CN104383527A - Preparation method of inactivated lyophilized vaccine for porcine circovirus type 2 - Google Patents
Preparation method of inactivated lyophilized vaccine for porcine circovirus type 2 Download PDFInfo
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Abstract
The invention provides a preparation method of an inactivated lyophilized vaccine for porcine circovirus type 2. The preparation method comprises the following steps: successively carrying out clarification procedure, ultrafiltration concentration and molecular sieve chromatography column purification on a virus culture solution, so that the impurity content of an antigen used for preparing the vaccine is effectively ensured to be reduced to the minimum, thus avoiding the phenomenon that side effects are generated after the product is used, and also being beneficial to ensuring the immune effect of the vaccine. In addition, according to the preparation method provided by the invention, as the inactivated antigen is directly prepared in a lyophilizing mode without adding an oil adjuvant, the problems of difficulty in injection and absorption do not exist. The PCV 2 inactivated lyophilized vaccine prepared by the preparation method is high in antigen content, immunogenicity and antigen purity, is good in safety, is low in side effect, is capable of rapidly generating antibody protection without an adjuvant and is easily injected and absorbed.
Description
Technical field
The present invention relates to vaccine preparation technology field, be specifically related to a kind of preparation method of porcine circovirus 2 type deactivation freeze dried vaccine.
Background technology
Porcine circovirus desease is by porcine circovirus 2 type (Porcine circovirus type2, PCV2) a series of diseases caused, comprise pmws (Postweaning multisystemie wastingsyndrome, PMWS), Corii Sus domestica inflammation and nephrotic syndrome (Porcine dermatitis and nephrophathysyndrome, PDNS), porcine respiratory disease complex (Porcine respiratory disease complex, PRDC).
1997, Clark etc. were separated to porcine circovirus 2 type first.In the 20th international swine diseases conference in 2008, circovirus disease is listed in the No.1 epidemic disease of current harm pig industry development.China confirms that in 2000 there is PCV2 in swinery infects, and the PWMS caused infected by PCV2 for 2002 is outbreak of epidemic in the whole nation.It is slow that Porcine circovirus desease causes pig to grow, and the price of deed is low, and immunity of organisms declines, and causes serious threat and tremendous economic loss to pig industry.According to investigations, PCV2 reaches more than 80% at the positive rate of China.
Vaccine is one of main method of this disease of prevention.At present, the porcine circovirus type 2 vaccines production technology of Chinese commodity is generally by the direct deactivation of virus liquid of results, then adds mineral oil adjuvant emulsifying and makes.This method is due to the direct production carrying out finished product Seedling with the virus liquid of cell proliferation, therefore the impact of the impurity such as cell breakage product, medium component, serum albumin, products of cellular metabolism can be subject to, easily anaphylaxis is there is after causing vaccine immunity animal, the side effect such as exothermic reaction, also immune effect can be affected simultaneously, and mineral oil adjuvant vaccine ubiquity injections difficult, injection site easily produces lump.
Summary of the invention
The present invention is intended to the technological deficiency for prior art, provides a kind of preparation method of porcine circovirus 2 type deactivation freeze dried vaccine, and to solve, its side reaction rate is high, immune effect is poor, injections difficult and the technical problem that not easily absorbs.
For realizing above technical purpose, the present invention by the following technical solutions:
A preparation method for porcine circovirus 2 type deactivation freeze dried vaccine, comprises the following steps:
1) get porcine circovirus 2 type virus-culturing fluid and carry out clarifying treatment, obtain virus liquid;
2) to step 1) virus liquid that obtains carries out ultrafiltration and concentration;
3) use molecular sieve chromatography to step 2) concentrated after virus liquid carry out purification;
4) beta-propiolactone is utilized to step 3) venom after purification carries out deactivation;
5) to step 4) virus liquid after deactivation implements lyophilizing, preparation.
Preferably, step 1) described clarifying treatment comprises the following steps: 6 ~ 10 layers of filtered through gauze first used to virus-culturing fluid, then filters with the double-layered filtration film of 0.5 ~ 2 μm.
Preferably, step 2) described concentrated multiple is 20 ~ 50 times.
Preferably, step 2) described ultrafiltration is cross-flow ultrafiltration, can be preferably 30 ~ 50K on this basis to the filter sizes that it uses.
Preferably, step 3) described molecular sieve chromatography chromatographic resin used be selected from Sepharose 4 FastFlow, Sepharose 6Fast Flow, Sepharose CL-2B or Sepharose CL-4B wherein a kind of, more excellent, select Sepharose 6Fast Flow resin.
Preferably, step 3) when utilizing column chromatography purification, by 5 ~ 8% (v/v) loading of column volume.
Preferably, step 3) virus titer>=10 in virus liquid after purification
8.5tCID
50/ ml, total protein concentration≤300ug/mL.
Preferably, step 4) described deactivation comprises the following steps: get beta-propiolactone dilution 700 ~ 900 times, by the beta-propiolactone solution after dilution with 1:(3 ~ 8) ratio of (v/v) joins step 3) in virus liquid after purification, deactivation 1 ~ 3h; On this basis can more preferably: inactivation process carries out under 35 ~ 40 DEG C of conditions.
Preferably, step 5) when implementing lyophilizing, freeze drying protectant used is grouped into by the one-tenth of following percetage by weight: trehalose 1 ~ 3%; Sucrose 5 ~ 8%; Lactose 1 ~ 2%; Polyvinylpyrrolidone 1 ~ 5%; Gelatin 3 ~ 6%; Glycine 1 ~ 3%; Alanine 0.5 ~ 1%; Sorbitol 1 ~ 10%; Bovine serum albumin 1 ~ 2%; Water surplus.
Preparation method can be:
(1) take trehalose by described weight proportion, sucrose, lactose, skimmed milk be dissolved in a small amount of water for injection, for subsequent use after autoclaving;
(2) taking glycine by described weight proportion is dissolved in a small amount of water for injection, degerming with 0.22 μm of membrane filtration, and-20 DEG C frozen for subsequent use;
(3) the solution 1:1 that step (1), (2) obtain is mixed, add to the full amount of water for injection and get final product.
In above technical scheme, successively clarifying treatment, ultrafiltration and concentration, sieve chromatography column purification are carried out to virus-culturing fluid, effectively ensure that in the antigen for the preparation of vaccine, impurity content reduces to minimum, thus reduce the phenomenon producing side reaction after product uses, contribute to ensureing immune effect of vaccine simultaneously.In addition, due to the present invention to the direct lyophilized formulations of the antigen after deactivation, do not add oily adjuvant, therefore do not exist be difficult to inject, absorb problem.In optimal technical scheme, filtered through gauze is selected to contribute to ensureing good clarifying treatment effect in conjunction with the pattern of the double-layered filtration film filtration of 0.5 ~ 2 μm, convenient operation simultaneously.Ultrafiltration and concentration is preferably after cross-flow ultrafiltration considers execution speed and filter effect and obtains.
The PCV2 deactivation freeze dried vaccine antigenic content utilizing the present invention to prepare is high, and immunogenicity is high; Antigen purity is high, and safety is good, and side reaction is low; Without adjuvant, produce antibody protection fast; Easy injection, easily absorbs.
Detailed description of the invention
Below will be described in detail the specific embodiment of the present invention.In order to avoid too much unnecessary details, in the examples below to belonging to known structure or function will not be described in detail.
The approximating language used in following examples can be used for quantitative expression, shows to allow quantity to have certain variation when not changing basic function.Therefore, this exact value itself is not limited to the numerical value that the language such as " approximately ", " left and right " is revised.In certain embodiments, " approximately " represents and allows its numerical value revised to change in the positive and negative scope of 10 (10%), such as, and any numerical value that what " about 100 " represented can be between 90 to 110.In addition, in the statement of " about first numerical value is to second value ", revise the first and second numerical value two numerical value approximately simultaneously.In some cases, approximating language may be relevant with the precision of gauge.
Apart from outside definition, technology used in following examples and scientific terminology have the identical meanings generally understood with those skilled in the art of the invention.
Embodiment 1 (vaccine preparation method)
1 instrument and reagent
1.1 embodiment instruments are as follows:
The double-deck filter element of 0.5-0.2um of Milipore company; The special sleeve of section hundred; Milipore company laterally cuts and stays ultrafiltration and concentration film bag, 100KD; GE company XK50/100 pillar; GE company AKTA Primer plus tomographic system; The Protein Detection instrument of Bio-Rad company; The small-sized freeze dryer of Pudong.
1.2 embodiment agents useful for same are as follows:
(1) 100L porcine circovirus 2 type virus liquid, 10
7.2tCID
50/ ml, is produced by our company;
(2) 1M NaOH 100L; 0.1M PBS100L; High purity water 100L;
(3) trehalose, sucrose, lactose, skimmed milk, glycine.
2 operating procedures
The clarification of 2.1 porcine circovirus 2 type venom
Under aseptic condition, the venom of results is filtered through 8 layers of sterile gauze, by the venom of initial filter, clarifies through 0.5um-2um double-layered filtration film;
Concentrating of 2.2 porcine circovirus 2 type venom
The porcine circovirus 2 type venom of clarification by laterally cutting the mode staying ultrafiltration and concentration, concentrated 30 times of virus liquid, wherein laterally cutting and staying ultrafiltration and concentration membrane aperture to be 50K.
The purification of 2.3 porcine circovirus 2 type venom
Use Sepharose 6Fast Flow (6FF) sieve chromatography to carry out purification to virus liquid, step is as follows:
2.3.1 post is filled
Connection chromatography is lived and column filler is also vertically fixed on iron stand, close chromatographic column lower end outlet, open the 0.01M PBS that column filler top adds 1/3 column volume in post, then the gel of grout shape is slowly at the uniform velocity poured in post to column filler top by Sepharose 6Fast Flow (6FF) medium while stirring continuously.Open the outlet of chromatographic column, be connected on column filler top with tomographic system, regulate suitable flow velocity, post pressure is advisable for 0.13MPa, gel is settled down to till glue face no longer declines, disconnection layer analysis system.Column filler and chromatographic column are carefully separated (ensureing that gap glue face is neat), tightening nuts after chromatographic column glue face carefully adds a small amount of 0.01MPBS as far as possible, after the adapter on adjustable column top is depressed into glue face, close chromatographic column lower end outlet.
2.3.2 aseptic process chromatographic column
Open chromatographic column lower end outlet, regulate suitable flow velocity, post is pressed as 0.1MPa is advisable, with 1M NaOH online treatment chromatographic column 3 column volumes.It is neutral that 0.01M PBS (aseptic) processes chromatographic column to pH.
2.3.4 loading purification
Get 30 times of concentrating virus liquid, keep flow velocity constant, by 5 ~ 8% loadings of column volume.Be 0.01mol/L PBS eluting by pH7.2 concentration after loading, elution speed is 10mL/min, and UV-detector detects Fraction collection first peak.First protein peak is viral peak, after sterile collection, and-20 DEG C of preservations.
2.3.5 virus titer is measured
Detect with the virus titer of IFA method to each sample, concrete steps are as follows:
1) bed board of cell and cultivation
Well-grown detection porcine kidney cell (PK15) digests by the EDTA-pancreatin with 0.25%, resuspended for 2 × 10 with cell growth medium
5/ ml is also inoculated in 96 porocyte culture plates, 100ul/ hole, is placed in 37 DEG C, 5%CO
224h is cultivated in cell culture incubator.
2) gradient dilution of virus
By the venom sample of each collection, carry out 10 times of doubling dilutions, 10 gradients (10 with 0.01M PBS
-1~ 10
-10).
3) viral inoculation and cultivation
Each gradient virus liquid after dilution is inoculated in the detection cell described in step (1), and 100ul/ hole, each gradient 8 repetition, arranges the cell controls not connecing virus liquid, 37 DEG C simultaneously, maintain 72h in 5%CO2 cell culture incubator.
4) IFA detects
After maintain terminates, abandon maintenance medium in most plate, cell is fixed, 100ul/ hole ,-20 DEG C, 5 ~ 10min with fixative (methanol acetone mixes, 1:1).
Abandon most fixative, cell plates are carried out air-dry.
Bovine serum albumin (BSA) solution with 1% carries out sealing treatment, 100ul/ hole, 37 DEG C, hatches 30 ~ 60min.
3 times are washed, 200 μ l/ holes with 0.1M PBS.The PCV2-cap protein monoclonal antibody (primary antibodie) that 500 ~ 700 times are diluted is made an addition to Tissue Culture Plate, and 100 μ l/ holes, hatch 30 ~ 60min by 37 DEG C.
Abandon most primary antibodie solution, wash 5 times with 0.01M PBS, 200 μ l/ holes.Add the sheep anti-mouse igg antibody (two resist) of fluorochrome (FITC) labelling that 800-1000 doubly dilutes, 100 μ l/ holes, hatch 30 ~ 60min by 37 DEG C.
Abandon two anti-solution to the greatest extent, wash 5 times with 0.01M PBS, 200 μ l/ holes, fluorescence microscopy Microscopic observation.
5) add up and calculate virus titer
There is the number in specific fluorescence hole in statistics, calculates with the titre (TCID50) of Reed-Muench method to swine fever virus, and result confirms that purified virus titre reaches 10
8.5tCID
50/ ml, viral recovery reaches 77%.
2.3.6 purified virus liquid total protein concentration is measured
Detect the purified virus of Fraction collection.Protein content adopts ultraviolet spectrophotometry, measures the optical density value of 280nm and 260nm respectively, calculates by formula protein content (mg/mL)=1.45 × A280nm –, 0.74 × A280nm.
The deactivation of 2.4 porcine circovirus 2 type venom
With 4 DEG C of cell culture fluids, beta-propiolactone is diluted 800 times, more slowly add in virus liquid by 1:5,4 DEG C are stirred deactivation 48h, and rearmounted 37 DEG C of water-baths 2 hours, stop deactivation.
The preparation of 2.5 vaccines
2.5.1 the preparation of freeze drying protectant
1) take trehalose by described weight proportion, sucrose, lactose, skimmed milk be dissolved in a small amount of water for injection, for subsequent use after autoclaving;
2) taking glycine by described weight proportion is dissolved in a small amount of water for injection, degerming with 0.22 μm of membrane filtration, and-20 DEG C frozen for subsequent use;
3) by step 1), 2) obtained solution 1:1 mixes, adds to the full amount of water for injection and get final product.
2.5.2 lyophilizing
The venom of deactivation is added freeze drying protectant by 1:1, aseptic subpackaged, lyophilizing.
Embodiment 2 (vaccine safety inspection prepared by embodiment 1)
The PCV2 deactivation freeze dried vaccine 1 prepared with above-mentioned technique, PCV2 white-oil adjuvant inactivated vaccine 2 prepared by common process, aseptic 0.01M PBS solution is as experiment reagent; Get 20 age in days PCV2ELISA negative antibodies, the healthy susceptible piglet 15 of PCV2 antigen negative as laboratory animal.Concrete operation step is as follows:
1 animal grouping
15 piglets are divided into 3 groups at random, often organize 5.
2 vaccine immunities
Musculi colli injects two kinds of vaccines and PBS uses piglet in test, operates as follows:
First group, every piglet musculi colli injection 4ml PCV2 deactivation freeze dried vaccine 1, totally 5;
Second group, PCV2 white-oil adjuvant inactivated vaccine 2 prepared by every piglet musculi colli injection 4ml common process, totally 5;
3rd group, every piglet musculi colli injection 4ml PBS, totally 5.
Continuous Observation 14 days after vaccine immunity.
3 experimental results
1) during immunity, PCV2 deactivation freeze dried vaccine is easily injected, and PCV2 white-oil adjuvant inactivated vaccine viscosity prepared by common process is large, injections difficult.
2), after immunity, PCV2 deactivation freeze dried vaccine group injection site is without any exception, and lump appears in PCV2 white-oil adjuvant inactivated vaccine group injection site prepared by common process, or even festers.
3) after immunity, PCV2 deactivation freeze dried vaccine group piglet daily gain is better than PCV2 white-oil adjuvant inactivated vaccine group note prepared by common process, and the piglet body temperature PCV2 white-oil adjuvant inactivated vaccine group that without obviously fluctuating prepared by common process has small size rising, result is as shown in table 1.
Table 1 respectively group pig weight increases and body temperature record
Embodiment 3 (vaccine potency inspection prepared by embodiment 1)
1 mice challenge test
With PCV2 deactivation freeze dried vaccine 1, PCV2 white-oil adjuvant inactivated vaccine 2 prepared by common process, aseptic 0.01M PBS solution is as experiment reagent; Get the healthy Balb/C mice 30 in 6 week age only as laboratory animal.Specific experiment step is as follows:
1.1 animal groupings
30 Balb/C mices are divided into 3 groups at random, often organize 10.
1.2 vaccine immunity
Lumbar injection two kinds of vaccines and PBS are in test mice, as follows operation:
First group, every mouse peritoneal injection 0.2ml PCV2 deactivation freeze dried vaccine 1, totally 10;
Second group, PCV2 white-oil adjuvant inactivated vaccine 2 prepared by every mouse peritoneal injection 0.2ml common process, totally 10;
3rd group, every mouse peritoneal injection 0.2ml PBS, totally 10.
1.3 animal counteracting toxic substances
Latter 21 days of each counteracting toxic substances group immunity, with PCV2-ZJ/C strain (10
7.0tCID
50/ ml) attack, lumbar injection 0.45ml/ is only.After counteracting toxic substances 21 days, slaughter, get spleen and carry out virus purification, get supernatant inoculation PK15 cell after grinding, in blind passage 3 generation, detect whether occur specific fluorescence cell with IFA method, occur that specific fluorescence person is judged to the virus purification positive, statistical analysis is often organized Murine Virus and is separated positive rate.
1.4 experimental result
First group, 10 mices are all that virus purification is negative;
Second group, there are 7 in 10 mices for virus purification feminine gender;
3rd group, 10 mices are all that virus purification is positive.
2 piglet immunological tests
With PCV2 deactivation freeze dried vaccine 1, PCV2 white-oil adjuvant inactivated vaccine 2 prepared by common process, aseptic 0.01M PBS solution is as experiment reagent; PCV2ELISA antibody assay kit (purchased from Ruipu (Baoding) Biological Pharmaceutical Co., Ltd.) is utilized to test; Get 20 age in days PCV2ELISA negative antibodies, the healthy susceptible piglet 15 of PCV2 antigen negative as laboratory animal.Specific experiment step is as follows:
2.1 experimental animal groupings
30 piglets are divided into 3 groups at random, often organize 10.
2.2 vaccine immunity
Musculi colli injects two kinds of vaccines and PBS uses piglet in test, operates as follows:
First group, every piglet musculi colli injection 2ml PCV2 deactivation freeze dried vaccine 1, totally 10;
Second group, PCV2 white-oil adjuvant inactivated vaccine 2 prepared by every piglet musculi colli injection 2ml common process, totally 10;
3rd group, every piglet musculi colli injection 2ml PBS, totally 10.
2.3 blood sampling and antibody tests
Take a blood sample weekly once before immunity and after immunity, totally 6 times, separation of serum, PCV2ELISA antibody assay kit detects.
2.4 experimental result
Experimental result shows, by first group of piglet antibody of PCV2 deactivation freeze dried vaccine immunity turn the sun time early than second group about 2 weeks, and immunity afterwards the antibody horizontal of the 6th week higher than second group about 1 times.
Embodiment 4 (vaccine preparation method)
A preparation method for porcine circovirus 2 type deactivation freeze dried vaccine, comprises the following steps:
1) get porcine circovirus 2 type virus-culturing fluid and carry out clarifying treatment, obtain virus liquid;
2) to step 1) virus liquid that obtains carries out ultrafiltration and concentration;
3) use molecular sieve chromatography to step 2) concentrated after virus liquid carry out purification;
4) beta-propiolactone is utilized to step 3) venom after purification carries out deactivation;
5) to step 4) virus liquid after deactivation implements lyophilizing, preparation.
On the basis of above technical scheme, step 1) described clarifying treatment comprises the following steps: 6 layers of filtered through gauze first used to virus-culturing fluid, then filters with the double-layered filtration film of 0.5 μm and 1 μm.Step 2) described ultrafiltration is cross-flow ultrafiltration.Step 2) described concentrated multiple is 20 times.Step 2) aperture of filter membrane used is 30K.Step 3) described molecular sieve chromatography chromatographic resin used is Sepharose CL-2B.Step 3) when utilizing column chromatography purification, by 7% (v/v) loading of column volume.Step 3) virus titer is 10 in virus liquid after purification
8.7tCID
50/ ml, total protein concentration is 221ug/mL.Step 4) described deactivation comprises the following steps: get beta-propiolactone and dilute 700 times, the beta-propiolactone solution after dilution is joined step 3 with the ratio of 1:3 (v/v)) in virus liquid after purification, deactivation 1h at 35 DEG C.Step 5) implement lyophilizing time, freeze drying protectant used is grouped into by the one-tenth of following percetage by weight: trehalose 1%; Sucrose 5%; Lactose 1%; Polyvinylpyrrolidone 1%; Gelatin 3%; Glycine 1%; Alanine 0.5%; Sorbitol 1%; Bovine serum albumin 1%; Water surplus.
Embodiment 5 (vaccine preparation method)
A preparation method for porcine circovirus 2 type deactivation freeze dried vaccine, comprises the following steps:
1) get porcine circovirus 2 type virus-culturing fluid and carry out clarifying treatment, obtain virus liquid;
2) to step 1) virus liquid that obtains carries out ultrafiltration and concentration;
3) use molecular sieve chromatography to step 2) concentrated after virus liquid carry out purification;
4) beta-propiolactone is utilized to step 3) venom after purification carries out deactivation;
5) to step 4) virus liquid after deactivation implements lyophilizing, preparation.
On the basis of above technical scheme, step 1) described clarifying treatment comprises the following steps: 10 layers of filtered through gauze first used to virus-culturing fluid, then filters with the double-layered filtration film of 1 μm and 2 μm.Step 2) described ultrafiltration is cross-flow ultrafiltration.Step 2) described concentrated multiple is 50 times.Step 2) aperture of filter membrane used is 50K.Step 3) described molecular sieve chromatography chromatographic resin used is Sepharose CL-4B.Step 3) when utilizing column chromatography purification, by 4% (v/v) loading of column volume.Step 3) virus titer is 10 in virus liquid after purification
8.9tCID
50/ ml, total protein concentration is 276ug/mL.Step 4) described deactivation comprises the following steps: get beta-propiolactone and dilute 900 times, the beta-propiolactone solution after dilution is joined step 3 with the ratio of 1:8 (v/v)) in virus liquid after purification, deactivation 3h at 40 DEG C.Step 5) implement lyophilizing time, freeze drying protectant used is grouped into by the one-tenth of following percetage by weight: trehalose 3%; Sucrose 8%; Lactose 2%; Polyvinylpyrrolidone 5%; Gelatin 6%; Glycine 3%; Alanine 1%; Sorbitol 10%; Bovine serum albumin 2%; Water surplus.
Embodiment 6 (vaccine preparation method)
A preparation method for porcine circovirus 2 type deactivation freeze dried vaccine, comprises the following steps:
1) get porcine circovirus 2 type virus-culturing fluid and carry out clarifying treatment, obtain virus liquid;
2) to step 1) virus liquid that obtains carries out ultrafiltration and concentration;
3) use molecular sieve chromatography to step 2) concentrated after virus liquid carry out purification;
4) beta-propiolactone is utilized to step 3) venom after purification carries out deactivation;
5) to step 4) virus liquid after deactivation implements lyophilizing, preparation.
On the basis of above technical scheme, step 1) described clarifying treatment realized by filter-cloth filtering.Step 2) described ultrafiltration be hollow fiber column vertical current filter.Step 2) described concentrated multiple is 40 times.Step 3) described molecular sieve chromatography chromatographic resin used is Sepharose 4Fast Flow.Step 5) implement lyophilizing time, freeze drying protectant used is grouped into by the one-tenth of following percetage by weight: trehalose 2%; Sucrose 6%; Lactose 1.5%; Polyvinylpyrrolidone 3%; Gelatin 4%; Glycine 2%; Alanine 0.75%; Sorbitol 5%; Bovine serum albumin 1.5%; Water surplus.
Embodiment 7 (vaccine preparation method)
A preparation method for porcine circovirus 2 type deactivation freeze dried vaccine, comprises the following steps:
1) get porcine circovirus 2 type virus-culturing fluid and carry out clarifying treatment, obtain virus liquid;
2) to step 1) virus liquid that obtains carries out ultrafiltration and concentration;
3) use molecular sieve chromatography to step 2) concentrated after virus liquid carry out purification;
4) beta-propiolactone is utilized to step 3) venom after purification carries out deactivation;
5) to step 4) virus liquid after deactivation implements lyophilizing, preparation.
Above the embodiment of the present invention has 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 preparation method for porcine circovirus 2 type deactivation freeze dried vaccine, is characterized in that comprising the following steps:
1) get porcine circovirus 2 type virus-culturing fluid and carry out clarifying treatment, obtain virus liquid;
2) to step 1) virus liquid that obtains carries out ultrafiltration and concentration;
3) use molecular sieve chromatography to step 2) concentrated after virus liquid carry out purification;
4) beta-propiolactone is utilized to step 3) venom after purification carries out deactivation;
5) to step 4) virus liquid after deactivation implements lyophilizing, preparation.
2. method according to claim 1, is characterized in that step 1) described clarifying treatment comprises the following steps: 6 ~ 10 layers of filtered through gauze first used to virus-culturing fluid, then filters with the double-layered filtration film of 0.5 ~ 2 μm.
3. method according to claim 1, is characterized in that step 2) described concentrated multiple is 20 ~ 50 times.
4. method according to claim 1, is characterized in that step 2) described ultrafiltration is cross-flow ultrafiltration.
5. method according to claim 4, is characterized in that step 2) aperture of filter membrane used is 30 ~ 50K.
6. method according to claim 1, is characterized in that step 3) described molecular sieve chromatography chromatographic resin used be selected from Sepharose 4 Fast Flow, Sepharose 6 Fast Flow, Sepharose CL-2B or Sepharose CL-4B wherein a kind of.
7. method according to claim 5, is characterized in that step 3) when utilizing column chromatography purification, by 5 ~ 8% (v/v) loading of column volume.
8. method according to claim 1, is characterized in that step 3) virus titer>=10 in virus liquid after purification
8.5tCID
50/ ml, total protein concentration≤300ug/mL.
9. method according to claim 1, it is characterized in that step 4) described deactivation comprises the following steps: get beta-propiolactone dilution 700 ~ 900 times, by the beta-propiolactone solution after dilution with 1:(3 ~ 8) ratio of (v/v) joins step 3) in virus liquid after purification, deactivation 1 ~ 3h.
10. method according to claim 1, is characterized in that step 5) implement lyophilizing time, freeze drying protectant used is grouped into by the one-tenth of following percetage by weight:
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| CN106279376A (en) * | 2016-08-31 | 2017-01-04 | 天津瑞普生物技术股份有限公司 | A kind of pig circular ring virus antigen purification method |
| CN106754764A (en) * | 2016-12-20 | 2017-05-31 | 天津瑞普生物技术股份有限公司 | A kind of purification process of 2 porcine circovirus virus-like particle |
| CN109055323A (en) * | 2018-07-26 | 2018-12-21 | 武汉汇研生物科技股份有限公司 | A kind of chromatography method of separating and purifying high-purity pig annulus whole virus vaccine |
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