CN106039304A - Porcine parvovirus, porcine epizootic diarrhea and Escherichia coli triple-antigen vaccine - Google Patents

Porcine parvovirus, porcine epizootic diarrhea and Escherichia coli triple-antigen vaccine Download PDF

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CN106039304A
CN106039304A CN201610368958.5A CN201610368958A CN106039304A CN 106039304 A CN106039304 A CN 106039304A CN 201610368958 A CN201610368958 A CN 201610368958A CN 106039304 A CN106039304 A CN 106039304A
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porcine
vaccine
triple
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escherichia coli
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CN106039304B (en
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刘新文
杜元钊
王秀丽
郭玉广
陶晓珊
刘蕾
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Qingdao Yebio Bioengineering Co Ltd
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    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract

The invention provides porcine parvovirus, porcine epizootic diarrhea and Escherichia coli triple-antigen vaccine. The porcine parvovirus, porcine epizootic diarrhea and Escherichia coli triple-antigen vaccine contains antigens and vaccine adjuvants. The antigens are porcine parvovirus VP2 proteins and fimbrial antigens of porcine epizootic diarrhea viruses and Escherichia coli K88, K99 and 987P, the porcine parvovirus VP2 proteins are expressed by strains X33-VP2 with a preservation number of CCTCC M 2016098, and a preservation number of the porcine epizootic diarrhea viruses and the Escherichia coli K88, K99 and 987P is CGMCC No.8503. The porcine parvovirus, porcine epizootic diarrhea and Escherichia coli triple-antigen vaccine has the advantages that the porcine parvovirus, porcine epizootic diarrhea and Escherichia coli triple-antigen vaccine is good in immunogenicity, high in post-immunization antibody production rate, long in storage life and low in immunizing dose, produced antibodies are high in titer and long in hold time, the selected adjuvants are easy to inject, and three types of diseases can be prevented and treated by means of injection at one step; immune injection can be carried out prior to mating, accordingly, piglets laid by pregnant sow are excellent in passive immunity and firm in immunity and can resist virulent virus infection, and the survival rate of the piglets can be increased.

Description

A kind of pig parvoviral, porcine epizootic diarrhea, colon bacillus triple vaccine
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of pig parvoviral, porcine epizootic diarrhea, big Intestinal Escherichia triple vaccine.
Background technology
Pig parvoviral (Porcine parvovirus, PPV) is one of important pathogen causing sow breeding difficulty, can Cause Sow abortion, premature labor, product stillborn fetus, mummy tire, weak son and sow is sterile and newborn piglet mortality.China is from 20 generation Record after the eighties and be in succession separated to PPV from various places, further investigation reveals that the Seropositive rates of this disease is up to 85% in some swinerys Above.PPV often exists in inapparent infection, and the especially phenomenon of low dosage persistent infection often occurs.This virus is except causing sow Breeding difficulty, also can cause multiple disease with other pathogen synergism, cause the biggest economic loss to pig industry.Pig Parvovirus serotype is single, have higher immunogenicity, and vaccination is to control the effective measures that PPV infects.
Porcine epizootic diarrhea (Porcine epidemic diarrhea, PED) is caused by Porcine epidemic diarrhea virus A kind of high degree in contact infectious intestinal disease of pig, drops to basic feature with vomiting, diarrhoea and appetite, and primary disease is to all ages and classes Pig is the most susceptible, and especially suckling pig harm is the most serious, is a kind of high degree in contact sexually transmitted disease.This disease is starting in English in 1971 State, early 1980s there is primary disease in China successively.In recent years, whole nation porcine epizootic diarrhea was eruption and prevalence, to aquaculture Cause huge economic loss.This disease infects and there is presently no effective Therapeutic Method, with vaccine immunity prevention is mainly Main.At present, abroad it is mainly the vaccines such as the Attenuate vaccine of Korea S, the domestic transmissible gastroenteritis of swine mainly breathing out beast development, but Clinical application effect is the best.
It addition, yellow scour of piglet is the first cub caused by the pathogenic colon bacillus (E.coli) of specific O antigenic type The infectious disease being characterized with dysentery of pig.This disease mainly results in (mainly 1～3 age in days) piglet morbidity in 1 week old, to discharge Huang Color watery stools and progressive death are characterized, and are to affect scale kind pig farm and the important disease of pig piglet survival ratio is raised in rural area scattered Sick.But effectively do not prevent pig parvoviral, porcine epizootic diarrhea, colon bacillus triple vaccine in the market.
Summary of the invention
It is an object of the invention to provide a kind of pig parvoviral, porcine epizootic diarrhea, colon bacillus triple vaccine, from And make up the deficiencies in the prior art.
The pig parvoviral of the present invention, porcine epizootic diarrhea, colon bacillus triple vaccine, include antigen and vaccine Adjuvant, wherein antigen include PPV VP 2 protein, Porcine epidemic diarrhea virus and colon bacillus K88, K99, The fibrial antigen of 987P.
Wherein the aminoacid sequence of PPV VP 2 protein is SEQ ID NO:1, expresses PPV VP 2 protein Pichia Pastoris X33-VP2 (Pichia pastoris X33-VP2), be preserved in force on March 9th, 2016 The China typical culture collection center of the Chinese, deposit number is CCTCC NO:M 2016098.
Described Porcine epidemic diarrhea virus, embodiment is preferably Porcine epidemic diarrhea virus SD10 strain (pig epidemic Diarrhea virus Porcine Epidemic Diarrhea Virus), it is preserved in the Chaoyang District, Beijing City North Star on November 08th, 2013 No. 3 China Committee for Culture Collection of Microorganisms of Chinese Academy of Sciences common micro-organisms centers of West Road 1 institute, deposit number is: CGMCC No.8503。
Described vaccine adjuvant is aluminium hydroxide gel adjuvant.
PPV VP 2 protein content >=150 μ g/ml, Porcine epidemic diarrhea virus content in above-mentioned triple vaccine ≥106.0TCID50/ ml, K88 fibrial antigen content >=100 antigen unit/ml, K99 fibrial antigen content >=50 antigen list Position/ml, 987P fibrial antigen content >=50 antigen unit/ml.
The pig parvoviral of the present invention, porcine epizootic diarrhea, colon bacillus triple inactivated vaccine immunogenicity are good, exempt from After epidemic disease, antibody produces fast, and the antibody titer height of generation and length of holding time, long shelf-life, immunizing dose is little, and the adjuvant of selection is easy In injection, three kinds of diseases can be prevented and treated by a pin, before breeding, carry out inoculation the farrowed pig of farrowing sow can be made to obtain preferably Passive immunity, makes piglet produce strong immunity, it is possible to the attack of the strong poison of opposing, improves the survival rate of piglet.
Accompanying drawing explanation
Fig. 1 is the amplification qualification figure of embodiment of the present invention PPV VP 2 protein gene PCR,
Fig. 2 is the blast comparison diagram of the VP2 albumen of the present invention；
Fig. 3 is embodiment of the present invention recombinant bacterium fermentation inducement expression product SDS-PAGE electrophoresis rating diagram.
Detailed description of the invention
Below in conjunction with the accompanying drawings embodiments of the invention are specifically described.
Embodiment 1: the screening of pig parvoviral (PPV) VP2 gene
Within 2010, in Shandong Province, multiple plants occur in that the symptom of porcine parvovirus, and before Affected individuals pig Inject existing parvovirus vaccine, thus it is speculated that the virus of infection there occurs variation；Therefore from Affected individuals, pig is carried out tiny The screening of virus；Finishing screen have selected pig parvoviral PPV1.
For the antigenicity of the virus of checking screening, employ 5 separate sources of the PPV1 strain comprising screening Strain prepares vaccine as antigen, carries out challenge viral dosage with the virus liquid of screening after immunity SPF pig, and result shows compared to it Its swine parvovirus vaccine, the vaccine itself prepared has more preferable immune effect (p ＜ 0.05), it is thus determined that it there occurs Variation in heredity.
Antigenic characteristic according to PPV VP 2 protein and aminoacid sequence, design synthesis pair of primers, primer 1:5'-GCGGTACCATGTCTGAAAATGTTGAAGAAC-3', containing KpnI site；
Primer 2:5'-GCGGCCGCCTAATATAATTTTCTTGGAAT-3', containing NotI site.Take PPV strain virus base Because as template, PCR expands purpose fragment, product reclaims and connects pMD18-T carrier, converts and screening positive clone pMD18-T- VP2, through sequencing the amplification qualification figure of pig parvoviral VP2 gene PCR (Fig. 1 be)；Its nucleotides sequence is classified as SEQ ID NO:2, the sequence of the albumen of coding is SEQ ID NO:1.Compared with the pig parvoviral VP2 gene disclosed in NCBI, the highest Homology is 96%；In the case of existence 579 is amino acid whose, show that the PPV VP 2 protein of the present invention is with published Albumen exists no less than 23 amino acid whose differences (Fig. 2).
Embodiment 2: the preparation of recombination porcine parvovirus VP2 albumen
Comprise the following steps: a. construction of expression vector；B. construction expression bacterial strain；C. the induction of recombinant VP 2 albumen and extraction Purification.Specific as follows: by double with KpnI and NotI respectively to positive colony plasmid pMD18-T-VP2 and expression vector pPICZ α carrier Digestion products after 1.2% agarose gel electrophoresis, with DNA gel reclaim test kit reclaim, obtain respectively about 1.7kb and 3.3kb fragment, connects 16 DEG C of orientations and builds pPICZ α-VP2 expression vector, after enzyme action is identified correctly；By plasmid linearization After, electricity is transformed into Pichia sp. competent cell, builds Pichiapastoris expression strain X33-VP2, and extracts expression strain X33- VP2 genome, uses primer primer 1 and primer 2 to carry out PCR and identifies correct, be preserved in China on March 9th, 2016 Type Tissue Collection, deposit number is CCTCC M2016098；Carry out abduction delivering, the picking Dan Ke containing X33-VP2 Grand colony inoculation is in BMGY fluid medium, and 30 DEG C of shaken cultivation overnight, are centrifuged and collect thalline, after suspending with appropriate BMMY, Add 0.55% methanol, induce 96 hours for 30 DEG C.4 DEG C, 9000rpm is centrifuged 5min, retains supernatant, adds the ammonium sulfate of 40% After precipitation, 12000rpm is centrifuged 5min and collects albumen precipitation, with the PBS molten albumen of weight.Add protein electrophoresis sample-loading buffer, boiling water After boiling 8 minutes, the separation gel with 12% carries out SDS-PAGE qualification, and (Fig. 3 is the SDS-of recombinant bacterium fermentation inducement expression product PAGE electrophoresis rating diagram, in figure 1,2, precipitate for VP2 protein expressioning product, M is molecular weight marker proteins matter).
The Pichiapastoris expression strain X33-VP2 strain producing PPV VP 2 protein is preserved in Chinese Typical Representative cultivate Thing preservation center, deposit number is CCTCC M 2016098.
1. X33-VP2 strain is inoculated in the YPD fluid medium containing bleomycin by the preparation of seedling bacterium solution, and 30 DEG C shaken cultivation 16～18 hours.Then streak inoculation is in the YPD solid medium added with bleomycin, chooses colonies typical 2 ～3 be mixed in a small amount of YPD fluid medium, putting shaken cultivation 18 hours in 30 DEG C of shaking tables, quantitative separating, through purely inspection After, as first order seed.Taking first order seed to be inoculated in BMGY fluid medium, 30 DEG C of shaken cultivation 16～18 hours, through mirror After inspection, put 2～8 DEG C of preservations, as secondary seed.
2. the preparation of seedling albumen presses fermenter volume 60% (V/V) the addition incomplete fluid medium of BMGY, simultaneously Add defoamer by culture medium 0.1% (V/V), be passed through high-temp steam sterilizing 30 minutes, treat that culture medium temperature is down to 32 DEG C, add YNB and biotin, Pigs Inoculated parvovirus VP2 protein production Pichia sp. X33-VP2 secondary seed solution, fermentation tank parameter sets Put respectively mixing speed 800r/min, temperature 30 DEG C, maintain DO value (dissolved oxygen amount) 20%.Bacterium solution after cultivating 24 hours is mended Adding methanol, the speed of adding of methanol is that 2ml/h/L abduction delivering is cultivated, according to above ferment control parameter and technique abduction delivering 120 hours；Fermentation culture bacterium solution is centrifuged 30 minutes through tube centrifuge 10000r/min, the supernatant of results, adds ammonium sulfate and sinks After the albumen of shallow lake, it is centrifuged 30 minutes results precipitations by 12000r/min, adds appropriate physiological saline solution albumen precipitation.
3., in protein liquid is placed in inactivation bottle by inactivation, metering adds 10% formalin, with adding with shaking so that it is the most mixed Closing, the ultimate density of formalin is 0.1%.Pour in another inactivation bottle after adding formalin, to avoid adhering near bottleneck Virus fail contact inactivator.37 DEG C inactivation 16 hours after take out, put 2～8 DEG C of preservations.
4. the inspection of semifinished product
(1) steriling test carries out steriling test by existing " Chinese veterinary pharmacopoeia " annex.
(2) determining the protein quantity presses Bradford method detection protein content.
(3) protein liquid after inactivation is taken a small amount of inoculation YPD solid medium by inactivation inspection, is placed in 30 DEG C and continues training Support 72 hours.Observe without colony growth, sentence inactivation inspection qualified.
Embodiment 3: the preparation of subunit inactivated vaccine
Semi-finished product VP2 proteantigen through after the assay was approved carries out vaccine and prepares that (in following preparation, each liquid component is pressed Volume basis).
(1) oil phase preparation takes white oil for animals 95 parts, aluminium stearate 1 part, is placed in after being heated to 80 DEG C in oil phase preparation tank, Jia Siben-80 5 parts again, to temperature reach 115 DEG C time, maintain 30min, standby after cooling.
(2) PPV VP 2 protein is used normal saline dilution to become 150 μ g/0.1ml by aqueous phase preparation.After taking sterilizing 5 parts of tween 80s, add in Agitation Tank, be simultaneously introduced seedling protein liquid 95 parts, stir 20～30min, make tween 80 complete CL.
(3) emulsifying takes oil phase 2 parts and is put in high-speed shearing machine, starts the stirring of motor slow rotation, the most slowly adds water 1 part mutually, with 10000r/min, emulsifying 5 minutes.After emulsifying, take 10ml, be centrifuged 15 minutes with 3000r/min, the water separated out at the bottom of pipe Corresponding less than 0.5ml.
Vaccine product inspection
(1) character
Outward appearance vaccine should be milky Emulsion, free from admixture and outer package should be qualified.
Dosage form is water-in-oil type.Take a cleaning suction pipe, draw a small amount of vaccine and instill in cold water, in addition to the 1st, all should not Diffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, is centrifuged 15 minutes with 3000r/min, the aqueous phase separated out at the bottom of pipe 0.5ml should be less than.
Viscosity is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.
(2) loading quantity inspection is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.
(3) steriling test is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.
(4) safety verification 50 age in days piglet 5, every cervical region subcutaneous injection vaccine 1.0ml, sets comparison 5 simultaneously, Raising at identical conditions, Continuous Observation 14 days, record test pig is searched for food, is drunk water and clinical setting.Should occur without by vaccine Any locally and systemically untoward reaction caused.
(5) efficacy test first farrowing sow 5, every cervical region subcutaneous injection vaccine, 0.5ml/ only, separately takes 5 at the beginning of with age in days Produce sow to compare as the most immune.After insemination of sows, conceived to counteracting toxic substances when 38-40 days, result is from inoculation subunit vaccine The blood plasma of sow is not separated to virus, compares and the blood plasma of sow is then separated to virus.Counteracting toxic substances was slaughtered after 40 days, inoculation Sow cherishes 65 the first-born pigs altogether, is not the most separated to virus, and compares sow and cherish 60 the first-born pigs altogether from their internal organs, Qi Zhongyou The internal organs of 31 are separated to virus.Result shows, can resist disease with PPV VP 2 protein subunit vaccine immunity sow The attack of poison, occurs without clinical symptoms, and tire pig virus purification is feminine gender., there is slight clinical symptoms, tire swine diseases in matched group Poison separation positive rate, more than 50%, shows that pig parvoviral subunit inactivated vaccine can provide and is effectively protected.
The preparation method of the triple inactivated vaccine of the present invention, it is characterised in that comprise the following steps:
Embodiment 4, the preparation of triple vaccine
(1) Vero cell monolayer is prepared in the seedling preparation of Porcine epidemic diarrhea virus antigen, takes Porcine Epidemic Diarrhea Poison SD10 strain is inoculated into Vero cell monolayer by 1% inoculum concentration, puts 37 DEG C and adsorbs 1.5 hours, adds containing 2% new-born calf serum MEM Liquid is maintained to continue to cultivate；After connecing poison, results virus liquid, multigelation 3 times when cytopathy reaches more than 75%；
(2) taking the virus liquid after freeze thawing and measure viral level, every 0.1ml viral level is not less than 105.0TCID50；Will inspection Qualified cell venom is placed in sterilization container, adds 10% formalin solution extremely final concentration of 0.2%, and 37 DEG C of inactivations 18 are little Time；
(3) preparation of seedling fibrial antigen presses fermenter volume 70% (V/V) addition Minca fluid medium, simultaneously Add defoamer by culture medium 0.1% (V/V), be passed through high-temp steam sterilizing 30 minutes, treat that culture medium temperature is down to 35～37 DEG C, Adding culture in fermentation tank by 3% (V/V), regulation cultivation temperature is to 37 DEG C, and regulation melts oxygen concentration to 35%, cultivates 5～7 Hour prepare the bacterium solution of colon bacillus C83549 bacterial strain containing K88 fibrial antigen, the large intestine angstrom containing K99 fibrial antigen respectively The bacterium solution of uncommon Salmonella C83644 bacterial strain and the bacterium solution of the colon bacillus C83710 bacterial strain containing 987P fibrial antigen.
(4) each strain fermentation is cultivated bacterium solution and is centrifuged 30 minutes through tube centrifuge 10000r/min, and the bacterium mud of results is pressed 0.1g/ml ratio is resuspended in normal saline.Use cutter at 58～65 DEG C of large intestines to containing different fibrial antigens The bacteria suspension of Escherichia is sheared by the shear strength of 5000～6000r/min and is carried out each fibrial antigen respectively in 20～25 minutes Extract；
(5) press in every 1ml finished product Seedling, K88 fibrial antigen content >=100 antigen unit, K99 fibrial antigen content >=50 Individual antigen unit, 987P fibrial antigen content >=50 antigen unit, preparation mixing cilium liquid；
(6) preparation of seedling PPV VP 2 protein will produce the Pichia sp. table of PPV VP 2 protein (being preserved in China typical culture collection center on March 9th, 2016, deposit number is CCTCC M to reach the strain of strain X 33-VP2 The X33-VP2 bacterial strain of 2016098) it is inoculated in the YPD fluid medium containing bleomycin, 30 DEG C of shaken cultivation 16～18 are little Time.Then streak inoculation is in the YPD solid medium added with bleomycin, choose colonies typical 2～3 be mixed in a small amount of YPD In fluid medium, putting shaken cultivation 18 hours in 30 DEG C of shaking tables, quantitative separating, after purely inspection, as first order seed.Take First order seed is inoculated in BMGY fluid medium, 30 DEG C of shaken cultivation 16～18 hours, after microscopy, puts 2～8 DEG C of preservations, As secondary seed.
(7) press fermenter volume 60% (V/V) and add the incomplete fluid medium of BMGY, simultaneously by culture medium 0.1% (V/ V) add defoamer, be passed through high-temp steam sterilizing 30 minutes, treat that culture medium temperature is down to 32 DEG C, add YNB and biotin, inoculation (6) in, (deposit number is CCTCC M to the PPV VP 2 protein production Pichia sp. secondary seed X33-VP2 of preparation 2016098), fermentation tank parameter arranges and is respectively mixing speed 800r/min, and temperature 30 DEG C maintains DO value (dissolved oxygen amount) to exist 20%.Bacterium solution after cultivating 24 hours adds methanol, methanol add speed be 2ml/h/L abduction delivering cultivate, according to more than Ferment control parameter and technique abduction delivering 120 hours；Fermentation culture bacterium solution is centrifuged 30 points through tube centrifuge 10000r/min Clock, the supernatant of results, after adding ammonium sulfate precipitated protein, it is centrifuged 30 minutes results precipitations by 12000r/min, adds appropriate Physiological saline solution albumen precipitation.
(8), in protein liquid is placed in inactivation bottle by inactivation, metering adds 10% formalin, with adding with shaking so that it is the most mixed Closing, the ultimate density of formalin is 0.1%.Pour in another inactivation bottle after adding formalin, to avoid adhering near bottleneck Virus fail contact inactivator.37 DEG C inactivation 16 hours after take out, put 2～8 DEG C of preservations.
(9) press in every 1ml finished product Seedling, PPV VP 2 protein content >=150 μ g, prepare proteantigen.
(10) after the protein solution in the cilium solution in the virus liquid in (2), (5) and (8) being mixed in proportion, with epidemic disease Seedling adjuvant is prepared by mixing into vaccine.
Embodiment 2, the inspection of triple inactivated vaccine
(1) after character stands, upper strata is pale red clear liquid, and lower floor is yellow-white precipitation, in uniform suspendible after shaking Liquid.
(2) steriling test is tested according to " People's Republic of China's veterinary drug allusion quotation " annex, the 3 the most aseptic lifes of mass products vaccine Long.
(3) safety verification does not eats just suckling piglet 20 with 14 ages in days, is randomly divided into 4 groups, often group 5, the 1st, 2,3 components Other musculi colli injection pig parvoviral, porcine epizootic diarrhea, colon bacillus triple inactivated vaccine each 4ml/ head, observe 14 Day, 15 pigs vaccinating group are all strong alive, and no abnormality seen reaction occurs, matched group piglet no abnormality seen.
Table 1 vaccine character, steriling test, safety verification and residual formaldehyde testing result
(4) efficacy test
4.1 pig parvoviral part first farrowing sows 5, every cervical region subcutaneous injection vaccine, 0.5ml/ only, separately takes 5 together Age in days first farrowing sow compares as the most immune.After insemination of sows, conceived to counteracting toxic substances when 38-40 days, result is from inoculation subunit The blood plasma of the sow of vaccine is not separated to virus, compares and the blood plasma of sow is then separated to virus.Counteracting toxic substances killed after 40 days Killing, vaccination of sows cherishes 65 the first-born pigs altogether, is not the most separated to virus, and compares sow and cherish 60 the first-borns altogether from their internal organs Pig, wherein has the internal organs of 31 to be separated to virus.Result shows, with PPV VP 2 protein subunit vaccine immunity sow Can resist the attack of virus, occur without clinical symptoms, tire pig virus purification is feminine gender., there is slight clinic in matched group Symptom, tire pig virus purification positive rate, more than 50%, shows that pig parvoviral subunit inactivated vaccine can provide effective guarantor Protect.
4.2 porcine epizootic diarrhea vaccine fractions 8 porcine epizootic diarrhea negative antibodies, the cloudy antenatal 30-45 age in days of antigen In-pig, is randomly divided into 4 groups, often group 2, the 1st, 2,3 groups of difference musculi colli injection pig parvoviral, pig epidemics Rush down, colon bacillus triple inactivated vaccine 201501,201502,201503 batches, do not inject as a control group for 2, often group is each Take 5-7 age in days and do not eat just suckling piglet 5, porcine epidemic diarrhea poison 5ml/ head by force, observe clinical manifestation, according to morbidity result Judge the protection of vaccine.Counteracting toxic substances result shows, immune group 4/5,5/5,5/5 protection respectively, matched group 4/5 is fallen ill.
The efficacy test of 4.3 bacillus coli vaccine fractions takes finished product vaccine 5～10ml, repeatedly freezes in-20 DEG C Melt 8 times, be centrifuged 15min with 3000r/min, take the aqueous phase separated out at the bottom of pipe and measure the antigen list of tri-kinds of ciliums of K88, K99,987P Position, in the aqueous phase with precipitation, K88 fibrial antigen >=300 antigen unit/ml, K99 fibrial antigen >=150 antigen unit/ It is qualified that ml, 987P fibrial antigen >=150 antigen unit/ml is judged to vaccine potency inspection.
(5) testing result of 3 batches of inactivated vaccines of residual formaldehyde detection is respectively 0.096%, 0.087%, 0.091%, It is below the regulation (0.2%) of veterinary biologics general rule.
The pig parvoviral of the present invention, porcine epizootic diarrhea, colon bacillus triple inactivated vaccine immunogenicity are good, exempt from After epidemic disease, antibody produces fast, and the antibody titer height of generation and length of holding time, long shelf-life, immunizing dose is little, and the adjuvant of selection is easy In injection, three kinds of diseases can be prevented and treated by a pin, before breeding, carry out inoculation the farrowed pig of farrowing sow can be made to obtain preferably Passive immunity, makes piglet produce strong immunity, it is possible to the attack of the strong poison of opposing, improves the survival rate of piglet.And, contrast Experiment shows, carries out immunity with the tiny disease vaccine of pig sold in the market, then attacks with the PPV1 strain of present invention screening Poison experiment；Result shows that the immune effect of the vaccine sold in the market is far below the effect of the triple vaccine of the present invention；Push away Survey is to cause the immune effect of current vaccine bad owing to the VP2 protein vaccine of PPV1 strain morphs.

Claims (6)

1. a triple vaccine, it is characterised in that described vaccine includes antigen and vaccine adjuvant, and wherein antigen includes pig Parvovirus VP2 albumen, Porcine epidemic diarrhea virus and colon bacillus K88, the fibrial antigen of K99,987P.
2. triple vaccine as claimed in claim 1, it is characterised in that the aminoacid sequence of described PPV VP 2 protein It is classified as SEQ ID NO:1.
3. triple vaccine as claimed in claim 1 or 2, it is characterised in that described PPV VP 2 protein is by preservation The Pichia Pastoris of numbered CCTCC NO:M 2016098 expresses preparation.
4. triple vaccine as claimed in claim 1, it is characterised in that the deposit number of described Porcine epidemic diarrhea virus is CGMCC No.8503。
5. triple vaccine as claimed in claim 1, it is characterised in that described vaccine adjuvant is aluminium hydroxide gel adjuvant.
6. triple vaccine as claimed in claim 1, it is characterised in that PPV VP 2 protein content in described vaccine >=150 μ g/ml, Porcine epidemic diarrhea virus content >=106.0TCID50/ ml, K88 fibrial antigen content >=100 antigen unit/ Ml, K99 fibrial antigen content >=50 antigen unit/ml, 987P fibrial antigen content >=50 antigen unit/ml.
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