CN101773667A - Preparation method for vaccine of porcine circovirus II - Google Patents
Preparation method for vaccine of porcine circovirus II Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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Abstract
The invention discloses mass production of inactivated vaccine of porcine circovirus II (PCV2) and a preparation method thereof. The preparation method comprises the following technical steps: (1) high-density culture of cells for vaccine preparation; (2) reproduction of venom for vaccine preparation; and (3) addition of adjuvant to prepare the inactivated vaccine. Compared with the prior art, the invention has the advantages that the virus yield is high, the virus titer is high, the production scale is large, the yield of a single batch is high, the production cost is relatively low, the product quality is high and stable, the operation is convenient, the operation space is small, the technological parameters are controlled accurately and the like. The inactivated vaccine has high safety, can induce pig bodies to generate immune protection and fully satisfies the national biological product standards.
Description
Technical field
The invention belongs to the veterinary biologics technical field, relate to animal virus, more specifically relate to the preparation method of pig circular ring virus II type vaccine.
Background technology
Pig circular ring virus II type (being called for short PCV2) infects the pmws (PMWS), pig respiratory disorder syndrome (PRDC), Corii Sus domestica skin and the nephrotic syndrome multiple diseases such as (PDNS) that cause and (is generically and collectively referred to as the pig circular ring virus 2 viral disease, 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 is soaked into, lymphadenopathy and rarer hepatitis and lymphocyte or granulomatous nephritis.(Clark E.G. (1997) Proc.Am.Assoc.Swine Prac.499-501; La Semaine VeterinaireNo.26, supplement to La Semaine Veterinaire 1996 (834); La Semaine Veterinaire1997 (857): 54; 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, (.Interstitial pneumonia andlymphadenophathy associated with circoviral infection in a six week-old pig.ProcAm Assoc Yet Lab Diag such as Daft B appears to this disease in state or area in the U.S., France, Spain, Northern Ireland etc. in succession, 1996,39:32; .Porcine circovirus infectionin Northern Ireland.vet Rec such as Kennedy S, 1998,142:495 and 96; .Piglet wastingdisease.Tlet Rec such as LeCann P, 1997,141:660; .First report of post-weaningmultisystemic wasting syndrome in pigs in Spain.Vet Rec such as Segales J, 1997,141:600-660).At home, Lang Hongwu etc. (Lang Hongwu etc. wean pig multisystem asthenic syndrome Serum Antibody Detection. Chinese veterinary's science and technology, 2000,30:3-5) and Cao Shengbo etc. (Cao Shengbo etc. the full genomic clone and the sequence analysis of the Henan A strain of pig II type porcine circovirus. viral journal, 2000,18:137-141) show that by serology and nosetiology investigation the existing PCV2's of China is popular respectively.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 at 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 has only PCV2 one type simultaneously.And the biological characteristics of PCV2 is more special, and it breeds titre on cell very low, and do not cause cytopathy, and the difficulty of therefore developing the inactivated vaccine of PCV2 and attenuated vaccine is very big.
Produce at present both at home and abroad vaccine and mainly contain two kinds of technologies: (1) bioreactor: scale is less, generally is laboratory usefulness, can not large-scale production; It has stirring arm, with the carrier is to rely on cultured cell.The shortcoming of this method is: stir and produce shearing force, and have bubble to generate, influence the cell growth, 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 big, take up an area of big, differences between batches are big, production cost is high, single batch yield poorly, be difficult to carry out the quality control of standardized production.
Also do not have really effectively also through the PCV2 vaccine of Ministry of Agriculture's approval in China, main cause is that also antigen concentration is lower, and the growth titre of PCV2 in cell is lower, generally only is 10
4.0TCID
50About/ml.Therefore improving virus titer and output, reduce production costs, is a difficult problem that solves the industrialization of pig circular ring virus II type vaccine.
Summary of the invention
The technical problem to be solved in the present invention provides the preparation method of a kind of porcine circovirus II type (PCV2) vaccine.This method production technology is simple, easy to operate, products obtained therefrom viral level height, and differences between batches are little, and system easy to control the quality can significantly improve vaccine output and quality.
The inventive method comprises following operating procedure:
(1) seedling is inoculated in the carrier tank that contains cell growth usefulness culture fluid and microcarrier with host cell, and, cell is attached on the microcarrier above-mentioned cell and microcarrier mix homogeneously; Under suitable culture environment, provide above-mentioned cell growth enough nutrient and gaseous environments, make cell on above-mentioned microcarrier, grow to 5～40 times of inoculum density;
(2) cell growth medium is replaced by keep and uses culture fluid, and add pig circular ring virus II type kind poison synchronously, it is adsorbed on the cell, cultivate virus of proliferation then;
(3) cultivation after 24 hours discards cell maintenance medium, and the cell of virus inoculation adds D-glucosamine and hatches 30min with the PBS thorough washing of 0.01mol/L;
(4) discard D-glucosamine,, add cell maintenance medium and continue cultured cell PBS (pH7.4) thorough washing of cell with 0.01mol/L;
(5) continuous culture was gathered in the crops viral liquid after 10 days, in-20 ℃ of freeze thawing twice, obtained the viral liquid of pig circular ring virus II type (PCV2);
(6) viral liquid adds formalin-inactivated through after the assay was approved, and the pig circular ring virus II type (PCV2) after the deactivation adds conventional oil adjuvant or two-phase adjuvant, and stirring and evenly mixing makes oil emulsion vaccine or two-phase vaccine.
The method of inspection of seedling venom: the seedling venom is tested by the relevant regulations of 15,19,20 pages of " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005, meets fully, pig safety is had no side effect no antibacterial, mycete, mycoplasma and exogenous virus pollution.Measure tiring of virus by the IFA method, the every 1.0ml of cell seedling venom contains virus 〉=10
6.0TCID
50
The product inspection method: the relevant regulations by 15,19,20 pages of " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005 is tested, and meets fully, pig safety is had no side effect no antibacterial, mycete, mycoplasma and exogenous virus pollution.
In the technique scheme, described host cell be for can breeding the cell line of pig circular ring virus virus II type (PCV2), as PK15 cell line etc.
In the technique scheme, described carrier is a reticular fiber, as polyester fiber.
In the technique scheme, microcarrier consumption described in the step (1) is that the initial inoculation amount of every 500ml culture fluid interpolation 5.5g microcarrier, cell is 1.4～2.6 * 10
7The cells/g microcarrier is better.
In the technique scheme, cell density is 1.1～2.0 * 10 during virus inoculation described in the step (2)
8The cells/g microcarrier.
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, no O
2The supply restriction, the non-foam worry.This multiple motion 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 increment virus, and this kind technology is referred to as tidal type microcarrier suspension culture technology.
Compared with prior art, the method for preparing pig circular ring virus II type (PCV2) vaccine of the present invention has following beneficial effect:
(1) malicious valency height: the PCV2 virus titer of traditional rolling bottle explained hereafter only is 10
4.0TCID
50/ ml, and the present invention adopts new technical parameter, utilization tidal type microcarrier suspension culture technology high density is cultivated the virus titer of producing and can be reached 10
6.0TCID
50/ ml～10
7.0TCID
50/ ml, its malicious valency will exceed 100-1000 doubly.
(2) big, single batch of output height of production scale: present domestic employing stirring-type suspension culture technology is cultivated zooblast, and separate unit bioreactor maximum-norm is no more than 100L; And the present invention adopts new technical parameter, and utilization tidal type microcarrier suspension culture technology is cultivated zooblast, and separate unit bioreactor scale reaches 500L, and maximum can reach 1000L, and the separate unit scale improves 5-10 doubly.
(3) production cost is relatively low, product quality is high and stable: the PCV2 virus titer that traditional rolling bottle production technology is produced only is 10
4.0TCID
50/ ml will reach 10 of veterinary biologics rules requirement
5.0TCID
50/ ml need carry out concentration, and the production cost of every m1 is 3 yuan, and the PCV2 virus titer of explained hereafter of the present invention can reach 10
6.0TCID
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).
(4) easy to operate, the working place is little: the 500L working volume only needs 20m
2Operating area, only need 2 people just can finish whole operations, and 2000 big square vases of traditional handicraft needs or rolling bottle, the minimum 600m that needs
2Operating area, minimum needs 100 people just can finish.
(5) technological parameter control accurately: controllable parameter had temperature, pH value, dissolved oxygen amount, gas concentration lwevel, carrier concn when the present invention used tidal type microcarrier suspension culture technology to produce, the parameter that can realize on-line monitoring has glucose, lactic acid and ammonium concentration, batch steady quality, and traditional rolling bottle culture process only can be controlled temperature and rotating speed, and the different batches mass discrepancy is big.
(6) the present invention utilizes bioreactor, solve the problem that antigen concentration is low, production cost is high, labor intensity is big, and can continuous culture, take up an area of shearing force, the generation of no bubble, pair cell little, that production scale big, pair cell forms during no stirring-type suspension culture and do not have injury.
The specific embodiment
Embodiment 1 tidal type microcarrier suspension culture technology high density is cultivated PK15 cell and pig circular ring virus II type (PCV2)
Employed bioreactor is the TideCell-020 of U.S. CE SCO company, and employed carrier is the BioNocII polyester fiber of U.S. CE SCO company.
(1) with PK15 cell 4.0 * 10
9Cell is inoculated in the 20L carrier tank and adds carrier B ioNocII polyester fiber 2.2 * 10
2G, and working volume is that culture fluid is used in the DMEM growth that contains 6% calf serum of 500L, tidal type microcarrier suspension culture technology is cultured to total cellular score and reaches 4.0 * 10
10Cell; Start the attaching program during cultured cell earlier, change the cultivation program behind the 4h into; Cell attaching program is: up:2500mL/min hold1min, Down:2500mL/min hold 30s, setting maximum are changed liquid measure 18500mL; The cell culture program: up:1900mL/min hold 1min, Down 1900mL/min hold 1min, setting maximum are changed liquid measure 18000mL;
(2) cell growth all is replaced by with culture fluid contains keeping of 2% calf serum and use the DMEM culture fluid, and (M.O.I.=0.1) adds pig circular ring virus II type (PCV2) and plant poison synchronously, in 37 ℃ of cultivations, controls gas concentration lwevel 5%, pH value 7.2; Start viral absorption program during virus inoculation earlier, change the Virus culture program behind the 4h into; Virus absorption program is: up:1600mL/min hold 1min, Down:1600mL/minhold 30s, setting maximum are changed liquid measure 18500mL; The Virus culture program: up:1000mL/min hold1min, Down 1000mL/min hold 1min, setting maximum are changed liquid measure 18000mL;
(3) cultivation discarded keeping liquid in the carrier tank after 24 hours, cell PBS (pH7.4) thorough washing of 0.01mol/L, adding 300mmol/L D-glucosamine is hatched 30 minutes, 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 with cell with PBS (pH7.4) thorough washing of 0.01mol/L to remove D-glucosamine residual on the carrier, fill it up with cell maintenance medium and continue cultured cell;
(5) continuous culture is gathered in the crops viral liquid after 10 days, in-20 ℃ of freeze thawing twice, promptly obtains pig circular ring virus II type (PCV2).The viral liquid of results is carried out the inspection of semifinished product to confirm to meet every standard;
The inspection of semifinished product
(a) pure property check: test by 15,19,20 pages of relevant regulations of " People's Republic of China's veterinary drug allusion quotation " 2005 editions appendix, the result does not have antibacterial, mycete, mycoplasma and exogenous virus and pollutes.
(b) viral level is measured: virus is done 10 times of gradient series dilutions with containing 300mmol/L D-glucosamine and 2% calf 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 fixed is measured the hole count that each dilution factor contains PCV2 positive cell (green fluorescence) with immunofluorescence antibody (IFA) method, calculates viral TCID according to the Reed-Muench method
50, every 1.0ml contains virus 〉=10
6.0TCID
50
(c) specificity: measure with indirect immunofluorescence antibody (IFA) method.With virus inoculation 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,, add 80% acetone soln of 100 μ L pre-coolings then, 4 ℃ of fixing 30min with PBS buffer (pH7.4) washed cell of 0.01mol/L 2 times.Then with PBS washing 3 times; Discard and keep liquid, 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 observes under fluorescence microscope.Cell control well should not have specificity fluorescent and occurs, and the virus inoculation cell hole should have a large amount of specificity fluorescents to occur.
Embodiment 2
The preparation and the immune efficacy evaluation of pig circular ring virus II type (PCV2) inactivated vaccine
1. materials and methods
1.1 vaccine
The viral liquid of pig circular ring virus II type (PCV2) that embodiment 1 obtains adds 206 adjuvants (French SEPPIC company product) in 1: 1 ratio, and fully mix homogeneously gets promptly that (lot number is: 0803,0804,0805,0806 and 0807).The vaccine that makes is carried out product inspection to confirm to meet every standard;
The character check: this Seedling is a W/O/W two-phase pale red Emulsion, and the character check has all reached the regulation of " quality standard (draft) ".Get a cleaning suction pipe, draw a small amount of vaccine and drip in cold water, except that first, all indiffusion.Stability test: under 37 ℃ of conditions, place 21 daily not stratified, breakdown of emulsion not.Get vaccine 10ml and be loaded in the centrifuge tube, with the centrifugal 15min of 3000r/min, the pipe end not water breakthrough separate out mutually.The viscosity check: with 1ml suction pipe (the outlet internal diameter is 1.2mm, and internal diameter suitable for reading is 2.7mm), draw 25 ℃ of left and right sides vaccine 1.0ml, make its vertical outflow naturally, record flows out the 0.4ml required time, all in 5s.
The check of pure property: test by 15,19,20 pages of relevant regulations of " People's Republic of China's veterinary drug allusion quotation " 2005 editions appendix, the result does not have antibacterial, mycete, mycoplasma and exogenous virus and pollutes.
Residual formaldehyde is measured: carry out residual formaldehyde according to " People's Republic of China's veterinary drug allusion quotation " version in 2005 and measure, content of formaldehyde all meets the pertinent regulations of veterinary biologics general rule as a result.
1.2. experimental animal
7～25 age in week PCV2 ELISA antibody and PCV2 antigen negative piglet, available from Luoyang, Henan.
1.3. safety testing
A 1.3.1. single dose inoculation of minimum immune age in days piglet
Get 30 of 7 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; The 6th group is the blank group, and the back isolated rearing 30 days of numbering and weigh is observed piglet and had or not clinical symptoms.Inoculation back take temperature 1～7 day every day (rectal temperature).The piglet of 5 batches of vaccinations and matched group pig all do not have clinical abnormal response, and body temperature is normal.Immunity was weighed test group piglet body weight and matched group piglet body weight zero difference (P＞0.5) in back 30 days.
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 inoculation 2 weeks of back, the immunity of reuse same dose is once; The 6th group is the blank group.The numbering and the back isolated rearing 30 days of weighing are observed piglet and are had or not clinical symptoms.Inoculation back take temperature 1～7 day every day (rectal temperature) is weighed during off-test.
The piglet of 5 batches of vaccinations and matched group pig all do not have clinical abnormal response, and body temperature is normal.Immunity was for the second time weighed test group piglet body weight and matched group piglet body weight zero difference (P＞0.5) in back 30 days.
A 1.3.3. overdose inoculation of piglet
Get 30 of 25 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 back isolated rearing 30 days of numbering and weigh is observed piglet and had or not clinical symptoms.Inoculation back take temperature 1～7 day every day (rectal temperature) is weighed during off-test.The piglet and the matched group pig of 5 batches of vaccine 2 multiple doses inoculations all do not have clinical abnormal response, and body temperature is normal.Immunity was weighed test group piglet body weight and matched group piglet body weight zero difference (P＞0.5) in back 30 days.
1.4. antibody test and counteracting toxic substances protection test:
Select 35 15～18 age in days PCV2ELISA antibody and PCV2 antigen negative ablactational baby pig, be divided into 7 groups at random, 5/group, 1st, 0803,0804,0805,0806 and 0807 batch of inactivated vaccine of 2,3,4,5 groups of immunity, the musculi colli injecting immune, the 2ml/ head, two all backs are 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 3 weeks of back uses PCV2-SH virus attack, intramuscular injection 3 * 10
5.0TCID
50/ head, behind the counteracting toxic substances the 4th, 7 day respectively in two oxters of pig and 4 inoculations of two hip portion with the emulsive keyhole hemocyanin of incomplete Freund (KLH/ICFA, 0.5mg/mL), 4mL/ head, abdominal cavity inoculation thioglycollate medium, 10mL/ head; The the 11st, 19 day abdominal cavity inoculation thioglycollate medium only behind the counteracting toxic substances, the 10mL/ head.Detect index: (1) exempts from back blood sampling in 14,21,35 days respectively at head, measures ELISA antibody and NAT, observes antibody and produces dynamically.(2) 1～20 day take temperature behind the counteracting toxic substances is observed clinical symptoms.(3) dead pig carries out pathological anatomy, and during off-test, slaughters all pigs, observes pathological change.
1.5.ELISA antibody test
As antigen, determine that by the square formation burette test antigenic best bag is by concentration with the PCV2-ORF2 albumen of escherichia coli expression.Antigen diluent is wrapped by coated elisa plate after the concentration to best, 100 μ L/ holes, 4 ℃ of bags are spent 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; Add enzyme target SPA (1: 10000 times of dilution) then, 100 μ L/ holes, 37 ℃ of effect 1h; Washing; Add substrate solution TMB (3 ', 3 ', 5 ', 5 ' ,-tetramethyl benzidine) colour developing, use the H of 2mmol/L at last
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 dilute serum method.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 1000TCID
50PCV2-SH 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 cell contrast and virus control hole simultaneously.Cultivate 12h for 37 ℃, handle with the D-glucosamine of 300mmol/L, 37 ℃ are continued to cultivate 48 hours, 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 pathological anatomy according to a conventional method, observe the internal organs pathological change, and after gathering internal organs 4% formalin fixed such as lungs, spleen, lymph node, 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 a piglet single dose repeated inoculation and the overdose inoculation of piglet all that performance physically well develops, and the mental status, appetite are normal, no fervescence and other untoward reaction.Prove that this vaccine safety is good.The results are shown in Table 1～3.
A single dose inoculation of table 17 age in days piglets safety testing result
Table 2 piglet single dose repeated inoculation safety testing result
An overdose inoculation of table 3 piglet safety testing result
2.2. immune swine PCV2 antibody test
Back 14 days of immunity, immune group all can detect PCV2 antibody; Back 35 days of first immunity, vaccine immunity group ELISA antibody and NAT reach more than 1: 3200 and 1: 32 respectively, and, the horizontal basically identical of ELISA antibody and neutralizing antibody.The results are shown in Table 4.
Table 4 piglet counteracting toxic substances protection test antibody test result
2.3. counteracting toxic substances protection 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, no abnormal 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 there is no obviously unusual clinical manifestation.5 batches of vaccine protection efficient difference 100%, 100%, 100%, 100% and 100%.The results are shown in Table 5.
2.3.2. body weight change
In order to estimate the protection effect of vaccine to the pig body, we weigh to the pig body before counteracting toxic substances and when slaughtering respectively, calculate the average daily gain of pig.The relative daily gain of vaccine immunity group and blank group is respectively 0.0255,0.0250,0.0267,0.0245,0.0264 and 0.0268kg, show that the relative daily gain of vaccine immunity group pig is similar to the blank group, but apparently higher than non-immune counteracting toxic substances matched group (P＜0.05).The proof 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 that lungs are hemorrhage, the elasticity step-down, under groin, the ilium, under the jaw, mesenteric adenophyma expands, hemorrhage, spleen edge slight bleeding etc.During off-test, slaughter all test pig, carry out pathological anatomy and histopathological examination.Result of the test shows that non-immune 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.The results are shown in Table 6.
Pig clinical symptoms statistical result in 20 days behind table 5 counteracting toxic substances
※: disorderly thick by hair, palor, loss of appetite has the phenomena of mortality.
Table 6 counteracting toxic substances swine diseases reason changes statistical result
3. in addition, relatively with the present invention's " extensive tidal type cell microcarrier suspension culture system " and rolling bottle culture systems commonly used, cultivate the PK15 cell, with the propagation pig circular ring virus, the viral correlation ratio that two system cells are cultivated is more as shown in table 7.
The correlation ratio of the different culture systems propagation of table 7 pig circular ring virus
Remarks: the adherent area 1g=2400cm of BioNOCII carrier
2, 1 TideCell-020 microcarrier suspension culture system need add BioNOCII carrier 220g.
4. conclusion
This research is inoculated piglets with 5 batches of pig circular ring virus II type inactivated vaccines, all no abnormal clinical manifestation of result, and safety is fine; Produced ELISA antibody and neutralizing antibody in 14 days behind the piglet immunological, head exempts from back 35 days counteracting toxic substances, no abnormal clinical manifestation and pathological change, and immunoprotection efficient reaches 100%, and immune effect is good.And adopt new technological parameter utilization tidal type microcarrier cell suspension cultures system to produce no matter the pig circular ring virus inactivated vaccine is indexs such as antigenic content, output or production cost with the present invention, be much better than rolling bottle culture systems commonly used.
Claims (10)
1. pig circular ring virus II type vaccine production method comprises following operating procedure:
(1) seedling is inoculated in the carrier tank that containing growth usefulness culture fluid and microcarrier with host cell, and, cell is attached on the microcarrier above-mentioned cell and microcarrier mix homogeneously; Under suitable culture environment, provide above-mentioned cell growth enough nutrient and gaseous environments, make cell on above-mentioned microcarrier, grow to 5～40 times of inoculum density;
(2) cell growth is replaced by to keep with culture fluid uses culture fluid, and add pig circular ring virus II type kind poison synchronously, it is adsorbed on the cell, cultivate virus of proliferation then;
(3) cultivation after 24 hours discards cell maintenance medium, and the cell of virus inoculation adds D-glucosamine and hatches 30min with the PBS thorough washing of 0.01mol/L;
(4) discard D-glucosamine, cell with the PBS thorough washing that 0.01mol/L, pH are 7.4, is added cell maintenance medium and continues cultured cell;
(5) continuous culture was gathered in the crops viral liquid after 10 days, in-20 ℃ of freeze thawing twice, obtained pig circular ring virus II type virus liquid;
(6) viral liquid adds formalin-inactivated through after the assay was approved, and the pig circular ring virus II type after the deactivation adds conventional oil adjuvant or two-phase adjuvant, and stirring and evenly mixing makes oil emulsion vaccine or two-phase vaccine.
2. method according to claim 1 is characterized in that described host cell is for breeding the cell line of pig circular ring virus virus II type.
3. method according to claim 2 is characterized in that the enrichment procedure of cell, virus adopts tidal type microcarrier suspension culture mode.
4. method according to claim 3, change the cultivation program into after starting attaching program 4h when it is characterized in that in the step (1), (2) cultured cell, cell attaches program parameter and is: up:2300～2700mL/min hold40～75s, Down:2300～2700mL/min hold 30～60s, set maximum and change liquid measure 18500ml; The cell culture program parameter is: up:1700～2100mL/min hold 40～75s, Down1700～2100mL/min hold 40～75s, setting maximum are changed liquid measure 18000ml.
5. method according to claim 4, change the virus multiplication program into after starting viral absorption program 4h when it is characterized in that virus inoculation, virus absorption program parameter is: up:1400～1800mL/min hold40～75s, Down:1400～1800mL/min hold 30～60s, setting maximum are changed liquid measure 18500ml; The virus multiplication program parameter is: up:900～1200mL/min hold 40～75s, Down900～1200mL/min hold 40～75s, setting maximum are changed liquid measure 18000ml.
6. method according to claim 5 is characterized in that described microcarrier is a polyester fiber.
7. method according to claim 5 is characterized in that microcarrier consumption described in the step (1) is that every 500ml culture fluid adds the 5.5g microcarrier, and the initial inoculation amount of cell is 1.4～2.6 * 10
7The cells/g microcarrier.
8. method according to claim 5, the environment that it is characterized in that cell culture are 37 ℃ of temperature, CO
2Concentration is 5%, and medium pH value is 7.0～7.6.
9. method according to claim 5, cell density is 1.1～2.0 * 10 when it is characterized in that virus inoculation described in the step (3)
8The cells/g microcarrier.
10. method according to claim 5 when it is characterized in that virus inoculation described in the step (3) is 0.01～1 ratio in the viral infection plural number.
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- 2010-09-03 CN CN2010102750752A patent/CN101934074B/en active Active
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Also Published As
| Publication number | Publication date |
|---|---|
| CN101871941A (en) | 2010-10-27 |
| CN101934074A (en) | 2011-01-05 |
| CN101934074B (en) | 2013-03-06 |
| CN106645716A (en) | 2017-05-10 |
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