CN107899008A - Sick three subunit vaccines of a kind of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus - Google Patents

Abstract

The present invention provides a kind of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus sick three subunit vaccines, include antigen and vaccine adjuvant, wherein antigen is by the Porcine epidemic diarrhea virus S1 albumen for the S1 plants of expression of X33 PEDV that deposit number is CGMCC No.14372,1 albumen of pig infectious gastroenteritis virus S for the S1 plants of expression of X33 TGEV that deposit number is CGMCC No.14371, and the pig fourth type coronavirus S1 albumen for the S1 plants of expression of X33 PDCoV that deposit number is CGMCC No.14370.Sick three subunit vaccines of the pig epidemic diarrhea of the present invention, transmissible gastroenteritis of swine, pig fourth type coronavirus are safe, immunogenicity is good, antibody is produced quick and held time length compared with antibody titers after immune, vaccine long shelf-life, immunizing dose is small, the adjuvant of selection is easy to inject, can a pin prevent three kinds of main diarrhea related diseases viral diseases, improve the survival rate of piglet.

Description

A kind of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus disease three Subunit vaccine

Technical field

The invention belongs to veterinary biologics technical field, and in particular to a kind of pig epidemic diarrhea, pig transmissible stomach and intestine Scorching, sick three subunit vaccines of pig fourth type coronavirus

Background technology

Pig epidemic diarrhea (Porcineepidemicdiarrhea, PED) is the pig as caused by Porcine epidemic diarrhea virus A kind of high degree in contact enteric infectious disease, using diarrhea, vomiting and loss of appetite as essential characteristic, pig of this disease to all ages and classes Susceptible, especially suckling pig endangers most serious, is a kind of highly contagious disease.The disease is starting in Britain in 1971, China first appears the disease within 1986.Since 2010, national pig epidemic diarrhea was in outbreak of epidemic, popular field strain There is variation in strain before comparing 2010, and huge economic loss is caused to aquaculture.

Transmissible gastroenteritis of swine（Transmissible gastroenteritis, TGE) be pig a kind of acute intestinal Infectious disease, falls ill anxious, propagates soon, and the pig at various ages can infect, to cause piglet within 3-10 ages in days to vomit, seriously Diarrhea, become thin and high mortality（Usual 100%）Be characterized, morbid pig also often shows as acute watery diarrhea, excrement in yellow or Light green or canescence, are mixed with indigested ziega in some suckling pig excrement.The malicious pig of sick pig, band and its excreta, breast Juice, vomitus etc. are the important infection sources, and disease whole year can occur, and various regions are in distribute prevalence in recent years.

Pig fourth type coronavirus（Porcine Deltacoronavirus, PDCoV) belong to the more viraleses of Buddhist nun (Nidovirales), the coronal diseases of Delta of coronaviridae (Coronaviridae), coronavirus genus (Coronavirus) Poison, to there is the single strand plus RNA virus of cyst membrane.At the beginning of 2014, Ohio, USA reports a kind of new pig enteric coronavirus at first Viral PDCoV, the end of the year then the disease in 17 states in the U.S. it is popular.Then plus by also being reported with South Korea detect PDCoV, its positive rate are up to 25%.What is more important, the clinical inspections of the PDCoV of CONTINENTAL AREA OF CHINA between 2014-2016 Survey positive rate and be up to 23.4%, and corresponding vaccine is there is no for the disease.

Infection for pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus disease, there is presently no effective Treatment method, mainly puted prevention first with vaccine immunity.At present, the Attenuate vaccine of external mainly South Korea, the country is mainly breathed out TGE, PED and three attenuated live vaccines of rotavirus of beast development, the TGE-PED bigeminy attenuated live vaccines of great Bei agricultures company, The vaccines such as the TGE-PED bivalent inactivated vaccines of Wuhan predecessor company of section, but clinical application effect is limited；In addition, attenuated vaccine is with going out Live vaccine exists in production dissipates malicious risk, and the excessive use of attenuated vaccine, on the one hand returns strong risk, the opposing party there are virulence Face, may cause the restructuring of popular strain and vaccine strain to make a variation and make field viral prevalence situation increasingly complexization, and make above-mentioned The purification difficulty of several diseases further increases, so the research and development application of novel subunit vaccine is for ensureing animal and veterinary industry Sustainable and healthy development it is most important.

The content of the invention

Based on above-mentioned discovery and understanding, it is a primary object of the present invention to provide a kind of pig epidemic diarrhea, pig transmissible stomach and intestine Scorching, sick three subunit vaccines of pig fourth type coronavirus, antigenic component is Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine disease Poison and the respective antigen protein S1 of three kinds of viruses such as pig fourth type coronavirus, and three kinds of viral S1 protein contents need respectively >= 150 μg/ml（Head part）.

Preferably, the S1 albumen of Porcine epidemic diarrhea virus of the invention, 1 albumen of pig infectious gastroenteritis virus S and pig The mass ratio of the S1 albumen of fourth type coronavirus is 1：1: 1.

Wherein the amino acid sequence of Porcine epidemic diarrhea virus S1 albumen is SEQ ID NO:1, express pig epidemic diarrhea The Pichia Pastoris X33-PEDV-S1 of viral S1 albumen（Pichia pastoris X33-PEDV S1）, in It is preserved in China General Microbiological culture presevation administrative center on June 30th, 2017, deposit number is CGMCC NO:14372, protect Tibetan address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.

Wherein the amino acid sequence of 1 albumen of pig infectious gastroenteritis virus S is SEQ ID NO:2, express pig transmissible stomach The Pichia Pastoris X33-TGEV-S1 of enteritis virus S1 albumen（Pichia pastoris X33-TGEV S1）, China General Microbiological culture presevation administrative center is preserved on June 30th, 2017, deposit number is CGMCC NO:14371, Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.

Wherein the amino acid sequence of pig fourth type coronavirus S1 albumen is SEQ ID NO:3, express pig fourth type coronavirus The Pichia Pastoris X33-PDCoV-S1 of S1 albumen（Pichia pastoris X33-PDCoV S1）, in 2017 On June 30, in is preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC NO:14370, preservation Location is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.

The vaccine adjuvant is MF59 adjuvant.

The μ g/ml of Porcine epidemic diarrhea virus S1 protein contents in above-mentioned triple vaccine >=150（Head part）, pig transmissible The μ g/ml of S1 protein contents of marcy agent >=150（Head part）, the μ g/ml of the S1 protein contents of pig fourth type coronavirus >=150 （Head part）.

The sick three subunit vaccines peace of the pig epidemic diarrhea of the present invention, transmissible gastroenteritis of swine, pig fourth type coronavirus Quan Xinggao, immunogenicity is good, it is immune after antibody produce quick and hold time length compared with antibody titers, vaccine long shelf-life, exempts from Epidemic disease dosage is small, and the adjuvant of selection is easy to inject, can a pin prevent three kinds of main diarrhea related diseases viral diseases, improve depositing for piglet Motility rate.

Brief description of the drawings

Fig. 1 is Porcine epidemic diarrhea virus S1 of embodiment of the present invention protein gene, 1 protein gene of pig infectious gastroenteritis virus S With the amplification qualification figure of the PCR of pig fourth type coronavirus disease S1 protein gene,

Fig. 2 is that the blast of the Porcine epidemic diarrhea virus S1 albumen of the present invention compares figure；

Fig. 3 is the Phylogenetic tree schematic diagram of the Porcine epidemic diarrhea virus S1 albumen of the present invention；

The 1 albumen blast of pig infectious gastroenteritis virus S that Fig. 4 is the present invention compares figure；

Fig. 5 is the Phylogenetic tree schematic diagram of 1 albumen of pig infectious gastroenteritis virus S of the present invention；

Fig. 6 is that the blast of the pig fourth type coronavirus S1 albumen of the present invention compares figure；

Fig. 7 is the Phylogenetic tree schematic diagram of the pig fourth type coronavirus S1 albumen of the present invention；

Fig. 8 is recombinant bacterium fermentation inducement expression product SDS-PAGE electrophoresis detection figures of the embodiment of the present invention.

Embodiment

The embodiment of the present invention is specifically described below in conjunction with the accompanying drawings

Embodiment 1：Porcine epidemic diarrhea virus（PEDV）S1 genes, transmissible gastro-enteritis virus（TGEV）S1 genes and pig fourth Type coronavirus（PDCoV）The screening of S1 genes

From 2010, there is the grice diarrhoea state of an illness of large area in the multiple province ,city and areas in China's Mainland, and before site morbidity pig Commercially available diarrhea vaccine at that time has been injected, therefore has speculated Field epidemic virus for strain after variation, to 2013-2016 Between clinical sample detection after, isolated out of morbid pig body different Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine disease Poison and pig fourth type coronavirus, carry out genome sequencing and phylogenetic analysis（Fig. 3, Fig. 5 and Fig. 7）Afterwards, select KB2013-4 plants of PEDV, BD plants of TGEV and the SD plants of Reference Strains as current Field epidemic strain of PDCoV；Wherein PEDV KB2013-4 plants（Porcine epidemic diarrhea virus Porcine epidemic diarrhea virus）In 08 month 2016 23 Day is preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC NO:12663, preservation address is north The institute 3 of Jing Shi Chaoyang Districts North Star West Road 1, Institute of Microorganism, Academia Sinica；BD plants of TGEV（Transmissible gastroenteritis of swine disease Malicious Transmissible Gastroenteritis virus）Chinese common micro- life has been preserved on 06 07th, 2017 Thing culture presevation administrative center, deposit number are CGMCC NO:13856, preservation address is BeiChen West Road, Chaoyang District, BeiJing City 1 Institute 3, Institute of Microorganism, Academia Sinica；SD plants of PDCoV（Pig δ coronavirus Porcine Deltacoronavirus） China General Microbiological culture presevation administrative center was preserved on 06 07th, 2017, deposit number is CGMCC NO: 13853, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.

According to the antigenic characteristic and amino acid sequence of Porcine epidemic diarrhea virus S1 albumen, design synthesizes pair of primers, primer 1:5 '-GGATCCATGAAGTCTTTAACCTACTT-3 ', HI containing Bam site；Primer 2: 5’- AAGCTTAACACCTTCAAGTGGTTTAG-3 ', III containing Hind site；PEDV KB2013-4 strain virus genes are taken as mould Plate, PCR amplification purpose fragment（Fig. 1 is the amplification qualification figure of Porcine epidemic diarrhea virus S1 gene PCRs）, product recycling connection PEASY-T1 cloning vectors, conversion and screening positive clone pEASY-T1-PEDV-S1, through sequencing；Its nucleotides sequence is classified as SEQ ID NO:4, the sequence of the albumen of coding is SEQ ID NO:1.The popular pig between the 2010-2017 disclosed in NCBI Epidemic diarrhea virus S1 protein sequences are compared, and homology is between 96.5%-99.9%（Fig. 2）.

According to the antigenic characteristic and amino acid sequence of 1 albumen of pig infectious gastroenteritis virus S, design synthesizes pair of primers, primer 3:5 '-GGATCCATGAAAAAACTATTTGTG-3 ', HI containing Bam site；Primer 4: 5’- AAGCTTACCAACAGGACTCAACGAC-3 ', III containing Hind site；TGEV BD strain virus genes are taken to expand as template, PCR Increase purpose fragment（Fig. 1 is the amplification qualification figure of 1 gene PCR of pig infectious gastroenteritis virus S）, product recycling connection pEASY-T1 Cloning vector, conversion and screening positive clone pEASY-T1-TGEV-S1, through sequencing；Its nucleotides sequence is classified as SEQ ID NO:5, the sequence of the albumen of coding is SEQ ID NO:2.The popular pig transmissible between the 2010-2017 disclosed in NCBI 1 protein sequence of gastroenteritis virus S is compared, and homology is between 96.7%-99.9%（Fig. 4）.

According to the antigenic characteristic and amino acid sequence of pig fourth type coronavirus S1 albumen, design synthesizes pair of primers, primer 5:5 '-GGATCCATGCAGAGAGCTCTATTGAT-3 ', HI containing Bam site；Primer 6: 5’- AAGCTTACCATCACTACACACGCTGAT-3 ', III containing Hind site；PDCoV SD strain virus genes are taken as template, PCR amplification purpose fragment（Fig. 1 is the amplification qualification figure of pig fourth type coronavirus S1 gene PCRs）, product recycling connection pEASY- T1 cloning vectors, conversion and screening positive clone pEASY-T1-PDCoV-S1, through sequencing；Its nucleotides sequence is classified as SEQ ID NO:6, the sequence of the albumen of coding is SEQ ID NO:3.The popular pig fourth type between the 2013-2017 disclosed in NCBI Coronavirus S1 protein sequences are compared, and homology is between 96.8%-99.9%（Fig. 6）.

Embodiment 2：Porcine epidemic diarrhea virus S1 albumen, 1 albumen of pig infectious gastroenteritis virus S and pig fourth type is recombinated to be preced with The preparation of shape virus S1 albumen,

Comprise the following steps：A. construction of expression vector；B. structure expression bacterial strain；C. recombinate Porcine epidemic diarrhea virus S1 albumen, Induction and extraction purification of 1 albumen of pig infectious gastroenteritis virus S with pig fourth type coronavirus S1 albumen.It is specific as follows：By sun Property cloned plasmids pEASY-T1-PEDV-S1 or pEASY-T1-TGEV-S1 or pEASY-T1-PDCoV-S1 and expression vector PPICZ α carriers are returned respectively with Bam HI and Hind III double digestions products after 1.0% agarose gel electrophoresis with DNA gel Kits recycling is received, obtains 1.9 kb or 1.9 kb or 1.6 kb and 3.3 kb fragments respectively, in 16 DEG C of orientation connection structures PPICZ α-PEDV-S1 or pPICZ α-TGEV-S1 or pPICZ α-PDCoV-S1 expression vectors are built, after digestion identification is correct； After plasmid linearization, electricity is transformed into Pichia pastoris competent cell, build Pichiapastoris expression strain X33-PEDV-S1 or X33- TGEV-S1 or X33- PDCoV-S1, and extract expression strain X 33- PEDV-S1 or X33- TGEV-S1 or X33- PDCoV-S1 genomes, carry out PCR identifications correctly using primer primer 1/2 or primer3/4 or primer5/6 respectively, in On June 30th, 2017 is preserved in China General Microbiological culture presevation administrative center, and deposit number is respectively CGMCC No.14370；Carry out induced expression, monoclonal of the picking containing X33-PEDV-S1 or X33- TGEV-S1 or X33- PDCoV-S1 Colony inoculation is stayed overnight in BMGY fluid nutrient mediums, 30 DEG C of shaken cultivations, and thalline is collected by centrifugation, and after being suspended with suitable BMMY, is added Enter 0.55% methanol, when 30 DEG C of inductions 96 are small.4 DEG C, 9000rpm centrifugation 5min, retain supernatant, add 40% ammonium sulfate precipitation Afterwards, 12000rpm centrifuges 5min and collects albumen precipitation, with the molten albumen of PBS weights.Add protein electrophoresis sample-loading buffer, boiling water boiling 8 After minute, SDS-PAGE identifications are carried out with 12% separation gel（Fig. 8 is the SDS- PAGE electricity of recombinant bacterium fermentation inducement expression product Swim rating diagram, 1,2,3 be respectively PEDV-S1, TGEV-S1 and PDCoV-S1 protein expressioning products precipitation in figure, and M is molecular weight mark Quasi- protein）.

X33-PEDV-S1 plants of Pichiapastoris expression strain for producing Porcine epidemic diarrhea virus S1 albumen is preserved in China General Microbiological Culture preservation administrative center, deposit number are CGMCC No.14372.

During X33-TGEV-S1 plants of Pichiapastoris expression strain for producing 1 albumen of pig infectious gastroenteritis virus S is preserved in State's General Microbiological Culture preservation administrative center, deposit number are CGMCC No.14371.

X33-PDCoV-S1 plants of Pichiapastoris expression strain for producing pig fourth type coronavirus S1 albumen is preserved in China General Microbiological Culture preservation administrative center, deposit number are CGMCC No.14370.

1. X33-PEDV-S1 or X33- TGEV-S1 or X33- PDCoV-S1 strains are inoculated with by the preparation of seedling bacterium solution In the YPD fluid nutrient mediums containing bleomycin, when 30 DEG C of shaken cultivation 16-18 are small.Then streak inoculation is in mould added with rich Lay The YPD solid mediums of element, choose colonies typical 2-3 and are mixed in a small amount of YPD fluid nutrient mediums, put in 30 DEG C of shaking tables and vibrate Cultivate 18 it is small when, quantitative separating, through purely examine after, as first order seed.First order seed is taken to be inoculated in BMGY fluid nutrient mediums In, when 30 DEG C of shaken cultivation 16-18 are small, after microscopy, 2-8 DEG C of preservation is put, as secondary seed.

2. the preparation of seedling albumen adds the incomplete fluid nutrient mediums of BMGY by fermenter volume 60% (V/V), press at the same time Culture medium 0.1% (V/V) adds defoamer, is passed through high-temp steam sterilizing 30 minutes, treats that culture medium temperature is down to 32 DEG C, adds YNB And biotin, it is inoculated with production Pichia pastoris X33-PEDV-S1 or X33- TGEV-S1 or X33- PDCoV-S1 secondary seeds Liquid, fermentation tank parameter setting are respectively mixing speed 800r/min, and 30 DEG C of temperature, maintains D0 values (dissolved oxygen amount) 20%.Culture 24 Bacterium solution after hour adds methanol, and the speed of adding of methanol is 2ml/h/L induced expression cultures, according to above ferment control parameter And technique induced expression 120 it is small when；Fermented and cultured bacterium solution through tube centrifuge 10000r/min centrifuge 30 minutes, harvest it is upper Clearly, after adding ammonium sulfate precipitated protein, 30 minutes harvest precipitations is centrifuged by 12000r/min, add suitable physiological saline solution Albumen precipitation.

3. protein liquid is placed in inactivation bottle by inactivation, metered 10% formalin, with adding with shaking, is sufficiently mixed it, The ultimate density of formalin is 0.1%.Poured into after adding formalin in another inactivation bottle, the disease nearby adhered to avoid bottleneck Poison fails to contact inactivator.Taken out after when 37 DEG C of inactivations 16 are small, put 2-8 DEG C of preservation.

4. the inspection of semifinished product

4.1 steriling tests are by existing《Chinese veterinary pharmacopoeia》Annex carries out steriling test.

4.2 determining the protein quantity are by Bradford methods detection protein content.

4.3 inactivations are examined takes a small amount of inoculation YPD solid mediums by the protein liquid after inactivation, puts 30 DEG C and continues culture 72 Hour.Observation sentences inactivation and examines qualification without colony growth.

Embodiment 3：Sick three subunit vaccines of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus Prepare

Vaccine preparation is carried out by semi-finished product PEDV-S1, TGEV-S1 and PDCoV-S1 proteantigen after the assay was approved（Below Each liquid component is counted by volume in preparation）.

1 adjuvant prepares and the Arlacel-85 of 0.5% volume is dispersed in the squalene phase of 5% volume, with the tween of 0.5% volume- 80 are mixed into the 10mM sodium citrate buffer solutions of 94% volume, and then mixed at high speed obtains thick lotion.Then by this thick lotion Iterate through high pressure homogenizer（1200bar, 6-8 circulations）, produce the lotion with homogeneous droplet size；Lotion is filtered 0.22 μm of filter membrane, to remove larger oil droplet, obtain lotion average droplet size kept at 4 DEG C at least 3 years it is constant.

Prepared by 2 antigens is preced with Porcine epidemic diarrhea virus S1 albumen, 1 albumen of pig infectious gastroenteritis virus S and pig fourth type Shape virus S1 albumen uses normal saline dilution into 150 μ g/0.1ml respectively.

3 emulsifications take 1 part of oil phase to be put in 50L-200L high-speed shearing machines, start the stirring of four leaf paddle slow rotations, while Xu Xu adds 1 part of water phase, with 300-500r/min, emulsifies 5 minutes.After emulsification, 10mL vaccines are taken to centrifuge 15 points with 3000r/min Clock, the water that tube bottom separates out accordingly are no more than 0.5ml.

4 vaccine product inspections

4.1 character

Appearance vaccine should be milky emulsion, and it is qualified that free from admixture and outer packing are answered.

Formulation is oil-in-water type.A cleaning suction pipe is taken, a small amount of vaccine is drawn and instills in cold water, it is uniform after gently rocking It is dispersed in water.

Stability is drawn vaccine 10ml and is added in centrifuge tube, is centrifuged 15 minutes with 3000r/min, and the water that tube bottom separates out is corresponding No more than 0.5ml.

Viscosity is by existing《Chinese veterinary pharmacopoeia》Annex carries out, and should meet regulation.

4.2 loading quantity inspections are by existing《Chinese veterinary pharmacopoeia》Annex carries out, and should meet regulation.

4.3 steriling tests are by existing《Chinese veterinary pharmacopoeia》Annex carries out, and should meet regulation.

4.4 safety verifications are with 3-5 ages in days piglet 10（Corresponding sow should be later stage of pregnancy detection pig epidemic diarrhea, pass Metachromia gastroenteritis and pig fourth type coronavirus disease neutralize antibody titers≤1:4）, neck intramuscular injection vaccine 1.0ml after every ear, Control 5 is set at the same time, is raised under the same conditions, continuous observation 14 days, the feeding of record test pig, drinking-water and clinical setting. Any locally and systemically adverse reaction as caused by vaccine should be occurred without.

4.5 efficacy test

4.5.1 examine immune piglet serum neutralizing antibody to be inoculated with the piglet of 1 times of immunizing dose, neutralizing antibody is used within 2 weeks after immune Test method(s) examines the serum for Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig fourth type coronavirus disease to neutralize Antibody, 10 piglets at least should turn sun by 8 piglets, for three kinds of viral neutralize antibody titers >=1:32, it can reach good Immune effect；

4.5.2 the piglet immunological agent 15 of Immunization experiment 1 multiple dose of inoculation is done to piglet, is divided into 3 groups within 3 weeks after immune （5/group）, together with the piglet of the same age 15 of non-vaccine inoculation（It is also classified into 3 groups, 5/group）, with 10^5.0 TCID₅₀/ ml pigs are popular Property diarrhea virus, transmissible gastro-enteritis virus and the strong poison of pig fourth type coronavirus disease oral challenge respectively, son is immunized after attacking poison Pig should have 4/5 protection respectively, and control pig 5/5 falls ill, or immune piglet 5/5 is protected, and control piglet 4/5 falls ill, and be judged to immune conjunction Lattice.

The testing result that 4.6 residual formaldehydes detect 3 batches of three subunit vaccines is respectively 0.092%, 0.085%, 0.086%, it is below the regulation of veterinary biologics general rule（0.2%）.

The pig epidemic diarrhea manufactured experimently in embodiment 4, embodiment 3, transmissible gastroenteritis of swine, pig fourth type coronavirus disease three Connection subunit vaccine is with the serum neutralizing antibody of commercially available inactivated vaccine and attenuated live vaccines compared with immune protective effect

Sick three subunit vaccines of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus in 1 materials Example 3 Preproduction；Transmissible gastroenteritis of swine, pig epidemic diarrhea, porcine rotavirus（G5 types）Trigeminal live vaccine（The weak magnificent strain+weak poison of poison CV777 plants+NX plants）, the production of Jilin decent job biological products limited company（Lot number 2016006）；Transmissible gastroenteritis of swine, pig Epidemic diarrhea bivalent inactivated vaccine, the production of Jilin decent job biological products limited company（Lot number 2016003）.

2 methods

2.1 animal experiments design

With 3-5 ages in days piglet 65（Corresponding sow should be later stage of pregnancy detection pig epidemic diarrhea, transmissible gastroenteritis and pig fourth Type coronavirus disease neutralize antibody titers≤1:4）, neck intramuscular injection vaccine 1.0ml after every ear, while control 15 is set, Raise, continuous observation 14 days, the feeding of record test pig, drinking-water and clinical setting, adopted respectively after 14d under the same conditions Blood, and in blood sampling same day strong virus attack.15 of negative control group are not immunized, and are divided into 3 groups, are respectively used to three kinds of viruses and attack The negative control of poison.Three vaccine groups are respectively equipped with one group（5）Blank control group, it is only immune without attacking poison.Specific packet Situation is shown in Table 1.

1 animal experiment of table is grouped situation

2.2 potency test

2.2.1 antibody determination

Efficacy test is carried out with reference to the 4.5.1 items method in embodiment 3, takes a blood sample respectively to immune piglet 14 days latter, separates blood Clearly, measure is directed to the neutralizing antibody of Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig fourth type coronavirus respectively Potency.

2.2.2 Immunization is protected

Carry out efficacy test with reference to the 4.5.2 items method in example 3 applied, to the piglets of 14 days after immune respectively with it is corresponding by force it is malicious into Row attack.Continuous observation 10 days, records the incidence of each group piglet.

3 results

3.1 antibody determination results

Manufactured experimently with sick three subunit vaccines of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus in embodiment 3 Product, transmissible gastroenteritis of swine, pig epidemic diarrhea, porcine rotavirus（G5 types）Trigeminal live vaccine（The weak magnificent strain+weak poison of poison CV777 plants+NX plants）（Lot number 2016006）And transmissible gastroenteritis of swine, pig epidemic diarrhea bivalent inactivated vaccine（Lot number 2016003）The susceptible piglet of 3~5 ages in days health is immunized respectively, the neutralizing antibody after 14d the results are shown in Table 2.The result shows that Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and the pig fourth type of being directed to is immunized in rear piglet serum in three subunit vaccines The neutralize antibody titers of coronavirus are not less than 1:32, and commercially available bivalent inactivated vaccine and commercially available three attenuated live vaccines are immunized The neutralize antibody titers of piglet are not higher than 1:16.Therefore, three subunit vaccines produced after being immunized for 3 kinds it is viral in It is better than commercially available three attenuated live vaccines and commercially available bivalent inactivated vaccine with antibody level.

After 2 three subunit vaccine preproduction of table is immunized with commercially available three attenuated live vaccines, commercially available bivalent inactivated vaccine Neutralize antibody titers compare

3.2 Immunizations protect result

Manufactured experimently with sick three subunit vaccines of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus in embodiment 3 Product, transmissible gastroenteritis of swine, pig epidemic diarrhea, porcine rotavirus（G5 types）Trigeminal live vaccine（The weak magnificent strain+weak poison of poison CV777 plants+NX plants）（Lot number 2016006）And transmissible gastroenteritis of swine, pig epidemic diarrhea bivalent inactivated vaccine（Lot number 2016003）The susceptible piglet of 3~5 ages in days health is immunized respectively, is attacked respectively with corresponding strong poison after 14d, continuously Observation 10 days, that attacks poison protection the results are shown in Table 3.

3 three subunit vaccine preproduction of table and commercially available three attenuated live vaccines, commercially available bivalent inactivated vaccine be immunized after attack poison Protecting effect compares

The result shows that the sick three subunit's epidemic diseases of the pig epidemic diarrhea of the present invention, transmissible gastroenteritis of swine, pig fourth type coronavirus Seedling is safe, and immunogenicity is good, it is immune after antibody produce quick and hold time length compared with antibody titers, vaccine storage life Long, immunizing dose is small, and the adjuvant of selection is easy to inject, can a pin prevent three kinds of main diarrhea related diseases viral diseases, improve son The survival rate of pig.

Sequence table

<110>Shaanxi Nowe Li Hua bio tech ltd

<120>Sick three subunit vaccines of a kind of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus

<130> 2017-11-08

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Met Lys Ser Leu Thr Tyr Phe Trp Leu Phe Leu Pro Val Leu Ser Thr

1 5 10 15

Leu Ser Leu Pro Gln Asp Val Thr Arg Cys Ser Ala Asn Thr Asn Phe

20 25 30

Arg Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val

35 40 45

Leu Gly Gly Tyr Leu Pro Ile Gly Glu Asn Gln Gly Val Asn Ser Thr

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Trp Tyr Cys Ala Gly Gln His Pro Thr Ala Ser Gly Val His Gly Ile

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Phe Val Ser His Ile Arg Gly Gly His Gly Phe Glu Ile Gly Ile Ser

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Gln Glu Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala

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Thr Asn Gly Asn Thr Asn Ala Thr Ala Arg Leu Arg Ile Cys Gln Phe

115 120 125

Pro Ser Ile Lys Thr Leu Gly Pro Thr Ala Asn Asn Asp Val Thr Thr

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Gly Arg Asn Cys Leu Phe Asn Lys Ala Ile Pro Ala His Met Ser Glu

145 150 155 160

His Ser Val Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe

165 170 175

Ser Asp Lys Ile Tyr Tyr Phe Tyr Phe Lys Asn Asp Trp Ser Arg Val

180 185 190

Ala Thr Lys Cys Tyr Asn Ser Gly Gly Cys Ala Met Gln Tyr Val Tyr

195 200 205

Glu Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly

210 215 220

Ile Ser Tyr Gln Pro Cys Thr Ala Asn Cys Ile Gly Tyr Ser Ala Asn

225 230 235 240

Val Phe Ala Thr Glu Pro Asn Gly His Ile Pro Glu Gly Phe Ser Phe

245 250 255

Asn Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu Val His Gly Lys

260 265 270

Val Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro

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Lys Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn Gln Thr Ile Asp

290 295 300

Gly Val Cys Asn Gly Ala Ala Val Gln Arg Ala Pro Glu Ala Leu Arg

305 310 315 320

Phe Asn Ile Asn Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val

325 330 335

Leu His Thr Ala Leu Gly Thr Asn Phe Ser Phe Val Cys Ser Asn Ser

340 345 350

Ser Asp Pro His Leu Ala Thr Phe Ala Ile Pro Leu Gly Ala Ile Gln

355 360 365

Val Pro Tyr Tyr Cys Phe Leu Lys Val Asp Thr Tyr Asn Ser Thr Val

370 375 380

Tyr Lys Phe Leu Ser Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile

385 390 395 400

Thr Lys Tyr Gly Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu

405 410 415

Gly Leu Leu Asp Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp

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Asp Asp Val Ser Gly Phe Trp Thr Ile Ala Ser Thr Asn Phe Val Asp

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Ala Leu Ile Glu Val Gln Gly Thr Ala Ile Gln Arg Ile Leu Tyr Cys

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Asp Asp Pro Val Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu

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Asp Asp Gly Phe Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu

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Gln Pro Ile Ser Phe Val Ala Leu Pro Ser Phe Asn Asp His Ser Phe

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Leu Ile Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val

530 535 540

Asp Thr Arg Gln Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser

545 550 555 560

Tyr Gly Tyr Val Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu

565 570 575

Gln Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr

580 585 590

Ser Leu Leu Ala Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu

595 600 605

Phe Gly Ser Gly Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys

610 615 620

Gly Glu Leu Ile Thr Gly Thr Ser Lys Pro Leu Glu Gly Val

625 630 635

<210> 2

<211> 632

<212> PRT

<213>Artificial synthesized (transmissible gastro-enteritis virus)

<400> 2

Met Lys Lys Leu Phe Val Val Leu Val Val Met Pro Leu Ile Tyr Gly

1 5 10 15

Asp Asn Phe Pro Cys Ser Lys Leu Thr Asn Arg Thr Ile Gly Asn Gln

20 25 30

Trp Asn Leu Ile Glu Thr Phe Leu Leu Asn Tyr Ser Ser Arg Leu Pro

35 40 45

Pro Asn Ser Asp Val Val Leu Gly Asp Tyr Phe Pro Thr Val Gln Pro

50 55 60

Trp Phe Asn Cys Ile Arg Asn Asp Ser Asn Asp Leu Tyr Val Thr Leu

65 70 75 80

Glu Asn Leu Lys Ala Leu Tyr Trp Asp Tyr Ala Thr Glu Asn Ile Thr

85 90 95

Trp Asn His Arg Gln Arg Leu Asn Val Val Val Asn Gly Tyr Pro Tyr

100 105 110

Ser Ile Thr Val Thr Thr Thr Arg Asn Phe Asn Ser Ala Glu Gly Ala

115 120 125

Ile Ile Cys Ile Cys Lys Gly Ser Pro Pro Thr Thr Thr Thr Glu Ser

130 135 140

Ser Leu Thr Cys Asn Trp Gly Ser Glu Cys Arg Leu Asn His Lys Phe

145 150 155 160

Pro Ile Cys Pro Ser Asn Ser Glu Ala Asn Cys Gly Asn Met Leu Tyr

165 170 175

Gly Leu Gln Trp Phe Ala Asp Glu Val Val Ala Tyr Leu His Gly Ala

180 185 190

Ser Tyr Arg Ile Ser Phe Glu Asn Gln Trp Ser Gly Thr Val Thr Phe

195 200 205

Gly Asp Met Arg Ala Thr Thr Leu Glu Val Ala Gly Thr Leu Val Asp

210 215 220

Leu Trp Trp Phe Asn Pro Val Tyr Asp Val Ser Tyr Tyr Arg Val Asn

225 230 235 240

Asn Lys Asn Gly Thr Thr Val Val Ser Asn Cys Thr Asp Gln Cys Ala

245 250 255

Ser Tyr Val Ala Asn Val Phe Thr Thr Gln Pro Gly Gly Phe Ile Pro

260 265 270

Ser Asp Phe Ser Phe Asn Asn Trp Phe Leu Leu Thr Asn Ser Ser Thr

275 280 285

Leu Val Ser Gly Lys Leu Val Thr Lys Gln Pro Leu Leu Val Asn Cys

290 295 300

Leu Trp Pro Val Pro Ser Phe Glu Glu Ala Ala Ser Thr Phe Cys Phe

305 310 315 320

Glu Gly Ala Gly Phe Asp Gln Cys Asn Gly Ala Val Leu Asn Asn Thr

325 330 335

Val Asp Val Ile Arg Phe Asn Leu Asn Phe Thr Thr Asn Val Gln Ser

340 345 350

Gly Lys Gly Ala Thr Val Phe Ser Leu Asn Thr Thr Gly Gly Val Thr

355 360 365

Leu Glu Ile Ser Cys Tyr Thr Val Ser Asp Ser Ser Phe Phe Ser Tyr

370 375 380

Gly Glu Ile Pro Phe Gly Val Thr Asp Gly Pro Arg Tyr Cys Tyr Val

385 390 395 400

His Tyr Asn Gly Thr Ala Leu Lys Tyr Leu Gly Thr Leu Pro Pro Ser

405 410 415

Val Lys Glu Ile Ala Ile Ser Lys Trp Gly His Phe Tyr Ile Asn Gly

420 425 430

Tyr Asn Phe Phe Ser Thr Phe Pro Ile Asp Cys Ile Ser Phe Asn Leu

435 440 445

Thr Thr Gly Asp Ser Asp Val Phe Trp Thr Ile Ala Tyr Thr Ser Tyr

450 455 460

Thr Glu Ala Leu Val Gln Val Glu Asn Thr Ala Ile Thr Lys Val Thr

465 470 475 480

Tyr Cys Asn Ser His Val Asn Asn Ile Lys Cys Ser Gln Ile Thr Ala

485 490 495

Asn Leu Asn Asn Gly Phe Tyr Pro Val Ser Ser Ser Glu Val Gly Leu

500 505 510

Val Asn Lys Ser Val Val Leu Leu Pro Ser Phe Tyr Thr His Thr Ile

515 520 525

Val Asn Ile Thr Ile Gly Leu Gly Met Lys Arg Ser Gly Tyr Gly Gln

530 535 540

Pro Ile Ala Ser Thr Leu Ser Asn Ile Thr Leu Pro Met Gln Asp His

545 550 555 560

Asn Thr Asp Val Tyr Cys Ile Arg Ser Asp Gln Phe Ser Val Tyr Val

565 570 575

His Ser Thr Cys Lys Ser Ala Leu Trp Asp Asn Ile Phe Lys Arg Asn

580 585 590

Cys Thr Asp Val Leu Asp Ala Thr Ala Val Ile Lys Thr Gly Thr Cys

595 600 605

Pro Phe Ser Phe Asp Lys Leu Asn Asn Tyr Leu Thr Phe Asn Lys Phe

610 615 620

Cys Leu Ser Leu Ser Pro Val Gly

625 630

<210> 3

<211> 546

<212> PRT

<213>Artificial synthesized (pig fourth type coronavirus)

<400> 3

Met Gln Arg Ala Leu Leu Ile Met Thr Leu Leu Cys Leu Val Arg Ala

1 5 10 15

Lys Phe Ala Asp Asp Leu Leu Asp Leu Leu Thr Phe Pro Gly Ala His

20 25 30

Arg Phe Leu His Lys Leu Thr Ser Asn Ser Ser Ser Phe Tyr Ser Arg

35 40 45

Ala Asn Asn Phe Asp Val Gly Val Leu Pro Gly Tyr Pro Thr Glu Asn

50 55 60

Val Asn Leu Phe Ser Pro Leu Thr Asn Ser Thr Leu Pro Ile Asn Gly

65 70 75 80

Leu His Arg Ser Tyr Gln Pro Leu Met Leu Asn Cys Leu Thr Lys Ile

85 90 95

Thr Asn Thr Leu Ser Met Tyr Leu Leu Pro Ser Glu Ile Gln Thr Tyr

100 105 110

Ser Cys Gly Gly Ala Met Val Lys Tyr Gln Thr His Asp Ala Val Arg

115 120 125

Ile Ile Leu Asp Leu Thr Val Thr Asp His Ile Ser Val Glu Val Val

130 135 140

Gly Gln Arg Gly Glu Asn Tyr Val Phe Val Cys Ser Glu Gln Phe Asn

145 150 155 160

Tyr Thr Thr Ala Leu His Asn Ser Thr Phe Phe Ser Leu Asn Ser Glu

165 170 175

Leu Tyr Cys Phe Thr Asn Asn Thr Tyr Leu Gly Ile Leu Pro Pro Asp

180 185 190

Leu Thr Asp Phe Thr Val Tyr Arg Thr Gly Gln Phe Tyr Ala Asn Gly

195 200 205

Tyr Leu Leu Gly Thr Leu Pro Ile Thr Val Asn Tyr Val Arg Leu Tyr

210 215 220

Arg Gly His Leu Ser Ala Asn Ser Ala His Phe Ala Leu Ala Asn Leu

225 230 235 240

Thr Asp Thr Leu Ile Thr Leu Thr Asn Thr Thr Ile Ser Gln Ile Thr

245 250 255

Tyr Cys Asp Lys Ser Val Val Asp Ser Ile Ala Cys Gln Arg Ser Ser

260 265 270

His Glu Val Glu Asp Gly Phe Tyr Ser Asp Pro Lys Ser Ala Val Arg

275 280 285

Ala Arg Gln Arg Thr Ile Val Thr Leu Pro Lys Leu Pro Glu Leu Glu

290 295 300

Val Val Gln Leu Asn Ile Ser Ala His Met Asp Phe Gly Glu Ala Arg

305 310 315 320

Leu Asp Ser Val Thr Ile Asn Gly Asn Thr Ser Tyr Cys Ala Thr Lys

325 330 335

Pro Tyr Phe Arg Leu Glu Thr Asn Phe Met Cys Thr Gly Cys Thr Met

340 345 350

Asn Leu Arg Thr Asp Thr Cys Ser Phe Asp Leu Ser Ala Val Asn Asn

355 360 365

Gly Met Ser Phe Ser Gln Phe Cys Leu Ser Thr Glu Ser Gly Ala Cys

370 375 380

Glu Met Lys Ile Ile Val Thr Tyr Val Trp Asn Tyr Leu Leu Arg Gln

385 390 395 400

Arg Leu Tyr Val Thr Ala Val Glu Gly Gln Thr His Thr Gly Thr Thr

405 410 415

Ser Val His Ala Thr Asp Thr Ser Ser Val Ile Thr Asp Val Cys Thr

420 425 430

Asp Tyr Thr Ile Tyr Gly Val Ser Gly Thr Gly Ile Ile Lys Pro Ser

435 440 445

Asp Leu Leu Leu His Asn Gly Ile Ala Phe Thr Ser Pro Thr Gly Glu

450 455 460

Leu Tyr Ala Phe Lys Asn Ile Thr Thr Gly Lys Thr Leu Gln Val Leu

465 470 475 480

Pro Cys Glu Thr Pro Ser Gln Leu Ile Val Ile Asn Asn Thr Val Val

485 490 495

Gly Ala Ile Thr Ser Ser Asn Ser Thr Glu Asn Asn Arg Phe Thr Thr

500 505 510

Thr Ile Val Thr Pro Thr Phe Phe Tyr Ser Thr Asn Ala Thr Thr Phe

515 520 525

Asn Cys Thr Lys Pro Val Leu Ser Tyr Gly Pro Ile Ser Val Cys Ser

530 535 540

Asp Gly

545

<210> 4

<211> 1914

<212> DNA

<213>Artificial synthesized (Porcine epidemic diarrhea virus)

<400> 4

atgaagtctt taacctactt ctggttgttc ttaccagtac tttcaacact tagcctacca 60

caagatgtca ccaggtgctc agctaacact aattttaggc ggttcttttc aaaatttaat 120

gttcaggcgc ctgcagttgt tgtactgggc ggttatctac ctattggtga aaaccagggt 180

gtcaattcaa cttggtactg tgctggccaa catccaactg ctagtggcgt tcatggtatc 240

tttgttagcc atattagagg tggtcatggc tttgagattg gcatttcgca agagcctttt 300

gaccctagtg gttaccagct ttatttacat aaggctacta acggtaacac taatgctact 360

gcgcgactgc gcatttgcca gtttcctagc attaaaacat tgggccccac tgctaataat 420

gatgttacaa caggtcgtaa ttgcctattt aacaaagcca tcccagctca tatgagtgaa 480

catagtgttg tcggcataac atgggataat gatcgtgtca ctgtcttttc tgacaagatc 540

tattattttt attttaaaaa tgattggtcc cgtgttgcga caaagtgtta caacagtgga 600

ggttgtgcta tgcaatatgt ttacgaaccc acctattaca tgcttaatgt tactagtgct 660

ggtgaggatg gtatttctta tcaaccctgt acagctaatt gcattggtta ttctgccaat 720

gtatttgcta ctgagcccaa tggccacata ccagaaggtt ttagttttaa taattggttt 780

cttttgtcca atgattccac tttggtgcat ggtaaggtgg tttccaacca accattgttg 840

gtcaattgtc ttttggccat tcctaagatt tatggactag gccaattttt ctcctttaat 900

caaacgatcg atggtgtttg taatggagct gctgtgcagc gtgcaccaga ggctctgagg 960

tttaatatta atgacacctc tgtcattctt gctgaaggct caattgtact tcatactgct 1020

ttaggaacaa atttttcttt tgtttgcagt aattcctcag atcctcactt agccaccttc 1080

gccatacctc tgggtgctat ccaagtaccc tattattgtt ttcttaaagt ggatacttac 1140

aactccactg tttataaatt cttgtctgtt ttacctccta ccgtcaggga aattgtcatc 1200

accaagtatg gtgatgttta tgtcaatggg tttggctact tgcatctcgg tttgttggat 1260

gctgtcacaa ttaatttcac tggtcatggc actgacgatg acgtttctgg tttttggacc 1320

atagcatcga ctaattttgt tgatgcactt atcgaagttc aaggaactgc cattcagcgt 1380

attctttatt gtgatgatcc tgttagccaa ctcaagtgtt ctcaggttgc ttttgacctt 1440

gacgatggtt tctaccctat ttcttctaga aaccttctga gtcatgaaca gccaatttct 1500

tttgttgctt tgccatcatt taatgatcat tcttttgtta acattactgt ctctgcgtcc 1560

tttggtggtc atagtggtgc caaccttatt gcatctgaca ctactatcaa tgggtttagt 1620

tctttctgtg ttgacactag acaatttacc atttcactgt tttataacgt tacaaacagt 1680

tatggttatg tgtctaaatc acaggacagt aattgccctt tcaccttgca atctgttaat 1740

gattacctgt cttttagtaa attttgtgtt tccaccagcc ttttggctag tgcctgtacc 1800

atagatcttt ttggttaccc tgagtttggt agtggtgtta agtttacgtc cctttacttt 1860

caattcacaa agggtgagtt gattactggc acgtctaaac cacttgaagg tgtc 1914

<210> 5

<211> 1896

<212> DNA

<213>Artificial synthesized (transmissible gastro-enteritis virus)

<400> 5

atgaaaaaac tatttgtggt tttggtcgta atgccattga tttatggaga caattttcct 60

tgttctaaat tgactaatag aactataggc aaccagtgga atctcattga aaccttcctt 120

ctaaactata gtagtaggtt accacctaat tcagatgtgg tgttaggtga ttattttcct 180

actgtacaac cttggtttaa ttgcattcgc aatgatagta atgaccttta tgttacactg 240

gaaaatctta aagcattgta ttgggattat gctacagaaa atatcacttg gaatcacaga 300

caacggttaa acgtagtcgt taatggatac ccatactcca tcacagttac aacaacccgc 360

aattttaatt ctgctgaagg tgctattata tgcatttgta agggctcacc acctactacc 420

accacagaat ctagtttgac ttgcaattgg ggtagtgagt gcaggttaaa ccataagttc 480

cctatatgtc cttctaattc agaggcaaat tgtggtaata tgctgtatgg cctacaatgg 540

tttgcagatg aggttgttgc ttatttacat ggtgctagtt accgtattag ttttgaaaat 600

caatggtctg gcactgtcac atttggtgat atgcgtgcga caacattaga agtcgctggc 660

acgcttgtag acctttggtg gtttaatcct gtttatgatg tcagttatta tagggttaat 720

aataaaaatg gtactaccgt agtttccaat tgcactgatc aatgtgctag ttatgtggct 780

aatgttttta ctacacagcc aggaggtttt ataccatcag attttagttt taataattgg 840

ttccttctaa ctaatagctc cacgttggtt agtggtaaat tagttaccaa acagccgtta 900

ttagttaatt gcttatggcc agtccctagc tttgaagaag cagcttctac attttgtttt 960

gagggtgctg gctttgatca atgtaatggt gctgttttaa ataatactgt agacgtcatt 1020

aggttcaacc ttaattttac tacaaatgta caatcaggta agggtgccac agtgttttca 1080

ttgaacacaa cgggtggtgt cactcttgaa atttcatgtt atacagtgag tgactcgagc 1140

tttttcagtt acggtgaaat tccgttcggc gtaactgatg gaccacggta ctgttacgta 1200

cactataatg gcacagctct taagtattta ggaacattac cacctagtgt caaggagatt 1260

gctattagta agtggggcca tttttatatt aatggttaca atttctttag cacatttcct 1320

attgattgta tatcttttaa tttgaccact ggtgatagtg acgttttctg gacaatagct 1380

tacacatcgt acactgaagc attagtacaa gttgaaaaca cagctattac aaaggtgacg 1440

tattgtaata gtcacgttaa taacattaaa tgctctcaaa ttactgctaa tttgaataat 1500

ggattttatc ctgtttcttc aagtgaagtt ggtcttgtca ataagagtgt tgtgttacta 1560

cctagctttt acacacatac cattgttaac ataactattg gtcttggtat gaagcgtagt 1620

ggttatggtc aacccatagc ctcaacatta agtaacatca cactaccaat gcaggatcac 1680

aacaccgatg tgtactgtat tcgttctgac caattttcag tttatgttca ttctacttgc 1740

aaaagtgctt tatgggacaa tatttttaag cgaaactgca cggacgtttt agatgccaca 1800

gctgttataa aaactggtac ttgtcctttc tcatttgata aattgaacaa ttacttaact 1860

tttaacaagt tctgtttgtc gttgagtcct gttggt 1896

<210> 6

<211> 1641

<212> DNA

<213>Artificial synthesized (pig fourth type coronavirus)

<400> 6

atgcagagag ctctattgat tatgacctta ctttgtctcg ttcgagcaaa gtttgctgat 60

gatctactcg atttgctcac cttcccgggt gcacatcgct tcttacataa actcacgagt 120

aattccagca gtttctactc gcgggctaat aactttgatg ttggcgttct tcctggctac 180

cccactgaga acgttaacct cttctcacca cttactaact ctactttgcc aattaatggc 240

cttcatcgga gttaccaacc tcttatgctg aattgtctta ctaaaataac taaccamact 300

ctcagcatgt atctcctacc tagtgagata caaacttata gctgcggcgg tgccatggtt 360

aaataccaga cacatgatgc agttcgtatc attttagacc tcactgtcac tgaccacatc 420

tctgttgaag tcgttggcca acgtggtgaa aattatgtgt ttgtttgtag tgagcagttt 480

aactatacca ctgcattaca caactctacc ttcttctcac ttaattctga gctttattgc 540

tttactaata acacctactt aggtattctt ccacctgatt taactgactt tacggtctat 600

cgtactgggc agttttatgc taatggttac cttttaggta ctttacctat tacggttaac 660

tatgtaaggt tgtatcgggg tcatttgtcg gccaatagtg cccactttgc ccttgcaaac 720

ctaaccgata cactcataac acttaccaat actactatat cgcaaatcac ttattgtgat 780

aagtcagtag ttgattcaat agcatgccag cgctcttctc acgaagtgga ggatgggttt 840

tactccgacc ctaaatctgc cgttagagct aggcaacgta ctattgttac actacctaag 900

ctccctgagc ttgaagtagt gcagttaaat atttctgcac acatggattt tggcgaagcc 960

agacttgaca gcgttaccat caatggtaac acatcctatt gtgccactaa gccttacttc 1020

aggcttgaaa ctaactttat gtgtacaggt tgcactatga atctgcgcac tgatacctgt 1080

agttttgacc tgtcagcagt aaacaatggc atgtcattct ctcaattctg tctaagcact 1140

gaatctggtg cttgtgagat gaaaattatt gttacctacg tatggaatta cttgctaagg 1200

cagcgtttgt atgttacagc tgtagagggc cagactcaca ctggaaccac ttcagtacat 1260

gcaacagaca cttctagtgt aatcactgat gtctgcactg attacactat ctatggagtc 1320

tctggtactg gcattattaa gccatcagat ctcttattac ataatggcat agcattcacc 1380

tctccaacag gtgagctcta tgcatttaaa aatataacca ctggcaaaac ccttcaggtc 1440

ttaccgtgtg aaaccccttc tcaactgatt gtgataaaca acaccgttgt cggtgctatc 1500

acatccagta actcaactga aaataatagg tttactacta ctattgtcac acctactttc 1560

ttttattcca caaatgccac caccttcaac tgcaccaagc ctgttttgtc ctatggaccc 1620

atcagcgtgt gtagtgatgg t 1641

Claims (10)

1. sick three subunit vaccines of a kind of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus, its feature exist In the vaccine includes antigen and vaccine adjuvant, and wherein antigen includes Porcine epidemic diarrhea virus S1 albumen, pig is infected Property 1 albumen of gastroenteritis virus S and pig fourth type coronavirus S1 albumen.

2. triple vaccine according to claim 1, it is characterised in that the ammonia of the Porcine epidemic diarrhea virus S1 albumen Base acid sequence is SEQ ID NO:1.

3. triple vaccine according to claim 1 or 2, it is characterised in that 1 egg of pig infectious gastroenteritis virus S White amino acid sequence is SEQ ID NO:2.

4. according to the triple vaccine described in claim 1 or 2 or 3, it is characterised in that the pig fourth type coronavirus S1 albumen Amino acid sequence be SEQ ID NO:3.

5. triple vaccine according to claim 1, it is characterised in that the Porcine epidemic diarrhea virus S1 albumen be by Deposit number is CGMCC NO:Prepared by 14372 Pichia Pastoris expression.

6. triple vaccine according to claim 1, it is characterised in that 1 albumen of pig infectious gastroenteritis virus S is It is CGMCC NO by deposit number:Prepared by 14371 Pichia Pastoris expression.

7. triple vaccine according to claim 1, it is characterised in that the pig fourth type coronavirus S1 albumen is by protecting It is CGMCC NO to hide numbering:Prepared by 14370 Pichia Pastoris expression.

8. triple vaccine according to claim 1, it is characterised in that the vaccine adjuvant is aluminium hydroxide gel, mineral Oil, Gel 01（French SEPPIC）, propolis, ISA206, ISA760VG, MF59.

9. triple vaccine according to claim 1, it is characterised in that the vaccine adjuvant is MF59.

10. triple vaccine according to claim 1, it is characterised in that Porcine epidemic diarrhea virus S1 in the vaccine The μ g/ml of protein content >=150（Head part）, the μ g/ml of the S1 protein contents of transmissible gastro-enteritis virus >=150（Head part）, pig The μ g/ml of S1 protein contents of fourth type coronavirus >=150（Head part）, MF59（50%, V/V）.