CN101934074B - Porcine circovirus II vaccine and production method thereof - Google Patents
Porcine circovirus II vaccine and production method thereof Download PDFInfo
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- CN101934074B CN101934074B CN2010102750752A CN201010275075A CN101934074B CN 101934074 B CN101934074 B CN 101934074B CN 2010102750752 A CN2010102750752 A CN 2010102750752A CN 201010275075 A CN201010275075 A CN 201010275075A CN 101934074 B CN101934074 B CN 101934074B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention discloses a porcine circovirus II vaccine and a production method thereof. The production method comprises the following technical steps: (1) performing high-density culture of cells used for making the vaccine; (2) propagating a venom used for making the vaccine; and (3) adding an adjuvant to prepare the inactivated vaccine. Compared with the prior art, the method has the advantages of high virus yield, high toxic value, large production scale, high batch yield, low production cost, high and stable product quality and the like. The inactivated vaccine of the invention has high safety, induces immune protection effect in swine bodies and is in complete accordance with national standards for veterinary biological products.
Description
Technical field
The invention belongs to biological product technical field, relate to a kind of porcine circovirus II vaccine and production method thereof.
Background technology
Pig gyrate virus II type (being called for short PCV2) infects the various diseases such as pmws (PMWS), pig respiratory disorder syndrome (PRDC), porcine skin and nephrotic syndrome (PDNS) that cause and (is generically and collectively referred to as Porcine circovirus desease, PCVDs), its Clinical symptoms is that body weight alleviates gradually, and sign for example rapid breathing, dyspnea and jaundice.From pathology point, it shows as lymphocyte or granuloma infiltrates, lymphadenopathy and rarer hepatitis and lymphocyte or granulomatous nephritis.(Clark E.G. (1997) Proc.Am.Assoc.Swine Prac.499-501; La Semaine Veterinaire No.26, supplement to La Semaine Veterinaire 1996 (834); La Semaine Veterinaire 1997 (857): 54; The people such as Nayar (1997) Can.Vet.J.38:385-387).This disease was found (Clark EG.Post-weaning multisystemic wasting syndrome.Proc Am Assoc Swine Pract early than 1991 at Canada West, 1997,28:499-501), subsequently, this disease is in succession in the U.S., France, Spain, (the .Interstitial pneumonia and lymphadenophathy associated with circoviral infection in a six week-old pig.Proc Am Assoc Yet Lab Diag such as Daft B appears in the state such as Northern Ireland or area, 1996,39:32; The .Porcine circovirus infection in Northern Ireland.vet Rec such as Kennedy S, 1998,142:495 and 96; The .Piglet wasting disease.Tlet Rec such as LeCann P, 1997,141:660; The .First report of post-weaning multisystemic wasting syndrome in pigs in Spain.Vet Rec such as Segales J, 1997,141:600-660).At home, Lang Hongwu etc. (Lang Hongwu etc. the postweaning multsystemic wasting syndrome Serum Antibody Detection. Chinese veterinary's science and technology, 2000,30:3-5) and Cao Shengbo etc. (Cao Shengbo etc. full genomic clone and the sequence analysis of the A strain of pig B type circular virus Henan. viral journal, 2000,18:137-141) show by serology and Etiological respectively, the existing PCV2's of China is popular.At present, PCV2 has extensively existed also popular all over the world, caused sizable economic loss for global pig industry.
At present also do not treat and prevent the method for this disease.Yet multinomial evidence points out that pig circular ring virus is the pathogen (people (1998) Can.Vet.J.39:44-51 such as Ellis) of PMWS (pmws).Porcine circovirus reclaims from the pig that suffers from PMWS, and confirms in suffering from the pig body of this disease for the antibody of pig circular ring virus.Because this virus is a kind of DNA viruses, the virus variation rate is low, and worldwide pathogenic only has PCV2 one type simultaneously.And the biological characteristics of PCV2 is more special, and its viral titer on cell is very low, and does not cause cytopathy, and the difficulty of therefore developing the inactivated vaccine of PCV2 and attenuated vaccine is very large.
Produce at present both at home and abroad vaccine and mainly contain two kinds of techniques: (1) bioreactor: scale is less, generally is use for laboratory, can not large-scale production; It has stirring arm, take carrier as relying on cultured cell.The shortcoming of this method is: stir and produce shearing force, and bubble formation is arranged, affect Growth of Cells, there is technical bottleneck in large-scale production.(2) rolling bottle technology: at present suitability for industrialized production substantially all adopts this technology, when producing cell and virus labor intensity large, take up an area large, differences between batches are large, production cost is high, single batch yield poorly, be difficult to carry out the quality control of standardized production.
Also do not have the PCV2 vaccine of really effectively also ratifying through the Ministry of Agriculture, main cause also to be in China, the growth titre of PCV2 in cell is lower, generally only is 10
4.0TCID
50About/ml.Therefore improve viral titer, guarantee that good clinical effectiveness is the task of top priority that solves the porcine circovirus II vaccine industrialization.
Summary of the invention
Main purpose of the present invention provides a kind of production method of porcine circovirus II vaccine, comprises the steps:
1) in the microcarrier bioreactor, microcarrier density is 60～80g/L, and every gram carrier adds 1.4 * 10
7～2.6 * 10
7After individual passage cell and cell growth medium, active cell absorption program made microcarrier and the abundant combination of passage cell, active cell was cultivated program, subculture cell;
2) above-mentioned passage cell is cultured to 5.0 * 10
9Cells/L～8 * 10
10Using cell maintenance medium during cells/L instead, is M.O.I.=0.001～1 Pigs Inoculated circovirus type II according to infection multiplicity, starts the viruses adsorption program, and virus is fully contacted with passage cell, uses the Virus culture program instead, the amplification cultivation pig gyrate virus II type;
3) behind Virus culture 24～36h, discard cell maintenance medium, wash passage cell with buffer solution, adding is hatched agent and is hatched 30～45min, discards and hatches agent, washes passage cell with buffer solution, adds cell maintenance medium and continues to cultivate;
4) the results culture fluid is processed through cell breakage, obtains the Porcine Circovirus virus liquid.
5) virus liquid adds inactivator, adds adjuvant, emulsifying agent after the deactivation, makes the Porcine Circovirus vaccine.
Preferably, bioreactor of the present invention is the microcarrier suspension culture bioreactor.
More preferably, bioreactor of the present invention is tidal type microcarrier suspension culture bioreactor.
Preferably, microcarrier of the present invention is spherical, netted or lamellar microcarrier.
More preferably, microcarrier of the present invention is polyester, gelatin or polysaccharide.
Preferably, cell growth medium the method for the invention step 1) is the DMEM culture medium that contains 3%～10% Ox blood serum.
More preferably, the content of Ox blood serum of the present invention is 5%～7%
Most preferably, the content of Ox blood serum of the present invention is 6%
Preferably, cell maintenance medium of the present invention is the DMEM culture medium that contains 1%～5% Ox blood serum.
More preferably, the content of Ox blood serum of the present invention is 2%～3%
Most preferably, the content of Ox blood serum of the present invention is 2%
It is preferably, of the present invention that to hatch agent be D-glucosamine, interferon or lipopolysaccharide.
It is more preferably, of the present invention that to hatch agent be D-glucosamine.
Preferably, the condition of culture in the bioreactor of the present invention is 37 ℃ of temperature, and CO2 concentration is 5%, and medium pH value is 7.0～7.6.。
Preferably, the volume of Porcine Circovirus virus liquid is 2.5L～1000L the method for the invention step 4).
Another aspect of the invention is the pig gyrate virus II type virus liquid that uses the inventive method preparation.
Another aspect of the present invention is for using the porcine circovirus II vaccine of the inventive method preparation.
Technique effect
Compared with prior art, the production method of porcine circovirus II vaccine of the present invention has following beneficial effect:
(1) malicious valency is high: the PCV2 viral titer of traditional rolling bottle explained hereafter only is 10
4.0TCID
50/ ml, and the present invention adopts new technical parameter, uses tidal type microcarrier suspension culture technology high density to cultivate the viral titer of producing and can reach 10
6.0TCID
50/ ml～10
7.0TCID
50/ ml, its malicious valency will exceed 100-1000 doubly.
(2) production scale is large, single batch of output is high: present domestic employing stirring-type suspension culture technique is cultivated zooblast, and separate unit bioreactor maximum-norm is no more than 100L; And the present invention adopts new technical parameter, uses tidal type microcarrier suspension culture technology to cultivate zooblast, and separate unit bioreactor scale reaches 500L, and maximum can reach 1000L, and the separate unit scale improves 5-10 doubly.
(3) the continuous sealing enclosed is produced, gathered in the crops virus: method of the present invention can totally-enclosedly be produced, and certainly moves liquid, and harvesting approach is collected virus liquid continuously, has reduced the probability of polluting, the product quality stable homogeneous, and differences between batches are little.Solved traditional handicraft and only can control temperature and rotating speed, the problem that the different batches mass discrepancy is large, differences between batches are large.The inventive method, directly linear amplification for the production of.
(4) production cost is relatively low, product quality is high and stable: the PCV2 viral titer that traditional rolling bottle production technology is produced only is 10
4.0TCID
50/ ml will reach 10 of regulations requirement
5.0TCID
50/ ml need to carry out concentration, and the production cost of every ml is 3 yuan, and the PCV2 viral titer of explained hereafter of the present invention on average can reach 10
6.5TCID
50/ ml, the production cost of every ml are 0.5 yuan, 6 times of cost decreases, and quality improves 100 times (in malicious valency).Different culture systems propagation pig circular ring virus comparative results see Table 1.
The correlation ratio of the different culture systems propagation of table 1 pig circular ring virus
Remarks: the adherent area 1g=2400cm of carrier
2, the amount of 1 microcarrier suspension culture system adding carrier is pressed 1600g and is calculated.
Description of drawings
Fig. 1 is the cell inoculation microphotograph on the microcarrier in the time of 0 day;
Fig. 2 is virus inoculation microphotograph on the microcarrier in the time of 5 days;
Fig. 3 is virus inoculation microphotograph on the microcarrier in the time of 10 days;
Fig. 4 is tidal type microcarrier suspension culture bioreactor construction schematic diagram.
The specific embodiment
Used tidal type microcarrier suspension culture bioreactor among the embodiment, the microcarrier suspension culture bioreactor of other types, such as stirring-type, rotary or filling type microcarrier suspension culture bioreactor, all can use the present invention's method large-scale production PCV2 virus or vaccine.Preferably, the present invention uses tidal type microcarrier suspension culture bioreactor, and the culture medium in the time of can improving cultivation and the supply of dissolved oxygen are little without bubble and shearing force, and be little to cell damage.
The bioreactor that adopts in the embodiment of the invention is the tidal type bioreactor.Structural representation as shown in Figure 4.Wherein, each labelling is respectively: constant temperature stirring system 1, the culture medium constant temp cell body 2 of getting the raw materials ready, automatically present material system 3, constant incubator 4, passage cell 5, microcarrier 6, DO detector and pH controller 7, catcher 8.More preferably, adopted the tidal type bioreactor system that designs according to the morning and evening tides principle in the embodiment of the invention, culture systems is divided into two parts; One is the carrier bottle, and another is that culture medium stirs bag (groove).Cell is fixed on the carrier bottle, and media flow causes intermittent exposure and floods carrier between carrier bottle and agitator tank.In embodiments of the present invention, reactor used carrier bottle is long-pending for 5L, 10L, 20L, 50L, 100L, all can automatically control temperature, pH value, dissolved oxygen, gas concentration lwevel.
In the embodiment of the invention, passage cell has used PK15, the passage cell that other this areas are commonly used such as PKK cell (PK15 clone cell), RK cell (rabbit kidney cell), Vero cell (African green monkey kidney cell), ST cell (pig testis cell), Dulac cell (porcine kidney cell) also can be used for the present invention's method large-scale production PCV2 virus or vaccine.
In the method for the invention, in cell absorption microcarrier stage and cell culture stage, cell absorption program and cell culture program have been started, optimized the control parameter of reactor in the embodiment of the invention, but the inventive method is not limited only to the parameter among the embodiment, those skilled in the art can be according to technology enlightenment provided by the invention, according to different bioreactors, adjust corresponding parameter, reach microcarrier and cell fully in conjunction with after, the purpose of a large amount of amplifying cells.
In the method for the invention, in viruses adsorption microcarrier stage and Virus culture stage, viruses adsorption program and Virus culture program have been started, optimized the control parameter of reactor in the embodiment of the invention, but the inventive method is not limited only to the parameter among the embodiment, those skilled in the art can be according to technology enlightenment provided by the invention, according to different bioreactors, adjust corresponding parameter, reach virus and cell and microcarrier fully in conjunction with after, the purpose of a large amount of amplicon virus.
In the embodiment of the invention, used and hatched agent and be D-glucosamine, other types hatch agent, such as IFN (interferon), LPS (lipopolysaccharide), also can be used for hatching before the virus harvest.
It is 60～80g/L that the present invention has tested microcarrier density, and every gram microcarrier adds 1.4 * 10
72.6 * 10
7The passage cell of cells, more preferably, the microcarrier density of using in the embodiment of the invention is 80g/L, cell density is 2.0 * 10
7The cells/g microcarrier.
The density that the present invention has tested when passage cell reaches 5.0 * 10
9Cells/L～8.0 * 10
10During cells/L, when namely density reaches 5～40 times of initial density, use cell maintenance medium instead, initial viruses adsorption and cultivation program are good.
It is M.O.I.=0.001～1 inoculation PCV2 that the present invention has tested according to infection multiplicity, the pig gyrate virus II type poison valency of results is the highest, preferably, has used the M.O.I.=0.1 virus inoculation in the embodiment of the invention, start viruses adsorption program and Virus culture program, the amplification pig gyrate virus II type.
Cell growth medium of the present invention has used the DMEM culture medium that contains 3%～10% Ox blood serum; More preferably, to have used the content of Ox blood serum be 5%～7% in the present invention; The content of the Ox blood serum of the DMEM culture medium of most preferably, using in the embodiment of the invention is 6%
Cell maintenance medium of the present invention has used the DMEM culture medium that contains 1%～5% Ox blood serum; More preferably, to have used the content of Ox blood serum be 2%～3% in the present invention; Most preferably, having selected the content of Ox blood serum in the embodiment of the invention is 2% DMEM culture medium.
The present invention has tested after the Virus culture time is 24～36h, discards cell maintenance medium, washes passage cell with buffer solution; Preferably, use the Virus culture time of 24h in the embodiment of the invention, washed passage cell with PBS.
It is 30～45min that the present invention has tested the incubation time of hatching agent, and preferably, incubation time is 30min in the embodiment of the invention.
The buffer solution that the present invention washes passage cell is conventional PBS, and preferably, the embodiment of the invention has been used the PBS buffer of 0.01mol/L.Preferably, adopt the method for multigelation in the embodiment of the invention, make cell come off fully, disperse, thereby obtain virus liquid.The present invention also can use the method for other this areas smudge cells commonly used, obtains virus liquid.
The preparation method of vaccine adds oil adjuvant, emulsifying agent for adding the formalin-inactivated agent in the embodiment of the invention after the deactivation, makes porcine circovirus II vaccine.Ablation method or inactivator that other are commonly used such as β-third lactone, dimethyleneimine, also can be used for preparing vaccine of the present invention.The adjuvant of other vaccine preparations commonly used of this area such as white oil, other aqueous adjuvants etc., also can be used for preparing vaccine of the present invention.
Embodiment of the invention method comprises following operating procedure:
(1) seedling is inoculated in the carrier tank that contains Growth of Cells usefulness culture fluid and microcarrier with host cell, and with above-mentioned cell and microcarrier mix homogeneously, cell is attached on the microcarrier; Under suitable culture environment, the enough nutrients of above-mentioned Growth of Cells and gaseous environment are provided, make cell grow to 5～40 times of inoculum density at above-mentioned microcarrier;
(2) cell growth medium is replaced by keep and uses culture fluid, and add synchronously pig gyrate virus II type kind poison, it is adsorbed on the cell, then cultivate virus of proliferation;
(3) behind the cultivation 24h cell maintenance medium is discarded, the cell of virus inoculation fully washs with the PBS of 0.01mol/L, adds D-glucosamine and hatches 30min;
(4) discard D-glucosamine, with PBS (pH7.4) the fully washing of cell with 0.01mol/L, add cell maintenance medium and continue cultured cell;
(5) continuous culture is after 10 days, the results virus liquid, in-20 ℃ freezing, 20 ℃ melt, repeatedly twice, obtain pig gyrate virus II type (PCV2) virus liquid;
(6) virus liquid adds formalin-inactivated through after the assay was approved, and the pig gyrate virus II type after the deactivation (PCV2) adds conventional oil adjuvant or two-phase adjuvant, and stirring and evenly mixing makes oil emulsion vaccine or two-phase vaccine.
In the technique scheme, described host cell be for can breeding the cell line of pig circular ring virus virus II type (PCV2), such as PK15 cell line etc.
In the technique scheme, described carrier is polyester (or gelatin or polysaccharide) microcarrier of netted (or spherical or lamellar).
In the technique scheme, microcarrier consumption described in the step (1) is that the initial inoculum of 80g/L, cell is 1.4 * 10
7～2.6 * 10
7The cells/g microcarrier is better.
In the technique scheme, cell density is 5.0 * 10 during virus inoculation described in the step (2)
9Cells/L～8.0 * 10
10Cells/L.
In the technique scheme, the environment of cell culture is 37 ℃ of temperature, CO
2Concentration is 5%, and medium pH value is 7.0-7.6.
In the technique scheme, during virus inoculation described in the step (2) be 0.01～1 ratio in viral infection plural number (M.O.I.).
In the technique scheme, begin the cultivation program behind the general attaching 4h during cell culture described in the step (1).
In the technique scheme, beginning Virus culture program behind general absorption 4h during virus inoculation described in the step (2).
Cultured cell and virus in the inventive method can be carried out in bioreactor.Bioreactor mainly comprises carrier bottle, fluid reservoir, pH controller, DO monitor, input and output system.Work process is as follows: cell is attached in the carrier bottle grows on the carrier, and when the culture fluid of fluid reservoir was pumped to the carrier bottle, the culture fluid liquid level rose and to supply with nutrient and to promote the removal of cell metabolism product to cell; When the culture fluid of carrier bottle pumps into fluid reservoir, cultivate liquid level and descend thereupon, cell is ventilated, promote to breathe, reduce the cell tangential pressure, without O
2The supply restriction, non-foam is worried.The motion of this repetition makes the cell on the carrier can access enough nutrition and O
2, produced simultaneously metabolic waste is as CO
2Can effectively be discharged from, thus amplifying cells that can be a large amount of and virus of proliferation, and this kind technology is referred to as tidal type microcarrier suspension culture technology.
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention, NM concrete experimental technique in the following example carries out according to the normal experiment method usually.
Embodiment 1 tidal type microcarrier suspension bioreactor large-scale production pig gyrate virus II type (PCV2)
Employed bioreactor is tidal type microcarrier bioreactor in the present embodiment, and employed microcarrier is polyester fiber.Wide 5mm, long 10mm, the 1g carrier provides 2,400cm
2Adherent area, can provide 1.0 * 10
9The Growth of Cells of above quantity.
(1) with PK15 cell 3.2 * 10
10Cells is inoculated in the 20L carrier tank and adds carrier polyester fiber 1600g, and working volume is that culture fluid is used in the DMEM growth that contains 6% Ox blood serum of 500L, and tidal type microcarrier suspension culture technology is cultured to total cellular score and reaches 1.28 * 10
12Cells; Start first the attaching program during cultured cell, change the cultivation program behind the 4h into; Cell attaching program is: Up:2500m l/min; Hold 1min; Down:2500ml/min; Hold 30s sets maximum and changes liquid measure 18.5L; Cell culture program: Up:1900ml/min; Hold lmin; Down 1900ml/min; Hold 1min sets maximum and changes liquid measure 18L.
(2) Growth of Cells all is replaced by with culture fluid contains keeping of 2% Ox blood serum and use the DMEM culture fluid, and (M.O.I.=0.1) adds pig gyrate virus II type (PCV2) synchronously, in 37 ℃ of cultivations, control gas concentration lwevel 5%, pH value 7.2; Start first the viruses adsorption program during virus inoculation, change the Virus culture program behind the 4h into; The viruses adsorption program is: Up:1600ml/min; Hold 1min; Down:1600ml/min; Hold 30s, setting maximum are changed liquid measure 18.5L; Virus culture program: Up:1000ml/min; Hold 1min; Down 1000m1/min; Hold 1min, setting maximum are changed liquid measure 18L.Wherein, Up be culture fluid by the rate of climb of microcarrier, Down be culture fluid by the decrease speed of carrier, Hold is that microcarrier is immersed in the time in the culture fluid;
(3) behind the cultivation 24h maintenance medium in the carrier tank is discarded, cell fully washs with the PBS (pH7.4) of 0.01mol/L, adding 300mmol/L D-glucosamine is hatched 30min, and (the D-glucosamine addition is as the criterion just to flood carrier, device program suspends between incubation period, static hatching);
(4) discard D-glucosamine, the PBS (pH7.4) of cell with 0.01mol/L fully washed to remove D-glucosamine residual on the carrier, fill it up with cell maintenance medium and continue cultured cell;
(5) continuous culture was gathered in the crops virus liquid after 10 days, in-20 ℃ freezing, 20 ℃ melt, repeatedly after twice, carry out the virus liquid check:
(a) pure property check: test by " People's Republic of China's veterinary drug allusion quotation " 2005 editions appendix 15,19,20 pages of relevant regulations, the result pollutes without antibacterial, mycete, mycoplasma and exogenous virus.
(b) viral level is measured: virus liquid is done 10 times of gradient series dilutions with containing 300mmol/L D-glucosamine and 2% Ox blood serum cell maintenance medium, from 10
-1To 10
-6Each 8 hole of dilution factor inoculation sets up feminine gender not connect the poison contrast simultaneously, puts into 5%CO
2Cultivate 48～72h for 37 ℃ in the incubator, 80% acetone is fixed, and measures the hole count that each dilution factor contains PCV2 positive cell (green fluorescence) with immunofluorescence antibody (IFA) method, calculates virus liquid TCID according to the Reed-Muench method
50, every 1.0ml contains virus 〉=10
6.5TCID
50
(c) specificity: measure with indirect immunofluorescence antibody (IFA) method.Virus liquid is inoculated in 96 porocyte plate PK15 cells, each sample 4 hole, every hole 200 μ l set up negative control simultaneously, put into 5%CO
2Cultivate 48～72h for 37 ℃ in the incubator; Discard growth-promoting media, with PBS buffer (pH7.4) washed cell of 0.01mol/L 2 times, then add 80% acetone soln of 100 μ l pre-coolings, 4 ℃ of fixing 30min.Then with PBS washing 3 times; Discard maintenance medium, after PBS washed 3 times, every hole added 100 μ l PBS1: the anti-PCV2 serum of pig of 100 dilutions, 37 ℃ of effect 1h; With PBS washing 3 times, behind each 3min; Add and use PBS1: two anti-(IgG-FITC) of the anti-pig IgG of fluorescently-labeled rabbit of 100 dilutions, every hole 100 μ l, 37 ℃ of effect 1h; With PBS washing 3 times, each 3min is at the fluorescence microscopy Microscopic observation.Cell control well should occur without specificity fluorescent, and the virus inoculation cell hole should have a large amount of specificity fluorescents to occur.
Preparation and the immune efficacy evaluation of embodiment 2 pig gyrate virus II types (PCV2) inactivated vaccine
1. materials and methods
1.1 vaccine
The pig gyrate virus II type that embodiment 1 obtains (PCV2) virus liquid adds 206 adjuvants (French SEPPIC company product) in 1: 1 ratio, and fully mix homogeneously gets namely that (lot number is: 0803,0804,0805,0806 and 0807).
1.2. experimental animal
PCV2ELISA antibody and PCV2 antigen negative piglet are available from Luoyang, henan.
1.3. safety testing
A 1.3.1. single dose inoculation of minimum the age of immune piglet
Get 30 of 7 age in days suckling pigs, be divided into 6 groups, 5 every group, 5 batches of vaccines of l～5 group intramuscular injection, dosage of inoculation 2ml/ head; The 6th group is the blank group, and the rear isolated rearing 30 days of numbering and weigh is observed piglet and had or not clinical symptoms.Take temperature 1～7 day every day (rectal temperature) after the inoculation.
1.3.2. piglet single dose repeated inoculation
Get 30 of 15～18 age in days suckling pigs, be divided into 6 groups, 5 every group, 5 batches of vaccines of the 1st～5 group of intramuscular injection, dosage of inoculation 2ml/ head in 2 weeks after the inoculation, is used the same dose immunity once again; The 6th group is the blank group.Numbering and the rear isolated rearing 30 days of weighing are observed piglet and are had or not clinical symptoms.Take temperature 1～7 day every day (rectal temperature) after the inoculation.
A 1.3.3. overdose inoculation of piglet
Get 30 of 15～18 age in days suckling pigs, be divided into 6 groups, 5 every group, 5 batches of vaccines of the 1st～5 group of intramuscular injection, dosage of inoculation 4ml/ head; The 6th group is the blank group, and the rear isolated rearing 30 days of numbering and weigh is observed piglet and had or not clinical symptoms.Take temperature 1～7 day every day (rectal temperature) after the inoculation.
1.4. antibody test and protest test:
Select 35 15～18 age in days PCV2ELISA antibody and PCV2 antigen negative ablactational baby pig, be divided at random 7 groups, 5/group, 1st, 2,3,4,5 groups of immunity 0803,0804,0805,0806 and 0807 batch of inactivated vaccine, the musculi colli injecting immune, the 2ml/ head, two weeks are rear with identical immunizing dose booster immunization once; The 6th group is the counteracting toxic substances matched group; The 7th group is the blank group, only inoculates keyhole hemocyanin (KLH/ICFA) and thioglycollate medium.Two exempt from rear 3 weeks uses PCV2 virus attack, intramuscular injection, 3 * 10
5.0TCID
50/ head, the keyhole hemocyanin (KLH/ICFA, 0.5mg/ml) of using incomplete Freunds adjuvant emulsifying in two oxters and 4 inoculations of two hip portion of pig respectively on the 4th, 7 day behind the counteracting toxic substances, 4mL/ head, intraperitoneal inoculation thioglycollate medium, 10ml/ head; The the 11st, 19 day intraperitoneal inoculation thioglycollate medium only behind the counteracting toxic substances, the 10ml/ head.Detect index: blood sampling in 14,21,35 days after exempt from respectively at head (1), measure ELISA antibody and NAT, observe antibody and produce dynamically.(2) 1～20 day take temperature behind the counteracting toxic substances is observed clinical symptoms.(3) pathological study.
1.5.ELISA antibody test
With the PCV2-ORF2 albumen of escherichia coli expression as antigen, by the coated concentration of the best of square formation burette test defined antigen.With antigen diluent coated elisa plate after the best coated concentration, 100 μ l/ holes, 4 ℃ of coated spending the night behind 37 ℃ of effect 2h; Wash each 3-5min 3 times; Every hole adds the 0.15%BSA confining liquid sealing plank of 200 μ l, 37 ℃ of effect 2h; Washing; With serum to be checked PBS doubling dilution, each sample delegation, every hole adds 100 μ l, 37 ℃ of effect 1h; Washing; Then add enzyme target SPA (1: 10000 times of dilution), 100 μ l/ holes, 37 ℃ of effect 1h; Washing; Add substrate solution TMB (3 ', 3 ', 5 ', 5 ' ,-tetramethyl benzidine) colour developing, use at last the H of 2mol/L
2SO
4Cessation reaction.The result judges: serum OD to be checked
450Value/negative serum OD
450Value 〉=2.1 is positive.
1.6. serum neutralization test
Adopt fixed virus diluted blood therapy for clearing away heat.56 ℃ of serum to be checked heating 30min, the centrifugal 5min of 10000rpm, careful sucking-off supernatant carries out doubling dilution after doing dilution in 1: 2; Respectively with equivalent 100TCID
50The PCV2 virus liquid mixes, and 37 ℃ of 1h are inoculated in 96 orifice plates that contain the PK15 cell monolayer, 100 μ l/ holes, and each dilution factor is inoculated 4 holes, establishes simultaneously cell contrast and virus control hole.Cultivate 12h for 37 ℃, process with the D-glucosamine of 300mmol/L, 37 ℃ are continued to cultivate 48h, and 80% acetone fixed cell is measured the hole count that each dilution factor contains fluorescence with indirect immunofluorescence.Serum greatest dilution with the cell hole that can suppress 50% specificity fluorescent cell number is tired as the neutralization of serum to be checked, and calculates every cell mean.
1.7. pathological examination
Carry out according to a conventional method pathological anatomy, observe the internal organs pathological change, and gather internal organs 4% formalin such as lungs, spleen, lymph node fixing after, preparation paraffin section, HE dyeing, microscopic examination lesion tissue.
2. result
2.1. vaccine safety
The 5 batches of vaccines do respectively after a single dose inoculation of minimum age in days piglet and piglet single dose repeated inoculation and the overdose inoculation of piglet all that performance physically well develops, and the mental status, appetite are normal, without fervescence and other untoward reaction.Prove that this vaccine safety is good.The results are shown in Table 2～4.
A single dose inoculation of table 27 age in days piglets safety testing result
Table 3 piglet single dose repeated inoculation safety testing result
An overdose inoculation of table 4 safety testing result
2.2. immune swine PCV2 antibody test
Rear 14 days of immunity, immune group all can detect PCV2 antibody; Rear 35 days of first immunity, vaccine immunity group ELISA antibody and NAT reach more than 1: 3200 and 1: 32 respectively, and ELISA antibody and neutralizing antibody level are basically identical.The results are shown in Table 5.
Table 5 piglet protest test antibody test result
2.3. protest test
2.3.1. clinical symptoms
The 1st～3 all fervescence (＞40 ℃) behind all pig counteracting toxic substances of counteracting toxic substances matched group continue 3～6 days, occur, a loss of appetite phenomenon thick disorderly by hair behind the counteracting toxic substances on the 10th day, and 1 death was arranged on the 11st day; The blank pig remains normally at the interim body temperature of whole counteracting toxic substances, without unusual clinical manifestation.0803, the pig of 0804,0805,0806 and 0807 batch of vaccine immunity all had 2～3 pigs body temperature to occur and surpasses 40 ℃ in the 1st week behind the counteracting toxic substances, continue 1～2 day, but was showed no obviously unusual clinical manifestation.5 batches of vaccine protection efficient difference 100%, 100%, 100%, 100% and 100%.The results are shown in Table 6.
2.3.2. body weight change
In order to estimate vaccine to the protection effect of pig body; we weigh to the pig body before counteracting toxic substances and when slaughtering respectively; calculate and respectively organize the average relative daily gain; utilize statistics software SPSS17.0 to carry out statistical analysis; the result shows the relative daily gain of vaccine immunity group pig similar to the blank group (P=0.5＞0.05); but apparently higher than nonimmune counteracting toxic substances matched group (P=0.02＜0.05), prove that vaccine all has better immanoprotection action.
2.3.3. pathological change
Behind the counteracting toxic substances the 11st day, 1 death of counteracting toxic substances contrast pig, pathology is analysed and is shown as Pulmonary hemorrhage, elasticity step-down, under groin, the ilium, under the jaw, mesenteric adenophyma is swollen, hemorrhage, spleen edge slight bleeding etc.Behind the counteracting toxic substances the 20th day, i.e. during off-test, slaughter all test pig, carry out pathological anatomy and histopathological examination.Result of the test shows that nonimmune counteracting toxic substances matched group pig has obvious naked eyes pathological change, lymph node tissue medium-sized lymphocyte disappearance, macrophages infiltration, inclusion body pathological changes; The lungs tissue has monocyte infiltration; And the immune swine pathological change is very not obvious.Concrete outcome sees Table 7.
Pig clinical symptoms statistical result in 20 days behind table 6 counteracting toxic substances
※: disorderly thick by hair, palor, loss of appetite has the phenomena of mortality.
Table 7 counteracting toxic substances swine diseases reason changes statistical result
3. conclusion
This research is inoculated piglets with 5 batches of porcine circovirus type II inactivated vaccines, and all without unusual clinical manifestation, safety is fine for the result; Produced ELISA antibody and neutralizing antibody in 14 days behind the piglet immunological, head exempts from rear 35 days counteracting toxic substances, and without unusual clinical manifestation and pathological change, immunoprotection efficient reaches 100%, and immune effect is good.And adopt new technological parameter to use tidal type microcarrier cell suspension culture system to produce no matter the pig circular ring virus inactivated vaccine is the indexs such as antigenic content, output or production cost with the present invention, be much better than spinner culture system commonly used.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (16)
1. the production method of a Porcine Circovirus vaccine is characterized in that, comprises the steps:
1) in the microcarrier bioreactor, microcarrier density is 6080g/L, and every gram carrier adds 1.4 * 10
7～2.6 * 10
7After individual passage cell and cell growth medium, active cell absorption program made microcarrier and the abundant combination of passage cell, active cell was cultivated program, subculture cell;
2) above-mentioned passage cell is cultured to 5.0 * 10
9Cells/L～8.0 * 10
10Using cell maintenance medium during cells/L instead, is M.O.I.=0.001～1 inoculation Porcine Circovirus according to infection multiplicity, starts the viruses adsorption program, and virus is fully contacted with passage cell, uses the Virus culture program instead, the amplification cultivation Porcine Circovirus;
3) behind Virus culture 24～36h, discard cell maintenance medium, wash passage cell with buffer solution, adding is hatched agent and is hatched 30～45min, discards and hatches agent, washes passage cell with buffer solution, adds cell maintenance medium and continues to cultivate;
4) the results culture fluid is processed through cell breakage, obtains the Porcine Circovirus virus liquid;
5) virus liquid adds inactivator, adds adjuvant, emulsifying agent after the deactivation, makes the Porcine Circovirus vaccine.
2. method according to claim 1 is characterized in that, described bioreactor is the microcarrier suspension culture bioreactor.
3. method according to claim 1 is characterized in that, described bioreactor is for being tidal type microcarrier suspension culture bioreactor.
4. method according to claim 1 is characterized in that, described microcarrier is spherical, netted or lamellar microcarrier.
5. method according to claim 1 is characterized in that, described microcarrier is polyester, gelatin or polysaccharide.
6. method according to claim 1 is characterized in that step 1) described in cell growth medium be the DMEM culture medium that contains 3%～10% Ox blood serum.
7. method according to claim 6 is characterized in that, the content of described Ox blood serum is 5%～7%.
8. method according to claim 6 is characterized in that, the content of described Ox blood serum is 6%.
9. method according to claim 1 is characterized in that step 2) described in cell maintenance medium be the DMEM culture medium that contains 1%～5% Ox blood serum.
10. method according to claim 9 is characterized in that, the content of described Ox blood serum is 2%～3%.
11. method according to claim 9 is characterized in that, the content of described Ox blood serum is 2%.
12. method according to claim 1 is characterized in that step 3) described in the agent of hatching be D-glucosamine, interferon or lipopolysaccharide.
13. method according to claim 1 is characterized in that, the condition of culture of described bioreactor is 37 ℃ of temperature, CO
2Concentration is 5%, and medium pH value is 7.0～7.6.
14. method according to claim 1 is characterized in that: step 4) described in the volume of Porcine Circovirus virus liquid be 2.5L～1000L.
15. Porcine Circovirus virus liquid by the preparation of claim 1-14 any one method.
16. Porcine Circovirus vaccine by the preparation of claim 1-14 any one method.
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