CN101240264A - Porcine circovirus 2 type inactivated vaccine - Google Patents
Porcine circovirus 2 type inactivated vaccine Download PDFInfo
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- CN101240264A CN101240264A CNA2008100244231A CN200810024423A CN101240264A CN 101240264 A CN101240264 A CN 101240264A CN A2008100244231 A CNA2008100244231 A CN A2008100244231A CN 200810024423 A CN200810024423 A CN 200810024423A CN 101240264 A CN101240264 A CN 101240264A
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Abstract
The pig circular ring virus 2 type (PVC2) inactivated vaccine (SH individual plant) of the invention belongs to biotechnology field. The pig circular ring virus 2 type poisonous individual plant SH belongs to circular ring virus section circular ring virus genus which has been preserved in Wuhan institute of virology, Chinese academy of sciences. The shanghai separated individual plant SH of purified PCV2 virus is obtained by gathering raw material from hogpen which happened bad weaning piglet multisystem exhaustion failure syndrome in Shanghai in 2002 year, separating, appraising and purifying virus. The PCV2-SH plant is proliferated in mass in PK-15 cell, inactivated through methyl aldehyde and emulsified with liquid paraffine adjuvant to prepare conventional liquid paraffin(e) adjuvant immunomodulators for vaccines. The laboratory has trial-manufactured five lots vaccines successfully which are good safety and also can induce pig bring immune protection effect, made out a draft rules for vaccines production and testing. The inactivated vaccine proved by every aspects experiment has met state biological products standard completely.
Description
One. technical field
Two, technical background
PCV-2 is worldwide a kind of swine disease, has caused tremendous loss for the pig industry in the world.This virus mainly causes weanling pig multisystemic wasting syndrome and ephrosis dermatitis syndromes.Should disease also can cause immunosuppression to a certain extent simultaneously, cause the secondary or the accompanying infection of other disease easily, especially should disease and PRRS mutual synergy is arranged.Because this virus is a kind of dna virus, the aberration rate of virus is low, and worldwide pathogenic has only PCV2 one type simultaneously.Should good antigenicity be arranged virus self simultaneously, and can induce body generation high-level antibody and provide provide protection this disease.But should the virus separation difficulty, identify difficulty, cultivate difficulty, therefore go back the vaccine listing that the none effectively preventing should disease at present in China.In view of this, my laboratory dropped into great amount of manpower and material resources to this virus separate, work such as evaluation.And the strain isolated SH strain that obtains has been prepared inactivated vaccine carry out immunization experiment after large scale culturing, proving effect is good.
Three, summary of the invention
Technical problem the objective of the invention is to develop a kind of vaccine that can effectively control the pig circular ring virus 2 viral disease, and the PCV2 disease of the current generation of China is controlled effectively, thereby can reduce the loss on pig farm and increase economic efficiency.
Technical scheme embodiment of the present invention are as follows:
Above-mentioned porcine circovirus 2 type (PCV2) SH strain, by following acquisition:
(1) Bing Du separation and cultivate the pig farm that serious weanling pig multisystemic wasting syndrome took place from Shanghai area in 2002 and gather pathological material of disease, pathological material of disease is after homogenate, multigelation 3 times, and high speed centrifugation is after be inoculated in the PK-15 cell that covers with individual layer behind the 0.22um membrane filtration.The PK-15 cell of inoculating pathological material of disease is carried out continuous passage carry out the virus separation.
(2) evaluation of the positive strain isolated of porcine circovirus 2 type is after 6 generations of PK-15 cell continuous passage, gets wherein part cell extracts PCV-2 virus after freeze thawing DNA, uses the specific PCR primer at the viral Cap protein gene of PCV-2 that goal gene is increased.Extension amplification outcome is gone into the pMD-18T carrier and is checked order.
(3) purifying of PCV2-SH strain isolated has identified that the PCV2 strain isolated carries out viral purification through limiting dilution assay, and the worker carries out viral purification 3 times, has obtained the Shanghai strain isolated (SH) of the PCV2 virus of purifying.
The method that above-mentioned porcine circovirus 2 type (PCV2) SH strain prepares vaccine is:
The PCV2-SH strain is bred in the PK-15 cell in a large number, add white-oil adjuvant through formalin-inactivated and carry out emulsification, the white-oil adjuvant vaccine that preparation is conventional.The PCV2 (SH) that takes cryopreservation in enlarging the virus culture process is by 10
4TCID
50Inoculation grows up to the PK-15 cell of individual layer.Inoculate after 24 hours, discard and keep liquid, hatch, discard the D-glucosamine adding then and keep liquid, receive poison after 3 days with D-glucosamine.
The vaccine made from above-mentioned porcine circovirus 2 type SH strain:
With the continuous passage 50 times on the PK-15 cell of the PCV2 virus of purifying, in per 5 generations, measured tiring of virus by the IPMA method.Prove that the continuous passage performance in the PK-15 cell of this virus is stable, connect poison back cytopathy occurrence law, the malicious valency of planting poison is stabilized in 10
4.33TCID
50/ 1.0ml.
Beneficial effect
The present invention has proposed first with the inactivated vaccine of PCV2 isolated strain preparation at PCV2, and the research of this vaccine and preparation have solved the current defective that can't prevent this disease of China, can effectively solve current PC V2 sickness rate height, and big influence causes damage.Inactivated vaccine technology with the strain isolated preparation is simple, easy to operate, and the vaccine antigen of preparing is good, safe, and environment does not have any detrimentally affect to external world, easily by safety evaluation.
Evidence, the PCV2-SH strain inactivated vaccine of the present invention's preparation does not produce any untoward reaction to animal, the experimental safe height, and can induce immune animal and to produce high-caliber circulating antibody and neutralizing antibody, the ELISA antibody titer reaches, NAT reaches, and the animal after the immunity can effectively be resisted the attack of PCV2 virus.
At the present PCV2 sickness rate of China than higher, the preparation of this vaccine, production, application and popularize and will and popularly provide strong instrument for the generation of the PCV2 disease that prevents China.
Four, description of drawings
Fig. 1. the virus multiplication curve
Five, embodiment:
1, seed culture of viruses source and standard
Make this product with and the seed culture of viruses of check be the PCV2-SH strain, by the animal epidemic diagnosis of the Ministry of Agriculture of Agricultural University Of Nanjing and immune emphasis open laboratory 2002 from pig farm, Shanghai isolation identification, called after ShangHai strain isolated (abbreviation SH strain).
1.1 seed culture of viruses standard
Viral level: virus is kept liquid with PRM-1640 do 10 times of serial dilutions, get 10
-3, 10
-4, 10
-53 extent of dilution, each extent of dilution are inoculated 96 well culture plate PK-15 cell monolayers, 4 holes respectively, and every hole 0.2ml sets up negative control simultaneously, continue to cultivate 48 hours in 37 ℃ of incubators that contain 5%CO2.The dehydrated alcohol fixed cell is measured each extent of dilution with immunoperoxidase monolayer assay (IPMA) and is contained the PCV2 positive cell hole count of (being pale brown look), calculates viral TCID50 according to the KarberShi method, and every ml viral level answers 〉=10
5.0TCID50.
Specificity: measure with the IPMA method.With virus inoculation in 96 porocyte plate PK15 cells, each sample 2 hole, every hole 200 μ l establish blank cell control well simultaneously, continue to cultivate 24 hours in 37 ℃ of incubators that contain 5%CO2; Discard nutritive medium, with PBS damping fluid (0.01mol/L, pH value 7.0) washed cell 1 time, 37 ℃ of dryings 45 minutes ,-20 ℃ freezing 45 minutes, add the cold dehydrated alcohol of 100 μ l again, fix 45 minutes for 4 ℃, PBS washs 3 times; Add the pig anti-PCV2 serum of 50 μ l with 0.5mol/L NaCl and 0.5%Tween80 dilution in 1: 20 in different holes respectively, 37 ℃ act on 1 hour, wash 3 times with 0.15mol/L NaCl and 0.5%Tween80; The SPA-HRP that adds dilution in 1: 100, every hole 50 μ l, 37 ℃ act on 1 hour, wash 3 times; Add 50 μ l AEC substrate solutions (4mgAEC, 1mlDMF, 10 μ l H2O2), room temperature effect 20～30min; Discard AEC, add the 0.05mol/L sodium-acetate (pH value 5.0) of 50 μ l, observe in microscopically.Cell control well should not have pale brown color substance and dyes, and dyes and should there be a large amount of pale brown color substances in the virus inoculation cell hole cell.
Virulence: with 5 of the healthy piglets of 28～30 age in days PCV2-ELISA negative antibodies, weigh in, (contain 10 with the PCV2-SH strain
5.0TCID50/ml) attack, every incidence intramuscular injection 3ml, establish healthy 5 of the piglets of 28～30 age in days PCV2-ELISA negative antibodies simultaneously and do blank, isolated rearing, attack poison back the 4th, 7 day respectively in two oxters of pig and 4 inoculations of two hip portion with Freund's incomplete adjuvant emulsive keyhole hemocyanin (KLH/ICFA, 0.5mg/ml), the 4ml/ head, abdominal cavity inoculation thioglycollate medium, the 10ml/ head; Attack poison back abdominal cavity inoculation on the the 11st, 19 thioglycollate medium, the 10ml/ head.Attack the poison back and observed 32 continuously, detect according to clinical symptom, pathological change and virus and judge (seeing note 2), the control group pig should all not fall ill, and the virus inoculation group should have 3 hair diseases at least.
Immunogenicity
(1) mouse immuning test: after preparing oil emulsion inactivated vaccine by these rules, with 5～6 age in week 5 of PCV2-ELISA negative antibody Healthy female cleaning level mouse, every intramuscular injection vaccine 0.2ml, establish simultaneously 5～6 age in week 5 of mouse of PCV2-ELISA negative antibody Healthy female cleaning level do blank, isolated rearing is observed, back 30 days sterile blood samplings of immunity, separation of serum, measure serum ELISA antibody (note 1), control group should be all negative, and vaccine inoculation group antibody titer answers 〉=1: 1600.
(2) piglet immunological test: after 1. ELISA antibody prepares oil emulsion inactivated vaccine by these rules, with 5 of the healthy piglets of 28～30 age in days PCV2-ELISA negative antibodies, every incidence intramuscular injection vaccine 2ml, establish healthy 5 of the piglets of 28～30 age in days PCV2 negative antibodies simultaneously and do blank, isolated rearing is observed, back 30 days sterile blood samplings of immunity, separation of serum, measure serum ELISA antibody (note 1), control group should be all negative, and vaccine inoculation group antibody titer answers 〉=1: 1600.2. piglet is attacked after malicious protection test prepares oil emulsion inactivated vaccine by these rules; with 5 of the healthy piglets of 28～30 age in days PCV2-ELISA negative antibodies; every incidence intramuscular injection vaccine 2ml; establish healthy 5 of the piglets of 28～30 age in days PCV2-ELISA negative antibodies simultaneously and do blank; isolated rearing is observed, and back 30 days of immunity (contains 10 with the PCV2-SH strain
5.0TCID50/ml) attack, every incidence intramuscular injection 3ml, isolated rearing, attack poison back the 4th, 7 day respectively in two oxters of pig and 4 inoculations of two hip portion with Freund's incomplete adjuvant emulsive keyhole hemocyanin (KLH/ICFA, 0.5mg/ml), the 4ml/ head, abdominal cavity inoculation thioglycollate medium, 10ml/ head; Attack poison back abdominal cavity inoculation on the the 11st, 19 thioglycollate medium, the 10ml/ head.Attack the poison back and observed 32 continuously, can judge according to one of following two standards: (1) is detected according to clinical symptom, pathological change and virus and is judged that the contrast pig has 3 hair diseases at least, and immune swine should be protected 4 at least.Judge according to the relative day weight gain of pig that (2) the relative day weight gain of immune group pig is apparently higher than control group (P＜0.05).Pure property: test for 15,19,20 pages by " Chinese veterinary drug allusion quotation " appendix, should not have bacterium, mould, mycoplasma and exogenous virus and pollute.In subalgebra: F11～F20 generation, are planted on the basis.Seed culture of viruses is preserved: below-50 ℃, preservation period is 12 months.
Quality index in 2 production technique
2.1 PCV2-SH strain proliferation conditions, has obtained a cover PCV2 rolling bottle cultural method by discussion and optimization to PK15 cell culture condition and virus multiplication method and rule thereof, that is: with 10
6～10
7The PK15 cell inoculation of individual/ml concentration adds the RPM-1640 nutritive medium contain 8%～10% calf serum in rolling bottle cell bottle, rotating speed be 12 change/hour, cultivated 1 day for 37 ℃, cell covers with individual layer, cellular form is better.Inoculation PCV2-SH strain, 37 ℃ adsorbed 30 minutes, added the RPM-1640 that contains 3%～4% calf serum, and the D-glucosamine with 300mM after 12 hours is handled, and cultivates 4 days for 37 ℃, can obtain higher titre virus, and virus titer can reach 10
5.0TCID50/ml more than, detailed results sees Table 1, table 2, table 3, table 4 and Fig. 1.
The different basic nutrient solution cultures of table 1 recuperate malicious titer determination as a result the different serum of table 2 cultivate the virus titer measurement result
The different adsorption methods of table 3. are cultivated virus titer measurement result table 4 D-glucosamine to cultivating the influence of virus titer
2.2 it is the single-phase oil emulsion inactivated vaccine of water-in-oil-type that proterties is checked this seedling.Prepared 10 batches of these seedlings altogether, wherein laboratory trial-production is 5 batches, 5 batches of middle test manufactures, and the proterties check has all reached the regulation of " quality standard (draft) ".Stability test: under 37 ℃ of conditions, place not stratified, breakdown of emulsion not on the 21st.Get vaccine 10ml and be loaded in the centrifuge tube, with 3000r/min centrifugal 15 minutes, 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 8 seconds.
2.3 steriling test carries out steriling test according to " Chinese veterinary drug allusion quotation ", has done the steriling test of 10 batches of finished products altogether, every batch of seedling is taken out 5 bottles at random, tests respectively.With T.G substratum 50ml, inoculation sample 1.0ml puts 37 ℃ of cultivations, draws culture after 3 days in bottle.Inoculate each 2 on T.G tubule and G.A inclined-plane respectively, every 0.2ml puts 37 ℃ for 1; Put 25 ℃ for 1, other gets 0.2ml, inoculates 1 G.P tubule and puts 25 ℃, all cultivates after 5 days and judges.10 batches of equal asepsis growths of inactivated vaccine illustrate that strict aseptic technique all can reach the requirement of asepsis growth as long as in the vaccine manufacturing processed as a result.
Carry out residual formaldehyde mensuration 2.4 residual formaldehyde is measured according to " Chinese veterinary drug allusion quotation ", done the check of 10 batches of finished products (5 batches of laboratory trial-productions, 5 batches of middle test manufactures) altogether, formaldehyde content all meets the regulation of veterinary biologics general rule as a result.
2.5 the vaccine safety check is 5 batches of inactivated vaccines of 1: 2 emulsive with the water-oil ratio example of laboratory trial-production, carries out safety testing research.
2.5.1 30 of 7 age in days sucking pigletss are got in a single dose inoculation of minimum immune age in days piglet, are divided into 6 groups, and 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, cuts overbit and the back isolated rearing 30 days of weighing, and observes piglet and has or not clinical symptom.Inoculation back take temperature 1～7 day every day (rectal temperature).The piglet of 5 batches of vaccine inoculations and control group pig all do not have clinical abnormal response, and body temperature is normal.Immunity was weighed test group piglet body weight and control group piglet body weight indifference (P＞0.5) in back 30 days.
A single dose inoculation of table 57 age in days piglets safety testing result
*Post-immunized day
2.5.2 piglet single dose repeated inoculation is got 30 of 30 age in days sucking pigletss, is divided into 6 groups, and 5 every group, the 1st～5 group of intramuscular injection vaccine, dosage of inoculation 2ml/ head in inoculation 2 weeks of back, is used the same dose immunity once again; The 6th group is the blank group.After cutting overbit and weighing, isolated rearing 30 days is observed piglet and is had or not clinical symptom.Inoculation back take temperature 1～7 day every day (rectal temperature).Weigh during off-test.
All do not have clinical abnormal response behind 5 batches of vaccines of 30 age in days piglet intramuscular injection, body temperature is normal.Immunity was for the second time weighed test group piglet body weight and control group piglet body weight indifference (P＞0.5) in back 30 days.
Table 6 piglet single dose inoculation safety testing result
*Fate behind the second immunisation
2.5.3 30 of 30 age in days sucking pigletss are got in an overdose inoculation of piglet, are divided into 6 groups, and 5 every group, the 1st～5 group of intramuscular injection vaccine, dosage of inoculation 4ml/ head; The 6th group is the blank group, cuts overbit and the back isolated rearing 30 days of weighing, and observes piglet and has or not clinical symptom.Inoculation back take temperature 1～7 day every day (rectal temperature).Weigh during off-test.30 age in days piglets inoculate 2 multiple dose vaccines respectively, and the result does not all have clinical abnormal response, and body temperature is normal.Immunity was weighed immunization piglet body weight and blank group piglet body weight indifference (P＞0.5) in back 30 days.
2.5.4 18 of 85～90 days sows of pregnancy are got in the security of farrowing sow (production performance), are divided into 6 groups, and 3 every group, the 1st～5 group of musculi colli vaccinate, dosage of inoculation 6ml/ head; The 6th group in contrast, and each is organized sow and separately raises under the same conditions, observes to Farrowing, observes breeding difficultys such as having or not premature labor, stillborn foetus, mummy tire and weak son and occur.The 5 batches of vaccines all do not have clinical abnormal response with 60～70 days sows of pregnancy of 2 times of immunizing doses inoculations, and appetite is normal, and childbirth on schedule, and farrowing is normal, breeding difficulty phenomenons such as premature labor, mummy tire do not occur.Healthy piglet number, stillborn foetus are similar to control group with weak young number.The results are shown in Table 3.
Table 7.5 batch vaccine inoculation is to the safety testing result of farrowing sow
2.6 immune efficacy
This research produced ELISA antibody and neutralizing antibody in 10 days with 5 batches of PCV2 inactivated vaccine inoculation piglets behind the piglet immunological, and immunity was attacked poison in back 30 days, no abnormal clinical manifestation and pathological change, and immunoprotection efficient reaches more than 80%, and immune effect is good.
2.6.1 immune swine PCV2 antibody test
The results are shown in Table 1-2.Back 10 days of immunity, immune group all can detect PCV2 antibody; Back 30 days of immunity, vaccine immunity group ELISA antibody and NAT reach more than 1: 1600 and 1: 16 respectively, and, the horizontal basically identical of ELISA antibody and neutralizing antibody.
Table 8 PCV2 inactivated vaccine induces piglet to produce the dynamic change (0501 and 0502 batch of vaccine) of antibody
* NT: do not detect KLH: keyhole hemocyanin
Table 9 PCV2 inactivated vaccine induces piglet to produce the dynamic change (0503,0504 and 0505 batch of vaccine) of antibody
* NT does not detect
2.6.2 attack malicious protection test
2.6.2.1 clinical symptom
0501 and 0502 batch of vaccine test the results are shown in Table 10.Attack all pigs of malicious control group and attack poison back 1-3 week fervescence (＞40 ℃), continue 3-6 days, attack the poison back and occurred, an appetite stimulator phenomenon thick disorderly on the 10th day, 1 death was arranged on the 11st day by hair; The blank pig is attacked malicious interim body temperature and remains normally no abnormal clinical manifestation whole.The pig of 0501 and 0502 batch of vaccine immunity, attacking the poison back all had 2～3 pigs body temperature to occur above 40 ℃ in the 1st week, continued 1-2 days, but there is no obviously unusual clinical manifestation, wherein, 0502 batch of vaccine immunity group has 1 temperature of pig body above 40 ℃, continue 5 days, and unusual clinical manifestation occurs.2 batches of vaccine protection efficient difference 100% and 80%.
0503,0504 and 0505 batch of vaccine test the results are shown in Table 11.Attack malicious control group and attack poison back 1-3 week fervescence (＞40 ℃), continue 3-5 days, the 9th day part pig, appetite stimulator thick disorderly by hair; The blank pig is attacked malicious interim body temperature and remains normally no abnormal performance whole.The pig of 3 batches of vaccine immunities, attacking the poison back all had 2-3 pig body temperature to occur above 40 ℃ in the 1st～2 week, continued 1～3 day, the Non Apparent Abnormality clinical manifestation, appetite is normal, but 0504 batch of vaccine group has 1 to occur clinical unusual.3 batches of vaccines protection efficient is respectively, 100%, 80% and 100%.
Table 10 is attacked pig clinical symptom statistics (0501,0502 batch of vaccine) in malicious back 32 days
*The statistics of 4 pigs of this group survival,
*A and b letter expression significant difference inequality (P＜0.05); Letter identical table differential different not remarkable (P＞0.05).
Table 11 is attacked pig clinical symptom statistics (0503,0504 and 0505 batch of vaccine) in malicious back 32 days
*A and b letter expression significant difference inequality (P＜0.05); Letter identical table differential different not remarkable (P＞0.05);
2.6.2.2 body weight change
In order to estimate the protection effect of vaccine to the pig body, we weigh the average daily gain of calculating pig to the pig body before attacking poison and when slaughtering respectively.0501 and 0502 batch of vaccine test the results are shown in Table 10.The relative day weight gain of vaccine immunity group and blank group is respectively 0.0240,0.0245,0.0190 and 0.0263kg, shows that the relative day weight gain of vaccine immunity group pig is similar to the blank group, but attacks malicious control group (P＜0.05) apparently higher than non-immunity.
0503,0504 and 0505 batch of vaccine test the results are shown in Table 11.The relative day weight gain of vaccine immunity group and blank group is respectively 0.0224,0.0200,0.0170 and 0.0231kg, and the relative day weight gain of vaccine immunity group pig is similar to the blank group, but attacks malicious control group (P＜0.05) apparently higher than non-immunity.
Prove that 5 batches of vaccines all have better immanoprotection action.
2.6.2.3 viremia
Serum is gathered in after attacking poison the 4th, 7,14 and 21 day respectively, extracts viral DNA with DNAzol from serum, detects the existence whether PCV2 is arranged with PCR method.The result has 3-5 the pig PCV2 positive for 1. attacking in the malicious control group in the whole process after attacking poison; 2. blank pig PCR detected result is all negative; 3. in the immune group, attack the poison back the 4th, 7 day, have 2-4 pig to detect PCV2 in two immune group; Attacked the poison back the 14th day, 2-4 the pig PCV2 positive; Attacked the poison back the 21st day, 0502 and 0504 batch of vaccine immunity group has 1 pig PCV2 positive of clinical symptom, and other pig PCV2 detects all negative.Attacked poison the 32nd day, all vaccine immunity group PCV2 feminine genders, but not immunity is attacked malicious control group and is still had 2-3 the PCV2 positive.Show the lifetime (seeing Table 12,13) that can shorten viremia behind 5 batches of vaccine immunities.
Table 12 is attacked PCV2 detected result (0501,0502 vaccine) in the different time serum of poison back
A: the pig number of denominator for detecting, molecule is PCV2 number positive: b: attack dead 1 of poison back the 11st day
Table 13 is attacked PCV2 detected result (0503,0504 and 0505 vaccine) in the different time serum of poison back
A: the pig number of denominator for detecting, molecule is the PCV2 number positive
2.6.2.4 pathological change
Attacked poison back the 11st day, and attacked 1 death of poison contrast pig, pathology is analysed and is shown as that lungs are hemorrhage, the elasticity step-down, under inguinal region, 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, the results are shown in Table 14,15.Show that non-immunity attacks malicious control group pig obvious pathological change is arranged, and the immune swine pathological change is very not obvious.
PCV2 detection statistics result (0501 and 0502 vaccine) during table 14 is attacked the variation of malicious swine disease reason and organized
A. molecule is for there being pathology pig's head number, and denominator contains dead swine disease reason variation statistics for analysing total number of pig: b.
C. molecule is the positive pig's head number of PCV2, and denominator is total number of pig that PCR detects
PCV2 detection statistics result (0503,0504 and 0502 vaccine) during table 15 is attacked the variation of malicious swine disease reason and organized
A. molecule is the pig's head number that this pathology is arranged, and denominator is for analysing total number of pig;
C. molecule is the positive pig's head number of PCV2, and denominator is total number of pig that PCR detects.
2.6.2.5 PCV2 detects in the histoorgan
The results are shown in Table 14 and table 15.Attacked poison back 32 days, and slaughtered all test pig, get lung, spleen and the lymph node tissue balanced mix of fresh pig, extract viral DNA, detect PCV2 with PCR method.The result is, attack in lungs, spleen and the lymphoglandula that 4 pigs are arranged respectively in the malicious control group in non-immunity and all to have detected virus, have only 1 pig to detect PCV2 in 5 batches of vaccine immunity groups, the blank pig does not detect virus, shows the band poison that can effectively reduce the pig body behind the vaccine immunity.
2.7 5 batches of vaccines are adopted in this research of duration of immunity, once behind Mian Yi the piglet immunological the 30th day, ELISA and neutralizing antibody level reached the peak, and immune back 90 days ELISA and neutralizing antibody reach respectively more than 1: 1600 and 1: 12, and the result sees table 16,17 for details; Twice immunity, then immunity back is the 60th day, and ELISA and neutralizing antibody level reach the peak, and back 135 days ELISA of immunity and neutralizing antibody reach respectively more than 1: 1600 and 1: 14, and the result sees table 18,19 for details.Illustrate that duration of immunity of this vaccine immunity reaches 2.5 months; Twice immunity, duration of immunity reaches 4 months.So stipulating the duration of immunity of twice immunity of this vaccine is 4 months.
2.7.1 once immunity
The results are shown in Table 16 and 17.Behind the piglet immunological of 5 batches of vaccine immunities 30-60 days, the ELISA antibody horizontal reached the peak, immunity back 75-90 days, and antibody descends gradually, and the 105th day, antibody dropped to below 1: 1600.The neutralizing antibody of 5 batches of vaccine-induced generations and ELISA antibody horizontal basically identical.Duration of immunity of this vaccine immunity of this presentation of results reaches 2.5-3 month.
The ELISA antibody that immune induction of table 16. produces is held time
The neutralizing antibody that immune induction of table 17 produces is held time
2.7.2 twice immunity
The results are shown in Table 18 and 19.After the piglet first immunisation of 5 batches of vaccine immunities 45-60 days, the ELISA antibody horizontal reached the peak, and after the first epidemic disease 90 days, antibody descended gradually, and 120-135 days, the ELISA antibody titer still reached more than 1: 1600, exempted from back 150 days to head, and antibody drops to below 1: 1440.The neutralizing antibody of 5 batches of vaccine-induced generations and ELISA antibody horizontal basically identical.Twice immunity of this vaccine of this presentation of results, duration of immunity reaches 4 months.
The ELISA antibody that twice immune induction of table 18 produces is held time
2.8 5 batches of vaccines are adopted in this research of preservation period, preserved 1～13 month at 2～8 ℃ respectively, preserve all can induce behind the immune piglet in 13 months and produce PCV2 antibody, back 30 days ELISA of immunity and neutralizing antibody reach more than 1: 1600 and 1: 12 respectively, and preserved 14～15 months, PCV2ELISA and neutralizing antibody drop to respectively below 1: 1600 and 1: 10 behind the immune piglet, illustrate that this vaccine preservation period can reach 13 months, for safety, the spy stipulates that this vaccine is 12 months 2～8 ℃ preservation period.
Claims (4)
1. porcine circovirus 2 type (PCV2) vaccine strain SH, it is characterized in that, porcine circovirus 2 type strain SH belongs to PCV-II section (Circoviridae) PCV-II and belongs to (Circovirus), China Committee for Culture Collection of Microorganisms carries out preservation in the common micro-organisms center, preservation date: on March 4th, 2008, preserving number is CGMCC No.2389.
2. porcine circovirus 2 type vaccine strain according to claim 1 (SH) makes up by the following method and forms:
(1) Bing Du separation and cultivate the pig farm that serious weanling pig multisystemic wasting syndrome took place from Shanghai area in 2002 and gather pathological material of disease, pathological material of disease is after homogenate, multigelation 3 times, high speed centrifugation is after be inoculated in the PK-15 cell that covers with individual layer behind the 0.22um membrane filtration, the PK-15 cell of inoculation pathological material of disease is carried out successive cell go down to posterity and carry out virus separation;
(2) evaluation of the positive strain isolated of porcine circovirus 2 type is with after 6 generations of PK-15 cell continuous passage, get wherein part cell extracts PCV-2 virus after freeze thawing DNA, use the specific PCR primer at PCV-2 virus Cap protein gene that goal gene is increased, extension amplification outcome is gone into the pMD-18T carrier and is checked order;
(3) purifying of PCV2-SH strain isolated has identified that the PCV2 strain isolated carries out viral purification through limiting dilution assay, carries out viral purification altogether 3 times, has obtained the Shanghai strain isolated SH of the PCV2 virus of purifying.
3. use the vaccine of claim 1 or 2 described porcine circovirus 2 type vaccine strains (SH) preparation.
4. prepare the method for vaccine with claim 1 or 2 described porcine circovirus 2 type vaccine strains (SH), comprising:
The PCV2-SH strain is bred in the PK-15 cell in a large number, add white-oil adjuvant through formalin-inactivated and carry out emulsification, the white-oil adjuvant vaccine that preparation is conventional.
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Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101934074A (en) * | 2010-01-28 | 2011-01-05 | 洛阳普莱柯生物工程有限公司 | Porcine circovirus II vaccine and production method thereof |
| CN102145165A (en) * | 2010-02-04 | 2011-08-10 | 绿十字兽医药品株式会社 | Porcine circovirus type 2 and use thereof |
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2008
- 2008-03-21 CN CNA2008100244231A patent/CN101240264A/en active Pending
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| CN101934074B (en) * | 2010-01-28 | 2013-03-06 | 普莱柯生物工程股份有限公司 | Porcine circovirus II vaccine and production method thereof |
| CN106645716A (en) * | 2010-01-28 | 2017-05-10 | 普莱柯生物工程股份有限公司 | Method for testing efficacy of porcine circovirus 2-type vaccine |
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| CN102145165A (en) * | 2010-02-04 | 2011-08-10 | 绿十字兽医药品株式会社 | Porcine circovirus type 2 and use thereof |
| CN102973932A (en) * | 2011-09-07 | 2013-03-20 | 普莱柯生物工程股份有限公司 | Trivalent inactivated vaccine of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and porcine pseudorabies virus and preparation method thereof |
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| CN103421748A (en) * | 2013-09-06 | 2013-12-04 | 青岛易邦生物工程有限公司 | Porcine circovivus2 strain and application thereof |
| CN105148263A (en) * | 2015-09-30 | 2015-12-16 | 章红兵 | Porcine circovirus inactivated vaccine and porcine cytokine combined immune method |
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| CN109125719A (en) * | 2017-06-19 | 2019-01-04 | 普莱柯生物工程股份有限公司 | It is a kind of containing 3 type of pig circular ring virus, the immunogenic composition of porcine circovirus 2 type antigen and its application |
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| CN109134619A (en) * | 2017-06-19 | 2019-01-04 | 普莱柯生物工程股份有限公司 | Porcine circovirus 2 type antigen, the immunogenic composition of its preparation, preparation method and application |
| JP2020524182A (en) * | 2017-06-19 | 2020-08-13 | 普莱柯生物工程股▲ふん▼有限公司 | Immunogenic composition containing antigens of porcine circovirus type 3 and porcine circovirus type 2 and uses thereof |
| CN109134619B (en) * | 2017-06-19 | 2021-10-01 | 普莱柯生物工程股份有限公司 | Porcine circovirus type 2 antigen, immunogenic composition prepared from same, preparation method and application |
| CN109125720B (en) * | 2017-06-19 | 2021-11-09 | 普莱柯生物工程股份有限公司 | Immunogenic composition containing porcine circovirus type 3 antigen and application thereof |
| CN109125719B (en) * | 2017-06-19 | 2022-06-14 | 普莱柯生物工程股份有限公司 | Immunogenic composition containing porcine circovirus type 3 and porcine circovirus type 2 antigens and application thereof |
| CN107475205A (en) * | 2017-08-31 | 2017-12-15 | 浙江美保龙生物技术有限公司 | A kind of pig circular ring virus separation method |
| CN112595846A (en) * | 2020-12-07 | 2021-04-02 | 新乡学院 | IPMA antibody detection method of PCV2 |
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