 cell monolayer, supply cell maintenance medium (DMEM98%; New-born calf serum 2%, adjust pH is to 7.2-7.4.) put 37 DEG C, continue cultivation 48 ~ 72 hours containing 5%CO2 incubator, the specific lesions such as the refractive index of cell lowers, cell circle contracts, come off should be there is, when cytopathy reaches 80-90%, results Pestivirus suis gene recombinant adenovirus liquid, saves backup in-20 DEG C of freezers.
The recombinant adenovirus poisons steriling test of results and viral level measure by 2; the ratio of a qualified virus liquid part and the heat-resisting lyophilized protecting agent prepared 4:1 by volume mixes; another part mixes with 5% sucrose milk protective material; and this adds penicillin and streptomycin and anti-mycoplasma drug respectively in proportion; fully shake up; quantitative separating, carries out vacuum freezedrying rapidly.The vaccine cooing protective material freeze-drying of milk sucrose is control group conventional vaccine.
Point virus liquid installed loads in freeze-drying cabinet by 3, choose that pre-freezing temperature is-50 DEG C, pre-freeze time cryogenic hold-time be 2h, pre-freeze beforehand control at-15 DEG C, quick freezing after product release is antipyretic: sublimation stage product temperature is-33 DEG C, flaggy temperature is set as-8 DEG C, sublimation time is 11h; Desorption temperature is 26 DEG C, desorption time is 4h, and freeze-drying whole process is 24h, outlet of jumping a queue, and obtains heat resistant type Pestivirus suis gene recombinant adenovirus living vaccine.
The indices test of freeze-drying prods prepared by embodiment
Before and after 1 freeze-drying, viral level measures and seed culture of viruses is made 10 times of serial dilutions by finished product vaccine serum-free DMEM, gets 10
-3, 10
-4, 10
-5, 10
-6, 10
-75 each 0.1ml of extent of dilution, respectively inoculating cell density 2.5 × 10
5.0individual/ml37 DEG C, containing 5%CO
2cultivate the HEK293 cell (24 porocyte culture plate) of 2 ~ 3 hours, add containing appropriate cell growth medium, each extent of dilution inoculates 6 holes, establishes cell negative control hole simultaneously.After inoculation, put 37 DEG C, containing 5%CO
2cultivate 48 hours, then carry out indirect immunofluorescence assay (IFA, note 2), observation of cell specificity green fluorescence under inverted fluorescence microscope, occur that specificity green cells person is judged to infection, calculate IF Μ.Every part viral level should be not less than 10
7.0iF Μ.The results are shown in Table 1.
Viral level (IF Μ/head part) change before and after the freeze-drying of table 1 each embodiment heat-resisting lyophilized protecting agent living vaccine
| Group | Before freeze-drying | After freeze-drying |
| Embodiment 1 vaccine group | 10 8.0 | 10 7.8 |
| Embodiment 2 vaccine group | 10 8.1 | 10 8.0 |
| Embodiment 3 vaccine group | 10 7.9 | 10 7.8 |
| Conventional vaccine control group | 10 8.0 | 10 7.2 |
From table 1, before and after the Pestivirus suis recombinant adenovirus vaccine freeze-drying adopting heat resisting protective of the present invention to prepare, viral level loss is few, is no more than 0.2 titre; And comparatively large with the conventional vaccine viral level loss of milk sucrose freeze-drying, be 0.8 titre.Illustrate heat resisting protective of the present invention comparatively GPF (General Protection False agent have better provide protection to Pestivirus suis gene recombinant adenovirus.
Preserve under the vaccine prepared in three embodiments is placed-15 DEG C, 2 ~ 8 DEG C, 37 DEG C conditions together with conventional vaccine by 2 of storage tests respectively, regularly take out several bottles, measure its proterties, vacuum tightness, residual moisture and viral level.Detailed results is in table 2.
Table 2: each 37 DEG C, embodiment freeze-drying sample preserves different time proterties, vacuum tightness, residual moisture and viral level detected result.
Table 3: 2 ~ 8 DEG C, freeze-drying sample preserves proterties of lower time, vacuum tightness, residual moisture, bioactivity result
Table 4 :-15 DEG C, freeze-drying sample preserves different time proterties, vacuum tightness, residual moisture, bioactivity result
As can be seen from above test-results, freeze-drying lyophilized vaccine of the present invention has good provide protection to Pestivirus suis, is obviously better than the conventional vaccine with common protective material freeze-drying from proterties, residual moisture, vacuum tightness and several aspects protected effect of tiring.
3 immune durations and antibody dynamic regularity detect
3.1 sow immune durations and antibody dynamic regularity detect with healthy susceptible replacement gilt 10 in 6 ~ 7 week age; be divided into 2 groups at random; often organize 5; intramuscular injection is stored in 2 ~ 8 DEG C of frozen-dried protective vaccinating agents of the present invention of 36 months and control group conventional vaccine respectively; every pig muscle injection vaccine 1ml (containing 1 part); one group of blood separation serum is respectively adopted respectively at after immunity 2 weeks, 3 weeks, 4 weeks and 2 months, 4 months, 6 months, 7 months; with serum ELISA test determination antibody titer, carry out immune duration and antibody dynamic regularity detection.
Test-results: the healthy susceptible replacement gilt of vaccine immunity prepared by the embodiment of the present invention 2, after 2 weeks, serum antibody 4/5 is positive, and 3 weeks antibody total positiveses are still 5/5 to 7 months antibody positive rate; And conventional vaccine control group serum antibody only has 2/5 for 2 weeks for positive after exempting from, be 0/5 to positive rate when 7 months.The results detailed in Table 5.
Table 5: two kinds of vaccine inoculation sow immune duration test-results
The negative < 0.3 of note: S/P; 0.3≤suspicious≤0.4; 0.4 < is positive.
3.2 piglet immunological extended periods and antibody dynamic regularity detect with the healthy susceptible piglet 10 of 28 ~ 35 ages in days; be divided into 2 groups at random; often organize 5; intramuscular injection is stored in 2 ~ 8 DEG C of frozen-dried protective vaccinating agents of the present invention of 24 months and control group conventional vaccine respectively; every pig muscle injection vaccine 1ml (containing 1 part); one group of blood separation serum is respectively adopted respectively at after immunity 2 weeks, 3 weeks, 4 weeks, 2,4,6,7 months; measure ELISA antibody titer, carry out immune duration and antibody dynamic regularity detection.
Test-results with the healthy susceptible piglet of the embodiment of the present invention 2 vaccine immunity, serum antibody 4/5 positive after 2 weeks, 3 weeks 5/5 antibody positives are still 5/5 to 7 months antibody positive rate; And conventional vaccine control group serum antibody 2 weeks is only 2/5 positive, be only 1/5 to positive rate in July.The results detailed in Table 6.
The immune duration test-results of table 6 two kinds of vaccine inoculation piglets
The negative < 0.3 of note: S/P; 0.3≤suspicious≤0.4; 0.4 < is positive.
Test-results shows, vaccine of the present invention is preserved 36 months under 2 ~ 8 DEG C of conditions, vaccine still has good immunogenicity, immune effect is certain, and antibody produces fast, and antibody positive rate is high, sow and piglet get final product 100% generation antibody for 4 weeks after exempting from, immune duration is long, and antibody lasts up to 7 months, and immune effect is obviously better than conventional vaccine control group.
4 replacement gilt protest tests, 6 ~ 7 monthly ages healthy susceptible replacement gilt 10, is divided into 2 groups at random, often organizes 5, every intramuscular injection 1ml.Breed after 28 days and take a blood sample, separation of serum, ELISA measures blood antibody.After conceived 30 ~ 45 days, every strong malicious 1ml of pig muscle injection swine fever crossdrift system, observes until childbirth.Within after attacking poison 7,14 days, take a blood sample, detect Pestivirus suis.During childbirth, record institute produces strong young number and the young number of exception (weak young number, stillborn foetus and mummy tire), and every exception is young adopts Cord blood or tissue, and PCR detects Pestivirus suis.Have 1 Pestivirus suis positive at least with every sow litter, sentence the morbidity of this sow.The results detailed in Table 7.
Table 7 two kinds of vaccine first farrowing sow protest test results
Test-results shows, vaccine of the present invention is preserved 36 months under 2 ~ 8 DEG C of conditions, and immune swine 100% can produce very high antibody horizontal, and immune swine can resist the attack of strong poison well; And conventional vaccine control group is preserved 36 months under 2 ~ 8 DEG C of conditions, immune effect is poor, only has 20% pig to produce antibody and can resist strong malicious attack.This test illustrates that immune effect of vaccine of the present invention is obviously better than conventional vaccine control group.
In sum, the protected effect of Pestivirus suis gene recombinant adenovirus freeze dried vaccine prepared by the gelatin sucrose heat-resisting lyophilized protecting agent that the Pestivirus suis gene recombinant adenovirus that the present invention builds adopts is apparently higher than common lyophilized vaccine.
Claims (10)
1. energy is for the preparation of a gene fragment for Pestivirus suis gene recombinant adenovirus, it is characterized in that, the nucleotides sequence of described gene fragment is classified as SEQIDNO:1.
2. gene fragment as claimed in claim 1, it is characterized in that, the aminoacid sequence of described gene segment encodes is SEQIDNO:2.
3. gene fragment according to claim 1 is for the preparation of Pestivirus suis gene recombinant adenovirus.
4. a Pestivirus suis gene recombinant adenovirus, is characterized in that, gene fragment homologous recombination according to claim 1 is prepared to the genomic multiple clone site of people 5 type replication-defective adenoviral by described Pestivirus suis gene recombinant adenovirus.
5. Pestivirus suis gene recombinant adenovirus according to claim 4 is for the preparation of vaccine.
6. a freeze dried vaccine, is characterized in that, preparing as antigen with Pestivirus suis gene recombinant adenovirus according to claim 4 of described freeze dried vaccine.
7. freeze dried vaccine as claimed in claim 6, it is characterized in that, the vaccine protectant that described freeze dried vaccine uses composed as follows: 2.5 ~ 3.5%NZ-amine, 0.2 ~ 0.4% monopotassium glutamate salt, 15 ~ 25% sucrose, 1.5 ~ 2.5% lactoalbumin hydrolysates, 4 ~ 6% gelatin, 2 ~ 8% glycerine; Surplus is water.
8. freeze dried vaccine as claimed in claim 7, it is characterized in that, also add penicillin, Streptomycin sulphate and anti-mycoplasma drug in described protective material, its final concentration is respectively penicillin 100I Μ/ml, Streptomycin sulphate 80 μ g/ml, and anti-mycoplasma drug is 5 μ g/ml.
9. freeze dried vaccine as claimed in claim 7, it is characterized in that, described protectant preparation method is as follows:
1) first take NZ-amine, monopotassium glutamate salt, sucrose and lactoalbumin hydrolysate, after dissolving, filtered liquid is made in filtration sterilization; Millipore filtration aperture used is 0.1 μm;
2) taking gelatin joins in 70-80 DEG C of water for injection, stir until dissolve completely, add 2-8% glycerine together autoclaving make gelatin solution; Autoclave conditions is 116 DEG C, 20min;
3) by step 1) filtered liquid prepared and step 2) gelatin solution 4:1 mix after, make lyophilized vaccine.
10. freeze dried vaccine as claimed in claim 9, it is characterized in that, the concentration of described gelatin is 25%.
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