CN103421748B

CN103421748B - Porcine circovivus2 strain and application thereof

Info

Publication number: CN103421748B
Application number: CN201310404122.2A
Authority: CN
Inventors: 邹敏; 张恒; 刘新文; 申洪银; 宫晓; 刘蕾; 邹桂荣; 韩乃君; 陶晓珊
Original assignee: Qingdao Yebio Bioengineering Co Ltd
Current assignee: Qingdao Yebio Bioengineering Co Ltd
Priority date: 2013-09-06
Filing date: 2013-09-06
Publication date: 2015-01-07
Anticipated expiration: 2033-09-06
Also published as: CN103421748A

Abstract

The invention provides a porcine circovivus2 strain and application thereof. The novel porcine circovivus2 strain is good in productivity and good in immunogenicity. A vaccine prepared through inactivation of the porcine circovivus2 strain can effectively prevent the porcine circovivus disease. The preservation serial number of the porcine circovivus2 strain is CGMCC NO. 7707. According to the porcine circovivus2 strain (PCV2SD), protective efficacy is good, MPR cells used for strain reproduction can be reproduced well with conventional methods, inoculation can be achieved synchronously as well as with conventional methods, and the virus titer obtained after inoculated culture can reach more than 105.25 TCID/0.1ml; the vaccine prepared with the PCV2SD can well prevent the occurrence or prevalence of the porcine circovivus disease, and the protection rate can reach more than 90% if ELISA detection is carried out after immunity. Compared with other vaccines of the same kind in the domestic market in respect of protective efficacy, the vaccine prepared with the PCV2SD has the advantages of being fast in antibody production, long in protecting period, and capable of shortening the immunity blank area obviously, reducing immunity frequency, and improving economic performance.

Description

A kind of porcine circovirus type 2 strain and application thereof

Technical field

The invention belongs to livestock and poultry pathogenic bacterium triage techniques field, be specifically related to a kind of porcine circovirus type 2 strain and application thereof.

Background technology

Within 1991, Canadian John Harding reports pmws (Postweaning Multisystemic Wasting Syndrome, PMWS), by investigation extensively and profoundly and research, confirmed that pig circular ring virus (Porcine Circovirus, PCV) was main pathogen in 1998.Now confirm, Porcine circovirus desease (Porcine Circovirus Disease, PCVD) be by porcine circovirus 2 type (Porcine Circovirus2, PCV2) infect and a kind of clinical symptom of causing is gradual becoming thin, expiratory dyspnea, hurriedly, anaemia, diarrhoea, jaundice, interstitial pneumonia, the virus disease of lymphadenitis and ephritis, main infringement weanling pig in 5 ~ 12 week age, with the PMWS occurred in recent years, pigskin inflammation and nephritic syndrome (Porcine Dermatitis and Nephropathy Syndrome, PDNS), porcine respiratory syndrome (Porcine Respiratory Disease Complex, PRDC), the congenital chatter of A2 type (Congenital Tremor, CT), Hypertrophic and necrotizing pneumonia (the Porcine Proliferative and Necrotizing Pneumonia of pig, PNP), the diseases such as breeding difficulty (Reproductive Failure) have closely related.The harm of PCV2 is to make the immunologic function of infection pig suffer damage, and causes Abwehrkraft des Koepers to decline, and meanwhile, this disease is other bacterium of secondary, virus infection easily, makes pig sickness rate, and mortality ratio improves, and brings larger financial loss to raiser.Confirm through etiology and serosurvey, PCV2 is worldwide distribution, brings tremendous economic loss to world's pig industry.To this, carry out PCV2 vaccine research both at home and abroad, part has got the Green Light and has produced, and played certain immunoprophylaxis effect, but various places still time have this disease to occur, and can detect that cause of disease exists in healthy swinery, illustrate that existing PCV2 vaccine prevention effect is unsatisfactory, trace it to its cause, except cultivation, disease factor, also may lower with the antigenic content of vaccine (producing cell strain is that PK15, PCV2 can not cause it and produce CPE, cannot Accurate Determining virus titer), vaccine preparation technology is coarse, strain morphs etc., and factor is relevant.Therefore, screen the variation strain made new advances, for preparation PCV2 vaccine, just there is important effect.

Summary of the invention

The object of this invention is to provide a kind of porcine circovirus type 2 strain and application thereof, i.e. a kind of novel porcine circovirus type 2 strain, this strain proliferative ability is high, immunogenicity good, and vaccine prepared by its deactivation effectively can prevent Porcine circovirus desease.

Porcine circovirus 2 type (Porcine Circovirus2) the strain PCV2SD strain of seedling in the present invention, be deposited on May 31st, 2013 and be positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center of Institute of Microorganism, Academia Sinica, deposit number is CGMCC NO.7707.

PCV2SD strain of the present invention is for the preparation of vaccine;

The present invention also provides a kind of vaccine, and described vaccine includes antigen and vaccine adjuvant, and wherein antigen is the PCV2SD virus strain of deactivation.

Wherein prepared by the PCV2SD virus strain formalin-inactivated virus liquid of deactivation.

Vaccine prepared by the present invention is for preventing Porcine circovirus desease.

PCV2SD virus strain immune efficacy of the present invention is good, and the MPR cell for strain propagation can well be bred in conventional manner, and synchronously can connect poison and also ordinary method can connect poison, the virus titer gathered in the crops after inoculation culture is up to 10 ^5.25more than TCID/0.1ml, higher than the titre of the cell cultures such as routine PK15.The vaccine prepared by PCV2SD virus strain can prevent the generation of circovirus disease or popular well, carries out ELISA detection after immunity, and protection ratio can reach more than 90%; Carry out immune efficacy with other similar vaccines of domestic list marketing and compare discovery, vaccine of the present invention has antibody and produces fast, the protection period longer (reaching more than 5 months), can obviously shorten Blank immunization district, reduce immune time, increase economic efficiency.

Embodiment

Below in conjunction with embodiment, the present invention is described in detail.

One, the screening of porcine circovirus 2 type (Porcine Circovirus2) strain PCV2SD strain

1. the screening of strain

The virus strain be separated to the tissue such as lymphoglandula, spleen, lungs of the morbidity piglet on doubtful PMWS pig farm is suffered from 2005 from Shandong Province.By the fresh tissue sample of morbidity pig through fully grinding, dual anti-(blue or green, Streptomycin sulphate) process, 12000rpm/min4 DEG C of centrifugal 10min, after 0.22 μm of millipore filtration Entkeimung, through PCV2, PRV (Pseudorabies virus) (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), Pestivirus suis (CSFV), the PCR/RT-PCR such as pig parvoviral (PPV) detect, turn out to be PCV2 positive merely, Sample supernatants inoculation MPR cell after process is carried out separation and Culture, finally obtain the PCV2 strain of a plant height proliferative ability, called after PCV2SD strain, this virus stain send China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservation of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on May 31st, 2013, deposit number is: CGMMCC No.7707.Carrying out, on series of studies, evaluation of foundation, having prepared porcine circovirus 2 type inactivated vaccine to the physicochemical property of this strain and biological characteristics.

(1) animal Orthogonal Rotational Regressive Tests result shows, attacking malicious group has the pig of 4/5 to occur obvious PCV2 viremia, skin rubefaction and transient fervescence; Attacking poison to compel to kill, dissect after 21 days, there is enlargement and hyperemia in visible many places lymphoglandula, spleen, and lungs occur that oedema in various degree and meat become, and there is blutpunkte even necrosis region etc. on kidney jaundice and surface;

(2) PCV2SD strain can cause MPR cell and produce obvious cells characteristic pathology, and propagation better performances, cultivate according to a conventional method, its virus titer can reach 10 ^5.25tCID/0.1ml;

(3) have wider spectrotype, randomly draw and carry out neutralization test from the porcine blood serum of domestic more than 20 live pig main producing regions and this strain, can neutralize well, MPR cell does not produce characteristic pathology.

Specific as follows:

1) PCV2SD virus strain (is tired and is not less than 10 by cytopathic effect ^5.25tCID ₅₀/ 0.1ml) kind poison by 5%(V/V) amount be inoculated in the MPR cell of firm confluent monolayers, put 37 DEG C, 5%CO ₂cell culture incubator in cultivate, start to occur cytopathy, show as: cell aggregation, fragmentation, to come off that the specific lesions such as then occur big area cavity, draw in the net can be gathered in the crops greatly when 60 ~ 72h about after inoculation after 36.

2) PCV2SD virus strain cell maintenance medium (the MEM liquid of 2% new-born calf serum, purchased from GIBCO) is carried out 10 times of serial dilutions by virus titer, gets 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-74 extent of dilution, be inoculated in the MPR cell (96 porocyte plate) grown fine respectively, each extent of dilution inoculates 4 holes, every hole 0.1ml.Establish virus positive control hole and cell negative control hole, cultivate and observe 7 for 37 DEG C, occur that CPE person is judged to infection, measure its virus titer (TCID50/0.1ml) with Reed-Muench method, every 0.1ml viral level is not less than 10 simultaneously ^5.25tCID ₅₀.

3) animal Orthogonal Rotational Regressive Tests gets the healthy susceptible piglet of 21 ages in days (PCV2ELISA antibody and PCV2 antigen PCR detect and be feminine gender) 10, is divided into 2 groups at random, often organizes 5.Wherein, PCV2SD F5 is attacked for viral cultures, every collunarium 1mL, intramuscular injection 2mL for 1 group; 2 groups of only identical approach inoculating cell nutrient solutions; Clinical observation 21 days after inoculation.Period, can find, attacking malicious group has the pig of 4/5 can occur obvious PCV2 viremia, skin rubefaction and transient fervescence; Attacking poison to compel to kill, dissect after 21 days, there is enlargement and hyperemia in visible many places lymphoglandula, spleen, and lungs occur that oedema in various degree and meat become, and there is blutpunkte even necrosis region etc. on kidney jaundice and surface.

4) spectrotype PCV2SD virus strain carries out neutralization test with the pig PCV2 positive serum picking up from domestic 20 provinces of mainly raising pigs in MPR cell, all effectively can be neutralized by these positive serums.

5) pure asepsis growth, pollutes without mycoplasma and exogenous virus.

Sequencing carries out full genome mensuration to PCV2SD strain, and find that the genomic dna total length of this strain is 1767bp, its nucleotides sequence is classified as SEQ ID NO:1, and the aminoacid sequence of the albumen translated is SEQID NO:2.With the homology of known domestic PC V2 epidemic strain about 96.5%, it is new a kind of pcv2 virus strain.

2: PCV2SD strain of the present invention prepares mono-specific antiserum antibody (primary antibodie) as antigen

By the expressivity BL21 Host Strains that single cloning recombinant plasmids PET-ORF2 correct for sequencing result transforms, be inoculated in the LB bacteria culture medium that 5ml contains penbritin (100mg/L), 37 DEG C of jolting overnight incubation, taking out 1ml culture next day is inoculated in the LB substratum of 120ml, after 2.5h is cultivated in 37 DEG C of joltings, bacterium liquid shakes to translucent half muddy state, and absorption photometry records OD ₆₀₀=0.6 ~ 0.8,10ml is first got as contrast before induction before induction, and jolting temperature is become 30 DEG C add IPTG (final concentration is 1.0mmol/L) induction, induction 4h collects 10ml bacterium liquid, to induce the bacterium liquid of unconverted pet vector as blank, using the bacterium liquid of Induction Transformation pet vector as empty vector control.The bacterium liquid of abduction delivering is taken out, centrifugally abandon supernatant, use the resuspended thalline of 0.5ml1 × PBS again, after ultrasonic treatment disrupt bacteria, add 2 × loading Buffer by 1:1 and boil 10min, centrifuging and taking supernatant, supernatant 12%SDS-PAGE electroresis appraisal analysis obtains the target protein of about 27 ~ 28kDa, utilizes His tag purification kit to carry out purifying by predetermined operation to target protein.His-Cap target protein spectrophotometric standard measure (1.05mg/ml) good for purifying is added the Freund's complete adjuvant of equivalent, neck subcutaneous injection 10 BALB/C mice, every 0.2ml(about 105 μ g/ only), by the Cap protein antigen dorsal sc immunity second time containing Freund's incomplete adjuvant after 2 weeks, 0.2ml/ only, third time immunity (method is with second time) after 2 weeks again, wherein the BALB/C mice of 4 non-immunizing antigens is as negative control group.10d after third time immunity, Culling heart blood is lethal, and the immune serum of precipitation puts-20 DEG C of preservations.

2) the Sensitivity and Specificity qualification of the mono-specific antiserum prepared for PCV2SD strain

By PCV2SD strain isolated virus liquid (10 ^6.25tCID ₅₀/ ml) with the PK15 cell suspension just digested by 1:9 volume ratio combined inoculation to 24 porocyte culture plate (1ml/ hole), Tissue Culture Plate is taken out after maintaining 72h, abandoning supernatant, one time is washed with 1 × PBS, acetone: methyl alcohol (1:1) stationary liquid is in-20 DEG C of fixing 30min, 1 × PBS washes 3 times, used by mice serum 1 × PBS by 1:50 respectively, 1:100, 1:200, 1:500 makes four extent of dilution and is added on the PK15 cell that fixes, hatch 90min for 37 DEG C, 1 × PBS washes 3 times, the sheep anti-mouse igg (Sigma company) 37 DEG C adding the FITC mark of 1:200 dilution again hatches 60min, 1 × PBS washes 3 times, discard washings, again Kong Jiayi each in 24 orifice plates is dripped the glycerine of 50%, observation experiment result under fluorescent microscope, qualification result effect when 1:100 extent of dilution is better.Differentiation qualification is carried out in other 4 strain PCV2:SDId01 strain (HM535640) utilizing the mono-specific antiserum prepared for PCV2SD strain (positive control strain) Cap protein to be preserved laboratory by IiT (IFA), AH05 strain (FJ644556), GXWM strain (EF675241) and SH03733 strain (GQ358998), and result and this 4 strain PCV2 all react and occur specificity green fluorescence.Compared with the mono-specific antiserum prepared with the Cap protein prepared with the PCV2 reported, mono-specific antiserum antibody (primary antibodie) Sensitivity and Specificity height prepared by PCV2SD strain Cap protein of the present invention is all significantly improved (p≤0.05), and this may have the aminoacid sequence of PCV2SD strain of the present invention and the difference of the albumen reported to cause.

Two, the production of porcine circovirus 2 type inactivated vaccine and effect

1. vaccine preparation

1.1 cell preparations are taken out the MPR seed cell pipe preserved that freezes and are put fast melt in 37 DEG C of waters bath with thermostatic control from liquid nitrogen container, cell is moved in aseptic 10ml centrifuge tube, the centrifugal 5min of 1000r/min normal temperature, abandoning supernatant leaves cell precipitation, with containing MEM cell culture fluid (purchased from the GIBCO) re-suspended cell of 8% new-born calf serum, moves into that new cell training sample bottle is mid-contains 5%CO ₂37 DEG C of incubators in carry out adherent culture, 48h after cell grows up to good individual layer, carry out Secondary Culture with pancreas enzyme-EDTA peptic cell.

Deposit number is by 1.2 production seeds culture of viruses preparations: the strain seed culture of viruses of CGMMCC No.7707 by 5% amount be inoculated in go down to posterity after 12h, the MPR cell be in process of growth, 37 DEG C adsorb 1 hour, add the cell maintenance medium containing 2% new-born calf serum, continue cultivation 60 ~ 72h, whole cell culture is gathered in the crops when cytopathy reaches more than 75%, multigelation 3 times, is sub-packed in sterilising vessel.

1.3 antigen preparations

1.3.1 the seed cell of enlarged culturing is inoculated in rolling bottle by monolayer cell culture, 37 DEG C of cultivations.

1.3.2 connect poison and discard nutrient solution, the amount by 5% inoculates the MPR cell grown fine, and puts 37 DEG C of absorption 1h, adds maintenance medium and continues to cultivate.

1.3.3 observe after connecing poison with results, observe 2 every day, record cytopathy situation.Gather in the crops when cytopathy reaches more than 75%, multigelation 3 times.-20 DEG C of preservations.

The virus liquid be up to the standards imports in deactivation tank by 1.4 inactivation of virus, is metered into 10% formaldehyde solution, opens stirrer and stirs, make it fully mix, make the ultimate density of formaldehyde be 0.1%.Deactivation 16 hours is stirred through 37 DEG C.Virus liquid after deactivation puts 2 ~ 8 DEG C of preservations.

1.5 vaccine preparations

1.5.1 oil phase preparation gets injection white oil 94 parts, aluminum stearate 2 parts, mixes and after being heated to 80 DEG C in oil phase preparation tank, then adds Si Ben-806 parts, maintains 30min, after cooling, import in storage tanker for subsequent use when temperature reaches 125 DEG C.

1.5.2 the porcine circovirus 2 type antigen liquid 96 parts be up to the standards and the tween-80 4 parts of sterilizing import in aqueous phase preparation tank by aqueous phase preparation, and agitator motor stirs 20 ~ 30min, and tween-80 is dissolved completely.

1.5.3 emulsification is got in oil phase 2 parts importing emulsion tank, starts motor stirring at low speed, after slowly adding aqueous phase 1 part, then stirs 40 minutes with 4000rpm/min simultaneously.After emulsification, get vaccine 10ml and add in centrifuge tube, the centrifugal 15min of 3000rpm/min, after precipitation, aqueous phase is no more than 0.5ml.

1.5.4 packing quantitative separating, seals.

Two, vaccine safety test

1. porcine circovirus 2 type inactivated vaccine is to the safety testing of target animals non-usage age in days

The porcine circovirus 2 type inactivated vaccine prepared with the present invention 01,02 batch respectively to 9 age in days sodium selenites (PCV2 antigen, negative antibody) 15 (being divided into 3 groups at random), be in and produce the farrowing sow 8 (being divided into 3 groups at random) of first two weeks and carry out immunity, immune group 2ml/ head, the physiological saline of control group injection sample dosage.Security after the vaccination of viewing test pig (search for food, spirit, drinking-water take temperature), piglet Continuous Observation 14 days, sow is observed to childbirth, statistics sows farrowing situation.Piglet exempt from latter 14 days often group cut open and kill 2, observe injection site vaccine absorbing state.Result shows, immune group and control group piglet, pregnant sow search for food, spirit, ight soil are all normal, and injection site has no swelling, tubercle, inflammation; The normal labor of immune group sow, has no the phenomenons such as premature labor, miscarriage, stillborn foetus, weak son and occurs.Illustrate that the vaccine of trial-production is safe to non-usage age in days inoculation piglet and pregnant sow.In table 1, table 2.

Table 1: porcine circovirus 2 type inactivated vaccine (SD strain) inoculates the safety testing of piglet

Note: fervescence is no more than 1 DEG C, delays and is no more than 2; Or subtract food and be no more than 1, it is qualified to be judged to be.If any l head temperature of pig body more than more than 1 DEG C, but be no more than 1.5 DEG C, and delay and be no more than 2, it is qualified also can be judged to.

Table 2 porcine circovirus 2 type inactivated vaccine (SD strain) inoculates the safety testing of pregnant sow

2. the safety testing of a pair minimum use age in days target animals single dose intramuscular inoculation

2 batches of (01, the 02) porcine circovirus type 2 vaccines prepared with the present invention are to 14 age in days susceptible piglet 12,6 monthly age replacement gilt 12 intramuscular injection vaccine respectively, 2ml/ head, each control group injects the physiological saline of same dosage, within 4 ~ 6 weeks after immunity, breeds to test sow.Security after the vaccination of viewing test pig (search for food, spirit, drinking-water take temperature), piglet Continuous Observation 21 days, sow is observed to childbirth, statistics sows farrowing situation.Result shows, 8 piglets of immunity and 8 replacement gilts, it is searched for food, drink water, spirit is showed no exception, Temperature changing is not obvious, immunity sow common property 79 is strong young, 1 weak son, 1 weak young PCV2PCR detection and virus purification result are feminine gender, illustrate that vaccine single dose intramuscular inoculation piglet that the present inventor adopts technical solution of the present invention to manufacture experimently and sow are safe.Refer to table 3, table 4.

Table 3: the safety testing result of a single dose intramuscular inoculation minimum use age in days piglet

Table 4: test sows farrowing situation

3. the safety testing of a pair target animals overdose inoculation

The vaccine prepared with the present invention 2 batches (01,02 batch) respectively carries out overdose immunization respectively to 32 the healthy susceptible piglets of 21 ages in days, 15 Suprapubic arch sling and 9 herd boars, be divided at random 3 groups (specifically seeing the following form) separately, test group immunizing dose is 4ml/ head, the physiological saline of control group injection Isodose.Piglet and herd boar exempt from rear observation 21 days, and sow breeding in 4 ~ 6 weeks after immunity is observed to farrowing, every day the searching for food of viewing test pig, mental status, injection site has no adverse reaction.Result shows, this vaccine all has no adverse effects to the piglet inoculated, sow and herd boar, and injection site has no swelling, tubercle, inflammation, test pig Temperature changing is not obvious, and the sow of inoculation normally farrows, and common property 105 is strong young, 1 weak son, compared with contrast 5 sows, farrowing has no difference.Illustrate that the vaccine that the present inventor adopts technical solution of the present invention to manufacture experimently inoculates safety to the disposable overdose of piglet, Suprapubic arch sling and herd boar.In table 5, table 6.

Table 5: the proof test result of disposable overdose inoculation

Table 6: test broad sow farrowing situation

4. on the impact of sow immunologic function

Get 28 age in days sodium selenites (PCV2, swine fever antigen, antibody are feminine gender) 16 and be divided into 4 groups at random, often organize 4.The porcine circovirus 2 type inactivated vaccine prepared with the present invention 01 batch of intramuscular immunisation the 1st group, 2ml/ head; Swine fever spleen seedling intramuscular immunisation the 2nd group, 1 part/head; Porcine circovirus 2 type inactivated vaccine 2ml/ head and swine fever spleen seedling 1 part/head intramuscular immunisation the 3rd group simultaneously; 4th group of injecting normal saline compares, 2ml/ head, and four groups of pigs are raised all separately.Observe every day and record every pig body temperature, search for food.Blood sampling in 2nd, 4,8,12 week after inoculation, separation of serum, detects PCV2 and CSFV ELISA antibody.Result shows, body temperature after each immune group piglet immunological in 14 days, search for food, drink water, the mental status is all normal, the immunity simultaneously of porcine circovirus 2 type inactivated vaccine and swine fever spleen seedling, little to having an impact of CSFV antibody, as can be seen here, porcine circovirus 2 type inactivated vaccine (SD strain) on the immune effect of swine fever spleen seedling without impact.Refer to table 7, table 8.

Table 7: porcine circovirus 2 type inactivated vaccine and the independent immune result of swine fever spleen seedling

Note: porcine circovirus 2 type ELISA antibody criterion (Jeno Biotech): S/P value >=0.4 is positive; 0.3≤S/P value < 0.4 is suspicious; S/P value < 0.3 is negative; Swine fever ELISA antibody criterion (IDEXX): CSFV blocking-up rate >=40% is positive; 30% < blocking-up rate < 40% is suspicious; Blocking-up rate≤30% is negative.Following table is same.

Table 8 porcine circovirus 2 type inactivated vaccine and swine fever spleen seedling immune result simultaneously

Three, immune antibody and attack poison protect correlation test

For determining the immune antibody of porcine circovirus 2 type inactivated vaccine prepared by the present invention and attacking the malicious dependency protected; healthy susceptible piglet 20 is injected by various dose (0.125ml/ head, 0.25ml/ head, 0.5ml/ head, 1.0ml/ head) musculi colli with 01 batch of porcine circovirus 2 type inactivated vaccine of the present inventor's trial-production; be divided into 4 groups at random; often organize 5, separately establish 5 nonimmune control groups.After immunity 2nd ~ 5 weeks, flash liberation serum of taking a blood sample weekly, carried out PCV2ELISA antibody test.Result display (table 9): PCV2 deactivation vaccine exempts from latter 14 days, 0.125ml/ head and 0.25ml/ head dosage group, ELISA antibody equal 5/5 is negative; 0.5ml/ head dosage group ELISA antibody 1/5 is suspicious, and 4/5 is negative; 1.0ml/ head dosage group, ELISA antibody 1/5 is positive, and 1/5 is suspicious, and 3/5 is negative; Exempt from latter 21 days, 0.125ml/ head dosage group, ELISA antibody equal 5/5 is negative; 0.25ml/ head dosage group ELISA antibody 1/5 is positive, and 4/5 is negative; 0.5ml dosage group ELISA antibody 3/5 is positive, and 1/5 is suspicious, and 1/5 is negative; 1.0ml/ head dosage group, ELISA antibody 4/5 is positive, and 1/5 is suspicious; Exempt from latter 28 days, 0.125ml/ head dosage group ELISA antibody 1/5 is suspicious, and 4/5 is negative; 0.25ml/ head ELISA antibody 2/5 is positive, and 2/5 is suspicious, and 1/5 is negative; 0.5ml/ head dosage group ELISA antibody 4/5 is positive, and 1/5 is suspicious; 1.0ml/ head dosage group ELISA antibody 5/5 is positive; Exempt from latter 35 days, 0.125ml/ head dosage group, ELISA antibody 1/5 is positive, and 1/5 is suspicious, and 3/5 is negative; 0.25ml/ head dosage group, ELISA antibody 2/5 is positive, and 2/5 is suspicious, and 1/5 is negative; 0.5ml/ head dosage group ELISA antibody 4/5 is positive, and 1/5 is suspicious; 1.0ml/ head dosage group ELISA antibody equal 5/5 is positive.

Different time ELISA antibody test result after table 9 various dose vaccine immunity

Note: S/P value >=0.4 is positive (+); 0.3≤S/P value < 0.4 is suspicious (±); S/P value < 0.3 is negative (-).

According to the antibody test result of the 5th week after exempting from, test pig is divided into 3 groups, with PCV2FJ strain (viral level 10 by negative antibody, the suspicious and positive ^6.25tCID ₅₀/ mL) attack, every collunarium 1mL, intramuscular injection 2mL.Attack Continuous Observation 28 days after poison, all test pig are weighed the clinical response of calculating body weight increase rate, record test pig.Cut open and kill test pig, get inguinal lymph nodes, adopt quantitative fluorescent PCR to carry out viral nucleic acid carrying capacity mensuration.According to the result of nucleic acid load and body weight increase rate, ELISA antibody all can not be protected after attacking poison time negative, attacks malicious rear section protection (this test protection ratio is 25%) when ELISA antibody is suspicious, and ELISA antibody is all protected after attacking poison time positive.

The above results shows that vaccine prepared by porcine circovirus 2 type SD of the present invention strain effectively can prevent the Porcine circovirus desease caused by porcine circovirus 2 type.

Claims

1. a porcine circovirus type 2 strain, is characterized in that, described porcine circovirus type 2 strain is PCV2SD strain, and its deposit number is CGMCC NO.7707.

2. strain according to claim 1 is preparing the application in vaccine.

3. a vaccine, described vaccine includes antigen and vaccine adjuvant, and wherein antigen is the strain according to claim 1 of deactivation.

4. vaccine as claimed in claim 3, is characterized in that described strain formaldehyde carrys out deactivation.