CN104474542A - Preparation method of bi-combined inactivated vaccine - Google Patents
Preparation method of bi-combined inactivated vaccine Download PDFInfo
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Abstract
The invention provides a preparation method of a bi-combined inactivated vaccine. The prepared bi-combined inactivated vaccine provided by the invention can be used for preventing swine type O foot-and-mouth diseases and porcine circovirus type 2. The preparation method provided by the invention is simple, and the vaccine is high in antigen and is convenient to immunize; compared with graded immunization in the prior art, the immunization times can be reduced, the stress reaction can be reduced, and the immunization cost can be lowered; and secondly, comparisons between the effects of immunizing piglets by using a bi-combined vaccine and a monovalent vaccine show that two antigen components cannot interfere with each other in the same injection of the bi-combined vaccine, and moreover, the immune effect of injecting the bi-combined vaccine on the piglets for a single time is higher than the immune effect of sequentially injecting the two vaccines.
Description
Technical field
The present invention relates to technical field of vaccines, be specifically related to a kind of bivalent inactivated vaccine preparation method.
Background technology
Foot and mouth disease (foot-and-mouth disease, FMD) is the zoonosis being infected a kind of acute and high degree in contact caused by foot and mouth disease virus (foot-and-mouth diseasevirus, FMDV).It is the domestic and wild artiodactyl such as cattle, sheep, pig that FMDV infects object.Epidemiological study and infection experiment prove, about have 100 many animals can infect this virus.The Clinical symptoms of this disease is that spread speed block, Epidemic Scope are wide, and the places such as the oral mucosa of adults, hoof and breast blister occur and fester, and young animal is many causes higher mortality rate because of myocardial damage.Except animal dead causes direct economic loss, animal stops in the production of meat and milk during one's sickness, and age of sucking, milk yield reduced, and resistance to cross growth of animal slow, and Seed practical value is lost, and also can cause serious economic loss.Primary disease is distributed in all over the world, and in Asia, Africa, the area such as South America and the Middle East be widely current, and often there is the illustration again breaking out primary disease the countries and regions eliminating foot and mouth disease.In view of foot and mouth disease is very harmful to aspects such as development of world economy, ecological harmony and social safeties, World Organization for Animal Health (OIE) and FAO (Food and Agriculture Organization of the United Nation) (FAO) are classified as one of epidemic disease that must circulate a notice of, and China is also classified as zoonotic first an of class.
Pig circular ring virus (Porcine circovirus, PCV) is a kind of minimum animal virus found so far.Existing known PCV has two serotypes, i.e. PCV1 and PCV2.PCV1 is the virus of non-pathogenic, and PCV2 is pathogenic virus, PCV2 is except causing piglet multisystemic exhaustion syndrome (Postweaning MultisystemicWasting Syndrome, PMWS) outward, the breeding difficulty of sow can also be caused, PCVD is extensively present in swinery all over the world, causes very large economic loss.
In recent years, in China swinery, PCV2 and pig O type FMDV is popular very serious, and M & M also improves constantly, and causes tremendous economic to lose to pig industry.The current prevention and control to these 2 kinds of viral blights mainly adopt inactivated vaccine, but PCV2 inactivated vaccine exists the difficult reality of cultivating of virus, and FMDV inactivated vaccine exists because of the deactivation not thoroughly danger of the poison that falls apart.Research shows; the subunit vaccine utilizing the PCV2 of bioreactor culture virus can improve virus titer and recombiant protein development infects FMDV and has good immune effect; the structural protein Vp1 of FMDV comprises the major antigenic sites of FMDV; it is the principal immune protective antigen of virus; protectiveness neutralizing antibody can be produced by induced animal body, can be used for the development of FMDV subunit vaccine.Therefore, development prevents the bigeminy vaccine of this two-strain epidemic disease significant simultaneously.Be badly in need of the bivalent inactivated vaccine of FMDV and PCV2, the PMWS syndrome caused to prevent O type foot and mouth disease and PCV2 simultaneously.
Summary of the invention
The present invention is intended to the technological deficiency for prior art, a kind of bivalent inactivated vaccine preparation method is provided, the method adopts prokaryotic expression purification FMDV-Vp1 albumen, in conjunction with the infectious titer of existing PCV-2 inactivated virus vaccine bioreactor suspension culture, prepare pig O type foot and mouth disease VP1 subunit, PCV-2 bivalent inactivated vaccine.
In order to realize above technical purpose, the present invention by the following technical solutions:
A kind of bivalent inactivated vaccine preparation method, comprises the following steps:
1) get PCV2ZJ/C virus strain infection host cell and carry out virus amplification, guarantee virus titer>=10
7.0tCID
50/ ml, harvesting venom;
2) by step 1) the cell venom that obtains, utilize doughnut to filter post and carry out clarification concentration;
3) by step 2) virus after concentration adds beta-propiolactone deactivation in the ratio of 0.1 ~ 0.3% (v/v) under stirring, obtains the first antigen stock;
4) get pET28a-FMDV VP1 bacterial strain and carry out cultivation amplification, and the IPTG adding 0.1 ~ 1mmol/L in backward cultivating system induces, induction time is 4 ~ 6h, and inducing temperature is 25 ~ 37 DEG C, then collects thalline, breaking cellular wall, collects inclusion body;
5) CAPS lysate or Urea Lysis liquid is utilized with the resuspended step 4 of the concentration of 15 ~ 25mg/mL) inclusion body that obtains, collect supernatant, then use Ni
2+column purification albumen, recombinant protein, refilters degerming, namely obtains the second antigen stock;
6) by step 3) the first antigen stock and step 5 of obtaining) the second antigen stock of obtaining is mixed in proportion, and namely obtain hybrid antigen stock solution, above-mentioned is that in the hybrid antigen stock solution guaranteeing to obtain, PCV2 content is 10 in proportion
7.0tCID50/ head part, FMDV VP1 protein content are 80 μ g/ head parts or PCV2 protein content is 10
7.5tCID50/ head part, FMDV VP1 protein content are 100 μ g/ head parts;
7) by step 6) the hybrid antigen stock solution that obtains and vaccine adjuvant stirring and evenly mixing, namely obtain a kind of bivalent inactivated vaccine.
Preferably, step 1) idiographic flow as follows: getting PCV2ZJ/C strain by infection multiplicity is 0.005 ~ 0.02 be inoculated in PK-15 cell, 34 ~ 39 DEG C of absorption 25 ~ 35min, add the MEM cell maintenance medium of the D-glucosamine hydrochloric acid containing 2 ~ 5% calf serums and 0.5 ~ 2mmol/L, 34 ~ 39 DEG C of cultivations, then multigelation, guarantees virus titer>=10
7.0tCID
50/ ml, results virus.
Preferably, step 7) mixed proportion of hybrid antigen stock solution and vaccine adjuvant is 1:1 (v/v).
Preferably, step 2) first utilize luxuriant and rich with fragrance holder filter plate to carry out a step filtration treatment before filter post clarifying treatment.
Preferably, step 3) deactivation condition is: adds under beta-propiolactone is placed on 4 DEG C of conditions and stirs deactivation 48 ~ 72h, be then placed in 37 DEG C of water-bath 2h.
Preferably, step 4) described in amplification cultivation specifically comprise following flow process: get pET28a-FMDV VP1 bacterial strain line to Kan+ resistance culture plate, 37 DEG C of incubated overnight, picking single bacterium colony access 10mL contains in the LB liquid medium of Kan+, 37 DEG C of 220r/min shaken cultivation 11-16h.
Preferably, step 4) described in breaking cellular wall specifically comprise following flow process: with the resuspended thalline of washbuffer that 1/10 bacteria liquid is long-pending, add 100 μ g/mL lysozyme, in ultrasonication thalline on ice.
Preferably, step 4) specifically comprise following flow process: IPTG induced concentration is 0.8mmol/L, and induction time is 5h, and inducing temperature is, 37 DEG C.
Preferably, step 5) described in Ni
2+post is Ni-NTA post.
Preferably, step 5) described in refolding method be: dilution refolding is in conjunction with ultrafiltration and concentration.
Preferably, vaccine adjuvant described in step 7 is the one or more combination thing of ISA206, ISA201, Gel01, ISA28VG etc.; More preferably, be ISA201.Above-mentioned name is called trade name, and the adjuvant wherein with ISA printed words is the ISA series vaccine adjuvant that match BIC Corp manufactures.
PET28a-FMDV VP1 expression strain described in above technical scheme, its deposit number is CCTCCM2014406.
Bivalent inactivated vaccine prepared by the present invention is for preventing pig O type foot and mouth disease, porcine circovirus 2 type, and preparation method of the present invention is simple, and the content of tiring of vaccine is high, immunity is convenient, compared with gradation immunity of the prior art, decreases immune time, decrease stress, reduce immune cost.
Secondly, discovery is compared with the effect of univalent vaccine immunity piglet by bigeminy vaccine, two kinds of antigenic components not only mutually do not disturb in same pin bigeminy vaccine, and the immune effect of bigeminy vaccine single injection piglet is higher than the immune effect of two kinds of single Seedlings successively injection.
The i.e. bivalent inactivated vaccine of PCV2, PPV of the present invention, has following beneficial effect:
1), after bivalent inactivated vaccine immunity piglet, side reaction reduces, and safety is better, avoids repeatedly the untoward reaction that immunoprophylaxis occurs.
2), after bivalent inactivated vaccine immunity sow, by maternal antibody, piglet obtains passive immunity, significantly reduces the mortality rate of piglet, improves survival rate, add economic benefit.
4) bivalent inactivated vaccine immunity piglet, compared with gradation immunity of the prior art, reduces immune cost, has saved immune programme for children, more economically reliably.
Accompanying drawing explanation
Fig. 1 is the vaccine preparation flow figure of the embodiment of the present invention 1.
Detailed description of the invention
Embodiment 1 porcine circovirus 2 type, Schweineseuche VP1 subunit bivalent inactivated vaccine
The preparation of 1 production seed culture of viruses
The preparation of 1.1PCV2ZJ/C strain: seed culture of viruses viral dilution liquid (the MEM culture medium of serum-free) is suitably diluted, be 0.01 be inoculated in PK-15 cell culture by infection multiplicity (M.O.I.), 37 DEG C of absorption 30min, add the MEM cell maintenance medium of the D-glucosamine hydrochloric acid containing 3% (v/v) calf serum and 1mmol/L, cultivate 5 for 37 DEG C, multigelation 3 times, results virus, virus titer>=10
7.0tCID
50/ ml.
The activation of 1.2VP1 protein expression strain: the prokaryotic expression strain preserved is rule to Kan+ resistance culture plate, 37 DEG C of incubated overnight.Picking single bacterium colony access 10mL contains in the LB liquid medium of Kan+, and 37 DEG C of 220r/min shaken cultivation 11-16h, as abduction delivering strain.
The preparation of 2 seedling antigens
The preparation of 2.1PCV2 antigen
2.1.1 virus liquid is cultivated: select zooblast bioreactor 10L to 42L to digest amplification and connect poison, low serum suspension culture Technology, 0.25%EDTA-trypsinization, be prepared into 4 × 10 with the special cell growth medium of MEM containing 3% low serum
5the cell suspension of individual/mL, in 1% ratio inoculation production seed culture of viruses, 37 DEG C, speed of agitator 35rpm/h, after inoculation, every day observes 1-2 time, and be cultured to 72-96h and abandon nutritional solution, freeze thawing harvesting and culture fluid, save backup in-20 DEG C.
2.1.2 viral level measures
Well-grown PK15 cell is carried out digestion dispersion by the EDTA-pancreatin with 0.25%, and with cell growth medium resuspended (8%NBCS MEM) for concentration is for 2 × 10
5the cell suspension of individual/ml, is inoculated in 96 porocyte culture plates, 100ul/ hole, is placed in 37 DEG C, 5%CO
2in cell culture incubator.
Get virus liquid and carry out 10 times of multiple proportions gradient dilutions with serum-free MEM, each gradient (10 after diluting
-4~ 10
-7) virus liquid is inoculated in step 1 and added in 96 porocyte culture plates of cell suspension, 100ul/ hole, each gradient 6 or 8 repeating holes, arrange the positive and negative control, 37 DEG C, 5%CO
272h is cultivated in cell culture incubator.
After maintain terminates, abandon maintenance medium in most plate hole, with methanol/acetone fixative (1:1), cell is fixed, 100ul/ hole ,-20 DEG C, 5 ~ 10min.Abandon most fixative, cell plates are air-dry; Bovine serum albumin (BSA) solution with 1% carries out sealing treatment, 100ul/ hole, 37 DEG C, hatches 60min.3 times are washed, 200ul/ hole with 0.01M PBS.The primary antibodie that 1000 times are diluted is made an addition to Tissue Culture Plate, 100ul/ hole, 37 DEG C, hatches 60min.5 times are washed, 200ul/ hole time with 0.01M PBS.(two of FITC labelling resists, and 100ul/ hole, hatches 60min by 37 DEG C to add 200 times of fluorochromes diluted.Abandon most two to resist, wash 5 times with 0.01M PBS, 200ul/ hole, fluorescence microscopy Microscopic observation.There is the number in specific fluorescence hole in statistics, calculates virus titer (TCID by Reed-Muench method
50).
2.1.3 the clarification of antigen and purification
By cell venom qualified for PCV2ZJ/C strain, carry out clarifying treatment with luxuriant and rich with fragrance holder filter plate (clarifier) respectively, then clarify venom and use the hollow fiber purification system of GE company (300KDa hollow fiber column) to carry out foreign protein again to remove further and concentration.
2.1.4 the deactivation of virus liquid
Inactivation of virus: virus liquid is placed in deactivation container, slowly adds beta-propiolactone in 1:2000 ratio under stirring, stirs deactivation 48h under being placed in 4 DEG C of conditions, is then placed in 37 DEG C of water-bath 2h, and 2-8 DEG C of preservation, is no more than 1 month.
Deactivation is checked: get inactivation of viruses liquid 1 part and 9 parts of PK15 cell suspension (2 × 10
5individual/mL) fully mix, inoculate 5 and be not less than 25cm
2tissue Culture Flask, virus positive control is set simultaneously.In 37 DEG C, 5%CO
2incubator cultivates 72h, harvesting and culture fluid, freeze thawing 3 inoculums as blind passage, blind passage 2 generation, IFA method inspection product, and the inoculating cell of inactivation of viruses all should redgreen fluorescence, and the compared with control cells of virus inoculation all should have green fluorescence.
Steriling test: inactivation of viruses liquid inoculation TG tubule, each 2 of GA inclined-plane, often prop up 0.2mL, put 37 DEG C of cultivations for 1, one is placed in 25 DEG C of cultivations, separately gets 0.2mL and inoculates 1 GP tubule and be placed in 25 DEG C of cultivations, all cultivate 7d, answer asepsis growth.
The preparation of 2.2VP1 proteantigen
2.2.1VP1 the abduction delivering of albumen: inoculate fresh bacterium liquid to containing Kan in 1:100 ratio
+lB fluid medium, 37 DEG C, 180rpm/min, cultivate 3.5h, measure OD
600, add 0.8mmoL/L IPTG and induce, 37 DEG C, cultivate 5h.Collect thalline.With the resuspended thalline of wash buffer that 1/10 bacteria liquid is long-pending, add 100 μ g/mL lysozyme, in ultrasonication thalline on ice, collect inclusion body.
2.2.2VP1 the purification of albumen: CAPS lysate or Urea Lysis liquid cracking inclusion body: in the resuspended inclusion body of ratio lysate (needing ultrasonic mixing if desired) of 20mg/mL, collects the supernatant containing soluble protein.Ni
2+column purification albumen, dilution refolding albumen, obtains enough albumen in conjunction with ultrafiltration and concentration.Measure endotoxin content≤50EU, protein concentration reaches 1mg/mL.Degerming through 0.22um membrane filtration, save backup.
2.4 join Seedling
By 2 antigen liquids after concentrated, purification by proper ratio mixing, then mixed liquor mixes by 1: 1 (V/V) with ISA201 adjuvant (France matches Bick SEPPIC company and produces), with 300 revs/min of stirring 30min.
In the embodiment of the present invention 1, the above-mentioned method preparing bigeminy vaccine can refer to Fig. 1.
2.5 character inspections
Outward appearance: milky Emulsion, is long placed in rear, and upper strata has a small amount of oil to separate out, in even Emulsion after jolting.Dosage form: water-in-oil-in water.Get a clean suction pipe, draw a small amount of vaccine and drip in cold water surface, should the diffusion in cloud.Stability: draw vaccine 10mL and add in centrifuge tube, the centrifugal 3min of 2500rpm, emulsion is not clearly demarcated, namely reaches instructions for use.Viscosity: the vaccine 1mL drawing about 25 DEG C with 1mL suction pipe (lower internal diameter 1.2mm, upper internal diameter 2.7mm), makes its vertical natural flow out 0.4mL, and record required time, should within 8s.By 3 of above-mentioned preparation batches, (lot number is: 201401,201402,201403).Character assay meets the requirements.By 3 of above-mentioned preparation batches, (lot number is: 201401,201402,201403).Character assay meets the requirements.Character assay is in table 1.
2.6 steriling tests: undertaken by " People's Republic of China's veterinary drug allusion quotation " (version in 2010) annex, asepsis growth answered by vaccine.Steriling test the results are shown in Table 1, and result shows that (lot number is 3 batches of vaccines: 201401,201402,201403) asepsis growth, vaccine finished product is qualified.
The character inspection of table 13 batch bivalent inactivated vaccine, steriling test and safety examination result
2.7 safety verification
2.7.1 hoof-and-mouth disease subunit vaccine safety evaluatio part
Be 350-450g Cavia porcellus 2 by body weight, every subcutaneous injection vaccine 2mL; With body weight 18-22g mice 5, every subcutaneous injection vaccine 0.5mL., all must not there is the death because vaccinate causes or significantly untoward reaction in Continuous Observation 7 days.
With the healthy susceptible piglet (measuring without antibodies against foot-and-mouth disease virus (cell NAT≤1:8 or neonatal rat NAT≤1:4 or LPB-ELISA antibody titer≤1:8) through neonatal rat neutralization test) 2 of 30-40 age in days, after each two Herba Houttuyniae, muscle branch injects 2 part vaccines, observes 14 day by day.All must not there is foot and mouth disease symptom or significantly because of toxic action that vaccinate causes.
2.7.2 circovurus type 2 inactivated vaccine safety evaluatio part
With 14-21 age in days PCV2ELISA negative antibody, the healthy susceptible piglet 5 of PCV2 antigen negative, PRRSV ELISA negative antibody, each musculi colli vaccinate 4.0mL, Continuous Observation 14 days, should not have whole body or local untoward reaction that vaccine causes.The results are shown in Table 2.
The safety examination result of table 23 batch bivalent inactivated vaccine
2.8 efficacy test
2.8.1 the efficacy test of Schweineseuche VP1 subunit inactivated vaccine part
Hoof-and-mouth disease subunit vaccine effect evaluation: the susceptible feeder pig (measuring without antibodies against foot-and-mouth disease virus (cell NAT≤1:8 or neonatal rat NAT≤1:4 or LPB-ELISA antibody titer≤1:8) through neonatal rat neutralization test) 15 by body weight being about 40kg, be divided into 3 groups, often organize 5.Vaccine to be checked is divided into 1 part, 1/6 part, 1/12 part, 3 dosage groups, every dose injects 5 pigs respectively at auricularis posterior meat.Inoculate latter 28 days, together with contrast pig 2, after every basal part of the ear, the swine foot-and-mouth disease virus O type OR/80 strain of intramuscular injection 1000 ID50 is malicious by force, Continuous Observation 10 days.Contrast pig all should have at least a hoof to occur vesicle or ulcer.Isolate in time after there is morbidity pig.According to the protection number of immune swine, calculate the PD50 of tested vaccine by Reed-Muench method.Every part vaccine at least should contain 6 PD50.Assay as following table 3, result show 3 batches of vaccine product foot-and-mouth disease vaccine effect all >=6 PD50, hoof-and-mouth disease subunit vaccine effect part is qualified.
The effect evaluation result of table 3 hoof-and-mouth disease subunit vaccine part
2.8.2 the efficacy test of pig circular ring virus inactivated vaccine part
Mouse immune counteracting toxic substances method: with the healthy Balb/C mice 10 in 6-8 age in week, each lumbar injection vaccine 0.2mL, latter 21 days of immunity, together with control mice 10, attack with PCV2-ZJ/C strain inspection poison (107.0TCID50/mL), every mouse peritoneal injection 0.45mL.After counteracting toxic substances 21 days, cut open and kill, get spleen and carry out virus purification, be separated to virus and be namely judged to the virus purification positive.Control mice should at least 7 virus purification positives, and immune mouse should at least 7 virus purification feminine genders.The results are shown in Table 4:
Table 4PCV2 inactivated vaccine some animals challenge test result
Interpretation of result: as seen from the above, counteracting toxic substances matched group protective rate 20%, illustrate that counteracting toxic substances dosage 107.0TCID50/ml matched group sets up, 3 batches of equal immune protective rates of vaccine group, more than 70%, reach regulatory requirements.
Embodiment 2FMDV VP1 subunit, PCV2 bivalent inactivated vaccine compare with the immune effect being used alone two kinds of vaccines (Schweineseuche O-shaped synthetic peptide vaccine (polypeptide 2570+7309) and PCV2 inactivated vaccine) immune piglet
1. material
Pig O type foot and mouth disease VP1 subunit, PCV2 bivalent inactivated vaccine, select the laboratory products in embodiment 1 (lot number is 201401); PCV2 inactivated vaccine (ZJ/C strain), Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. produces (lot number is XXXX), and viral level to be at least before deactivation 10
7.0tCID
50/ ml; Schweineseuche O-shaped synthetic peptide vaccine (polypeptide 2570+7309), purchased from Shanghai Shen Lian Biological Co., Ltd. (lot number is XXXX), containing each at least 25ug/ml of Schweineseuche synthetic peptide.
2. animal experiment design
Select 21 ~ 28 age in days ablactational baby pig 90, be divided into 6 groups, often organize 15; 1st, 2 groups of every pig musculi colli injection pig O type foot and mouth disease VP1 subunit, PCV2 bivalent inactivated vaccine (lot number 201401) 2ml respectively; 3rd, 4 groups of every pig left neck intramuscular injection Schweineseuche O-shaped synthetic peptide vaccine 2ml respectively, right neck intramuscular injection PCV2 inactivated vaccine (ZJ/C strain) 2ml, does not inoculate for the 5th, 6 group.28d after first immunisation, respectively uses PCV2ZJ/C strain (containing 10 for the 1st, 3,5 group
7.0tCID50/ml) collunarium 1ml, intramuscular injection 2ml, slaughters, carries out the separation of circoviras antigen for after counteracting toxic substances the 14th day.2nd, 4,6 groups of immune latter 28 days of first times, booster immunization 1 time, intramuscular injection 2mL, rear 10d talent and learning separation of serum is exempted from 2, utilize foot and mouth disease O type LPB-ELISA detection kit to detect antibody titer, during ELISA antibody titer >=1:64, belong to more than 99% protection domain; During ELISA antibody titer≤1:4, belong to not protection domain, need to carry out fundamental immunity; Antibody titer is when 1:6 ~ 1:45, and belonging to the 50% protection scope of tiring needs to carry out booster immunization.
3 result of the tests
Result of the test is as table 5, and Combined vaccine and the counteracting toxic substances protection of pig circular ring virus deactivation list Seedling to PCV2ZJ/C strain all reach more than 70%, and the counteracting toxic substances protected effect of Combined vaccine is slightly better than pig circular ring virus deactivation list Seedling; Combined vaccine and Schweineseuche O-shaped subunit vaccine ELISA Antibody Efficacy testing result show, the piglet ELISA antibody of Combined vaccine immunity is better than Schweineseuche O-shaped subunit vaccine (as table 5).Therefore, the immune effect of Combined vaccine and single Seedling is substantially quite or higher than now selling inactivated vaccine; From dosage of inoculation comparatively speaking, Combined vaccine plays 2 pins, altogether 4ml, and two kinds of single Seedling couplings need play 4 pins, altogether 8ml, and apparent effect is that Combined vaccine use is more convenient, time saving and energy saving; From the view of safety, the safety of Combined vaccine is comparatively safe, and single Seedling coupling has the test pig of 1/15 ~ 2/15 to have untoward reaction, because the dosage of injection compares, Combined vaccine will increase by 1 times; Comprehensively state it, Combined vaccine is not only easy to use, safer, and immune effect and independent two kinds of single Seedling coupling effects are quite or slightly high, the results are shown in Table 5.
Table 5 bivalent inactivated vaccine and single Seedling immune effect comparative test result
Embodiment 3
1, a bivalent inactivated vaccine preparation method, is characterized in that comprising the following steps:
1) get PCV2ZJ/C virus strain infection host cell and carry out virus amplification, guarantee virus titer>=10
7.0tCID
50/ ml, harvesting venom;
2) by step 1) the cell venom that obtains, utilize doughnut to filter post and carry out clarification concentration;
3) by step 2) virus after concentration adds beta-propiolactone deactivation in the ratio of 0.1 ~ 0.3% (v/v) under stirring, obtains the first antigen stock;
4) get pET28a-FMDV VP1 bacterial strain and carry out cultivation amplification, and the IPTG adding 0.1 ~ 1mmol/L in backward cultivating system induces, induction time is 4 ~ 6h, and inducing temperature is 25 ~ 37 DEG C, then collects thalline, breaking cellular wall, collects inclusion body;
5) CAPS lysate or Urea Lysis liquid is utilized with the resuspended step 4 of the concentration of 15 ~ 25mg/mL) inclusion body that obtains, collect supernatant, then use Ni
2+column purification albumen, recombinant protein, refilters degerming, namely obtains the second antigen stock;
6) by step 3) the first antigen stock and step 5 of obtaining) the second antigen stock of obtaining is mixed in proportion, and namely obtain hybrid antigen stock solution, above-mentioned is that in the hybrid antigen stock solution guaranteeing to obtain, PCV2 content is 10 in proportion
7.0tCID50/ head part, FMDV VP1 protein content are 80 μ g/ head parts or PCV2 protein content is 10
7.5tCID50/ head part, FMDV VP1 protein content are 100 μ g/ head parts;
7) by step 6) the hybrid antigen stock solution that obtains and vaccine adjuvant stirring and evenly mixing, namely obtain a kind of bivalent inactivated vaccine.
Embodiment 4
1, a bivalent inactivated vaccine preparation method, is characterized in that comprising the following steps:
1) get PCV2ZJ/C virus strain infection host cell and carry out virus amplification, guarantee virus titer>=10
7.0tCID
50/ ml, harvesting venom;
2) by step 1) the cell venom that obtains, utilize doughnut to filter post and carry out clarification concentration;
3) by step 2) virus after concentration adds beta-propiolactone deactivation in the ratio of 0.2% (v/v) under stirring, obtains the first antigen stock;
4) get pET28a-FMDV VP1 bacterial strain and carry out cultivation amplification, and add IPTG in backward cultivating system and induce, IPTG induced concentration is 0.8mmol/L, and induction time is 5h, and inducing temperature is, 37 DEG C, then collects thalline, breaking cellular wall, collects inclusion body;
5) CAPS lysate or Urea Lysis liquid is utilized with the resuspended step 4 of the concentration of 20mg/mL) inclusion body that obtains, collect supernatant, then use Ni
2+column purification albumen, recombinant protein, refilters degerming, namely obtains the second antigen stock;
6) by step 3) the first antigen stock and step 5 of obtaining) the second antigen stock of obtaining is mixed in proportion, and namely obtain hybrid antigen stock solution, above-mentioned is that in the hybrid antigen stock solution guaranteeing to obtain, PCV2 content is 10 in proportion
7.0tCID50/ head part, FMDV VP1 protein content are 80 μ g/ head parts;
7) by step 6) the hybrid antigen stock solution that obtains and vaccine adjuvant stirring and evenly mixing, namely obtain a kind of bivalent inactivated vaccine.
In above technical scheme, step 1) idiographic flow as follows: getting PCV2ZJ/C strain by infection multiplicity is 0.01 be inoculated in PK-15 cell, 37 DEG C of absorption 30min, add the MEM cell maintenance medium of the D-glucosamine hydrochloric acid containing 4% calf serum and 0.1mmol/L, 37 DEG C of cultivations, then multigelation, guarantees virus titer>=10
7.0tCID
50/ ml, results virus.
Step 7) mixed proportion of hybrid antigen stock solution and vaccine adjuvant is 1:1 (v/v).
Step 2) first utilize filter plate to carry out a step filtration treatment before doughnut filter post clarifying treatment.
Step 3) deactivation condition is: adds under beta-propiolactone is placed on 4 DEG C of conditions and stirs deactivation 60h, be then placed in 37 DEG C of water-bath 2h.
Step 4) described in amplification cultivation specifically comprise following flow process: get pET28a-FMDV VP1 bacterial strain line to Kan+ resistance culture plate, 37 DEG C of incubated overnight, picking single bacterium colony access 10mL contains in the LB liquid medium of Kan+, 37 DEG C of 220r/min shaken cultivation 14h.
Step 4) described in breaking cellular wall specifically comprise following flow process: with the resuspended thalline of wash buffer that 1/10 bacteria liquid is long-pending, add 100 μ g/mL lysozyme, in ultrasonication thalline on ice.
Step 5) described in Ni
2+post is Ni-NTA post.
Step 5) described in refolding method be: dilution refolding is in conjunction with ultrafiltration and concentration.
Embodiment 5
1, a bivalent inactivated vaccine preparation method, is characterized in that comprising the following steps:
1) get PCV2ZJ/C virus strain infection host cell and carry out virus amplification, guarantee virus titer>=10
7.0tCID
50/ ml, harvesting venom;
2) by step 1) the cell venom that obtains, utilize doughnut to filter post and carry out clarification concentration;
3) by step 2) virus after concentration adds beta-propiolactone deactivation in the ratio of 0.1% (v/v) under stirring, obtains the first antigen stock;
4) get pET28a-FMDV VP1 bacterial strain and carry out cultivation amplification, and the IPTG adding 0.1mmol/L in backward cultivating system induces, induction time is 4h, and inducing temperature is 25 DEG C, then collects thalline, breaking cellular wall, collects inclusion body;
5) CAPS lysate or Urea Lysis liquid is utilized with the resuspended step 4 of the concentration of 15mg/mL) inclusion body that obtains, collect supernatant, then use Ni
2+column purification albumen, recombinant protein, refilters degerming, namely obtains the second antigen stock;
6) by step 3) the first antigen stock and step 5 of obtaining) the second antigen stock of obtaining is mixed in proportion, and namely obtain hybrid antigen stock solution, above-mentioned is that in the hybrid antigen stock solution guaranteeing to obtain, PCV2 protein content is 10 in proportion
7.5tCID50/ head part, FMDV VP1 protein content are 100 μ g/ head parts;
7) by step 6) the hybrid antigen stock solution that obtains and vaccine adjuvant stirring and evenly mixing, namely obtain a kind of bivalent inactivated vaccine.
Step 1 in above technical scheme) idiographic flow as follows: getting PCV2ZJ/C strain by infection multiplicity is 0.005 be inoculated in PK-15 cell, 34 DEG C of absorption 25min, add the MEM cell maintenance medium of the D-glucosamine hydrochloric acid containing 2% calf serum and 0.5mmol/L, 34 DEG C of cultivations, then multigelation, guarantees virus titer>=10
7.0tCID
50/ ml, results virus.
Step 7) mixed proportion of hybrid antigen stock solution and vaccine adjuvant is 1:0.8 (v/v).
Step 2) first utilize luxuriant and rich with fragrance holder filter plate to carry out a step filtration treatment before doughnut filter post clarifying treatment.
Step 3) deactivation condition is: adds under beta-propiolactone is placed on 4 DEG C of conditions and stirs deactivation 48h, be then placed in 37 DEG C of water-bath 2h.
Step 4) described in amplification cultivation specifically comprise following flow process: get pET28a-FMDV VP1 bacterial strain line to Kan+ resistance culture plate, 37 DEG C of incubated overnight, picking single bacterium colony access 10mL contains in the LB liquid medium of Kan+, 37 DEG C of 220r/min shaken cultivation 11-16h.
Step 4) described in breaking cellular wall specifically comprise following flow process: with the resuspended thalline of wash buffer that 1/10 bacteria liquid is long-pending, add 100 μ g/mL lysozyme, in ultrasonication thalline on ice.
Step 5) described in Ni
2+post is Ni-NTA post.
Step 5) described in refolding method be: dilution refolding is in conjunction with ultrafiltration and concentration.
Embodiment 6
1, a bivalent inactivated vaccine preparation method, is characterized in that comprising the following steps:
1) get PCV2ZJ/C virus strain infection host cell and carry out virus amplification, guarantee virus titer>=10
7.0tCID
50/ ml, harvesting venom;
2) by step 1) the cell venom that obtains, utilize doughnut to filter post and carry out clarification concentration;
3) by step 2) virus after concentration adds beta-propiolactone deactivation in the ratio of 0.3% (v/v) under stirring, obtains the first antigen stock;
4) get pET28a-FMDV VP1 bacterial strain and carry out cultivation amplification, and the IPTG adding 1mmol/L in backward cultivating system induces, induction time is 6h, and inducing temperature is 37 DEG C, then collects thalline, breaking cellular wall, collects inclusion body;
5) CAPS lysate or Urea Lysis liquid is utilized with the resuspended step 4 of the concentration of 25mg/mL) inclusion body that obtains, collect supernatant, then use Ni
2+column purification albumen, recombinant protein, refilters degerming, namely obtains the second antigen stock;
6) by step 3) the first antigen stock and step 5 of obtaining) the second antigen stock of obtaining is mixed in proportion, and namely obtain hybrid antigen stock solution, above-mentioned is that in the hybrid antigen stock solution guaranteeing to obtain, PCV2 content is 10 in proportion
7.0tCID50/ head part, FMDV VP1 protein content are 80 μ g/ head parts;
7) by step 6) the hybrid antigen stock solution that obtains and vaccine adjuvant stirring and evenly mixing, namely obtain a kind of bivalent inactivated vaccine.
Step 1 in above technical scheme) idiographic flow as follows: getting PCV2ZJ/C strain by infection multiplicity is 0.02 be inoculated in PK-15 cell, 39 DEG C of absorption 35min, add the MEM cell maintenance medium of the D-glucosamine hydrochloric acid containing 5% calf serum and 2mmol/L, 39 DEG C of cultivations, then multigelation, guarantees virus titer>=10
7.0tCID
50/ ml, results virus.
Step 7) mixed proportion of hybrid antigen stock solution and vaccine adjuvant is 1:1.2 (v/v).
Step 3) deactivation condition is: adds under beta-propiolactone is placed on 4 DEG C of conditions and stirs deactivation 72h, be then placed in 37 DEG C of water-bath 2h.
Step 4) described in amplification cultivation specifically comprise following flow process: get pET28a-FMDV VP1 bacterial strain line to Kan+ resistance culture plate, 37 DEG C of incubated overnight, picking single bacterium colony access 10mL contains in the LB liquid medium of Kan+, 37 DEG C of 220r/min shaken cultivation 16h.
Step 4) described in breaking cellular wall specifically comprise following flow process: with the resuspended thalline of wash buffer that 1/10 bacteria liquid is long-pending, add 100 μ g/mL lysozyme, in ultrasonication thalline on ice.
Above embodiments of the invention have been described in detail, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All make in application range of the present invention any amendment, equivalent to replace and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a bivalent inactivated vaccine preparation method, is characterized in that comprising the following steps:
1) get PCV2ZJ/C virus strain infection host cell and carry out virus amplification, guarantee virus titer>=10
7.0tCID
50/ ml, harvesting venom;
2) by step 1) the cell venom that obtains, utilize doughnut to filter post and carry out clarification concentration;
3) by step 2) virus after concentration adds beta-propiolactone deactivation in the ratio of 0.1 ~ 0.3% (v/v) under stirring, obtains the first antigen stock;
4) get pET28a-FMDV VP1 bacterial strain and carry out cultivation amplification, and the IPTG adding 0.1 ~ 1mmol/L in backward cultivating system induces, induction time is 4 ~ 6h, and inducing temperature is 25 ~ 37 DEG C, then collects thalline, breaking cellular wall, collects inclusion body;
5) CAPS lysate or Urea Lysis liquid is utilized with the resuspended step 4 of the concentration of 15 ~ 25mg/mL) inclusion body that obtains, collect supernatant, then use Ni
2+column purification albumen, recombinant protein, refilters degerming, namely obtains the second antigen stock;
6) by step 3) the first antigen stock and step 5 of obtaining) the second antigen stock of obtaining is mixed in proportion, and namely obtain hybrid antigen stock solution, above-mentioned is that in the hybrid antigen stock solution guaranteeing to obtain, PCV2 content is 10 in proportion
7.0tCID50/ head part, FMDV VP1 protein content are 80 μ g/ head parts or PCV2 protein content is 10
7.5tCID50/ head part, FMDV VP1 protein content are 100 μ g/ head parts;
7) by step 6) the hybrid antigen stock solution that obtains and vaccine adjuvant stirring and evenly mixing, namely obtain a kind of bivalent inactivated vaccine.
2. a kind of bivalent inactivated vaccine preparation method according to claim 1, it is characterized in that step 1) idiographic flow as follows: getting PCV2ZJ/C strain by infection multiplicity is 0.005 ~ 0.02 be inoculated in PK-15 cell, 34 ~ 39 DEG C of absorption 25 ~ 35min, add the MEM cell maintenance medium of the D-glucosamine hydrochloric acid containing 2 ~ 5% calf serums and 0.5 ~ 2mmol/L, 34 ~ 39 DEG C of cultivations, then multigelation, guarantees virus titer>=10
7.0tCID
50/ ml, results virus.
3. a kind of bivalent inactivated vaccine preparation method according to claim 1, is characterized in that step 7) mixed proportion of hybrid antigen stock solution and vaccine adjuvant is 1:1 (v/v).
4. a kind of bivalent inactivated vaccine preparation method according to claim 1, is characterized in that step 2) first utilize luxuriant and rich with fragrance holder filter plate to carry out a step filtration treatment before filter post clarifying treatment.
5. a kind of bivalent inactivated vaccine preparation method according to claim 1, is characterized in that step 3) deactivation condition is: adds under beta-propiolactone is placed on 4 DEG C of conditions and stirs deactivation 48 ~ 72h, be then placed in 37 DEG C of water-bath 2h.
6. a kind of bivalent inactivated vaccine preparation method according to claim 1, it is characterized in that step 4) described in amplification cultivation specifically comprise following flow process: get pET28a-FMDV VP1 bacterial strain line to Kan+ resistance culture plate, 37 DEG C of incubated overnight, picking single bacterium colony access 10mL contains in the LB liquid medium of Kan+, 37 DEG C of 220r/min shaken cultivation 11-16h.
7. a kind of bivalent inactivated vaccine preparation method according to claim 1, it is characterized in that step 4) described in breaking cellular wall specifically comprise following flow process: with the resuspended thalline of PBS buffer that 1/10 bacteria liquid is long-pending, add 100 μ g/mL lysozyme, in ultrasonication thalline on ice.
8. a kind of bivalent inactivated vaccine preparation method according to claim 1, is characterized in that step 4) specifically comprise following flow process: IPTG induced concentration is 0.8mmol/L, and induction time is 5h, and inducing temperature is, 37 DEG C.
9. a kind of bivalent inactivated vaccine preparation method according to claim 1, is characterized in that step 5) described in Ni
2+post is Ni-NTA post.
10. a kind of bivalent inactivated vaccine preparation method according to claim 1, is characterized in that step 5) described in refolding method be: dilution refolding is in conjunction with ultrafiltration and concentration.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107513524A (en) * | 2017-09-30 | 2017-12-26 | 中牧实业股份有限公司 | One plant of pig Sai Neijia paddy virus stain and its application |
| WO2018223668A1 (en) * | 2017-06-09 | 2018-12-13 | 普莱柯生物工程股份有限公司 | Bivalent vaccine composition for preventing and/or treating porcine circovirus infections and preparation method and use thereof |
| CN113521271A (en) * | 2021-05-27 | 2021-10-22 | 杭州佑本动物疫苗有限公司 | Porcine reproductive and respiratory syndrome bivalent inactivated vaccine and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1607004A (en) * | 2003-10-15 | 2005-04-20 | 北京迪威华宇生物技术有限公司 | Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine |
| CN102512671A (en) * | 2011-12-16 | 2012-06-27 | 厦门大学 | Anti-foot-and-mouth disease type O virus-like particle vaccine and preparation method thereof |
| CN103083663A (en) * | 2013-02-04 | 2013-05-08 | 江苏省农业科学院 | Immunity enhancing agent, inactivated vaccine, and preparation method thereof |
| CN104043117A (en) * | 2013-03-11 | 2014-09-17 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
-
2014
- 2014-11-18 CN CN201410659965.1A patent/CN104474542A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1607004A (en) * | 2003-10-15 | 2005-04-20 | 北京迪威华宇生物技术有限公司 | Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine |
| CN102512671A (en) * | 2011-12-16 | 2012-06-27 | 厦门大学 | Anti-foot-and-mouth disease type O virus-like particle vaccine and preparation method thereof |
| CN103083663A (en) * | 2013-02-04 | 2013-05-08 | 江苏省农业科学院 | Immunity enhancing agent, inactivated vaccine, and preparation method thereof |
| CN104043117A (en) * | 2013-03-11 | 2014-09-17 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 秦晓冰等: "2型猪圆环病毒与O型口蹄疫病毒二联重组鸡痘活疫苗的构建及实验免疫研究", 《人畜共患传染病防治研究新成果汇编》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018223668A1 (en) * | 2017-06-09 | 2018-12-13 | 普莱柯生物工程股份有限公司 | Bivalent vaccine composition for preventing and/or treating porcine circovirus infections and preparation method and use thereof |
| US11116836B2 (en) | 2017-06-09 | 2021-09-14 | Pulike Biological Engineering, Inc. | Bivalent vaccine composition for preventing and/or treating porcine circovirus infections and preparation method and use thereof |
| CN107513524A (en) * | 2017-09-30 | 2017-12-26 | 中牧实业股份有限公司 | One plant of pig Sai Neijia paddy virus stain and its application |
| CN113521271A (en) * | 2021-05-27 | 2021-10-22 | 杭州佑本动物疫苗有限公司 | Porcine reproductive and respiratory syndrome bivalent inactivated vaccine and preparation method thereof |
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