CN107899008B - 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, it include antigen and vaccine adjuvant, wherein antigen is the Porcine epidemic diarrhea virus S1 albumen expressed by the X33-PEDV-S1 strain that deposit number is CGMCC No.14372, the pig fourth type coronavirus S1 albumen for the X33-PDCoV-S1 strain expression that 1 albumen of pig infectious gastroenteritis virus S and deposit number for the X33-TGEV-S1 strain expression that deposit number is CGMCC No.14371 are CGMCC No.14370.Sick three subunit vaccines of pig epidemic diarrhea of the invention, transmissible gastroenteritis of swine, pig fourth type coronavirus are highly-safe, immunogenicity is good, antibody is generated quickly and is 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 needle prevent and treat 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 fields, 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 technique

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, causes huge economic loss to aquaculture.

Transmissible gastroenteritis of swine (Transmissible gastroenteritis, TGE) is a kind of acute intestinal of pig Infectious disease is fallen ill anxious, is propagated fastly, and the pig at various ages can infect, to cause piglet within 3-10 age in days to vomit, seriously Diarrhea, it is thin with high mortality (usual 100%) is characterized, and morbid pig also often shows as acute watery diarrhea, excrement be 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, cream 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) belongs to the more virales of Buddhist nun (Nidovirales), the coronal disease 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 current year disease in 17 states in the U.S. it is popular.Then plus by also being reported with South Korea detect PDCoV, positive rate are up to 25%.More importantly the PDCoV clinic of CONTINENTAL AREA OF CHINA is examined between 2014-2016 It surveys positive rate and is up to 23.4%, and there is no corresponding vaccine 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.Currently, external is mainly the Attenuate vaccine of 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 Da Bei Nong company, The vaccines such as the TGE-PED bivalent inactivated vaccine of Wuhan predecessor company, section, but clinical application effect is limited；In addition, attenuated vaccine with go out Live vaccine exists in production dissipates malicious risk, on the one hand the excessive use of attenuated vaccine has virulence and returns strong risk, another party Face may cause the recombination variation of popular strain and vaccine strain 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.

Summary of the invention

Based on above-mentioned discovery and understanding, the main purpose of the present invention is to provide a kind of pig epidemic diarrheas, pig transmissible Sick three subunit vaccines of gastroenteritis, pig fourth type coronavirus, antigenic component are Porcine epidemic diarrhea virus, pig transmissible stomach and intestine The respective antigen protein S1 of three kinds of viruses such as scorching virus and pig fourth type coronavirus, and three kinds of viral S1 protein contents need to divide Not >=150 μ g/ml(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, expresses pig epidemic diarrhea The Pichia Pastoris X33-PEDV-S1(Pichia pastoris X33-PEDV S1 of viral S1 albumen), in It is preserved in China General Microbiological culture presevation administrative center on June 30th, 2017, deposit number is CGMCC NO:14372, is protected Hiding 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, expresses pig transmissible stomach The Pichia Pastoris X33-TGEV-S1(Pichia pastoris X33-TGEV S1 of enteritis virus S1 albumen), 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, expresses pig fourth type coronavirus The Pichia Pastoris X33-PDCoV-S1(Pichia pastoris X33-PDCoV S1 of S1 albumen), in 2017 It is preserved in China General Microbiological culture presevation administrative center on June 30, 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.

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

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

Detailed description of the invention

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

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

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

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

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

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

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

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

Specific embodiment

The embodiment of the present invention is specifically described with reference to the accompanying drawing

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

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 site morbidity pig Commercially available diarrhea vaccine at that time has been injected before, therefore has speculated that Field epidemic virus is strain after variation, to 2013- After clinical sample detection between 2016, different Porcine epidemic diarrhea virus, pig transmissible stomach are isolated out of morbid pig body Enteritis virus and pig fourth type coronavirus, after carrying out genome sequencing and phylogenetic analysis (Fig. 3, Fig. 5 and Fig. 7), Selected PEDV KB2013-4 plants, BD plants of TGEV and SD plants of the PDCoV Reference Strains as current Field epidemic strain；Wherein KB2013-4 plants of PEDV (Porcine epidemic diarrhea virus Porcine epidemic diarrhea virus) is in 2016 It is preserved within 23 days 08 month China General Microbiological culture presevation administrative center, deposit number is CGMCC NO:12663, preservation Location is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；BD plants of (pig transmissible stomaches of TGEV Enteritis virus Transmissible Gastroenteritis virus) be preserved on 06 07th, 2017 China it is general Logical Microbiological Culture Collection administrative center, deposit number are CGMCC NO:13856, and preservation address is the Chaoyang District, Beijing City North Star The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica；SD plants of PDCoV (pig δ coronavirus Porcine Deltacoronavirus it) was preserved in China General Microbiological culture presevation administrative center on 06 07th, 2017, is protected Hiding number is CGMCC NO:13853, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences Object research institute.

According to the antigenic characteristic and amino acid sequence of Porcine epidemic diarrhea virus S1 albumen, design synthesizes pair of primers, The site primer 1:5 '-GGATCCATGAAGTCTTTAACCTACTT-3 ', HI containing Bam；Primer 2: 5'- The site AAGCTTAACACCTTCAAGTGGTTTAG-3 ', III containing Hind；Take PEDV KB2013-4 strain virus gene as mould Plate, PCR amplification target fragment (the amplification qualification figure that Fig. 1 is Porcine epidemic diarrhea virus S1 gene PCR), product recycling connection PEASY-T1 cloning vector, 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 are SEQ ID NO:1.The popular pig between 2010-2017 disclosed in NCBI Epidemic diarrhea virus S1 protein sequence is compared, homology 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, The site primer 3:5 '-GGATCCATGAAAAAACTATTTGTG-3 ', HI containing Bam；Primer 4: 5'- The site AAGCTTACCAACAGGACTCAACGAC-3 ', III containing Hind；Take TGEV BD strain virus gene as template, PCR expands Increase target fragment (the amplification qualification figure that Fig. 1 is 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 are SEQ ID NO:2.The popular pig transmissible between 2010-2017 disclosed in NCBI 1 protein sequence of gastroenteritis virus S is compared, homology 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, The site primer 5:5 '-GGATCCATGCAGAGAGCTCTATTGAT-3 ', HI containing Bam；Primer 6: 5'- The site AAGCTTACCATCACTACACACGCTGAT-3 ', III containing Hind；Take PDCoV SD strain virus gene as template, PCR amplification target fragment (the amplification qualification figure that Fig. 1 is pig fourth type coronavirus S1 gene PCR), product recycling connection pEASY- T1 cloning vector, 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 are SEQ ID NO:3.The popular pig fourth type between 2013-2017 disclosed in NCBI Coronavirus S1 protein sequence is compared, homology between 96.8%-99.9% (Fig. 6).

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

The following steps are included: a. construction of expression vector；B. building expression bacterial strain；C. Porcine epidemic diarrhea virus S1 egg is recombinated The induction of white, 1 albumen of pig infectious gastroenteritis virus S and pig fourth type coronavirus S1 albumen and extraction purification.It is specific as follows: will Positive colony plasmid pEASY-T1-PEDV-S1 or pEASY-T1-TGEV-S1 or pEASY-T1-PDCoV-S1 and expression vector PPICZ α carrier uses Bam HI and Hind III double enzyme digestion product after 1.0% agarose gel electrophoresis respectively, is returned with DNA gel Kits recycling is received, obtains 1.9 kb or 1.9 kb or 1.6 kb and 3.3 kb segments respectively, connects structure in 16 DEG C of orientations PPICZ α-PEDV-S1 or pPICZ α-TGEV-S1 or pPICZ α-PDCoV-S1 expression vector is built, after digestion identification is correct； After plasmid linearization, electrotransformation enters Pichia pastoris competent cell, construct 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 genome carries out PCR identification 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 inducing 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 medium, 30 DEG C of shaken cultivations, and thalline were collected by centrifugation, after being suspended with suitable BMMY, adds Enter 0.55% methanol, 30 DEG C induce 96 hours.4 DEG C, 9000rpm is centrifuged 5min, retains supernatant, 40% ammonium sulfate precipitation is added Afterwards, 12000rpm is centrifuged 5min and collects albumen precipitation, with the molten albumen of PBS weight.Protein electrophoresis sample-loading buffer, boiling water boiling 8 is added After minute, carrying out SDS-PAGE identification with 12% separation gel, (Fig. 8 is the SDS- PAGE electricity of recombinant bacterium fermentation inducement expression product It swims rating diagram, 1,2,3 be respectively PEDV-S1, TGEV-S1 and PDCoV-S1 protein expressioning products precipitating in figure, and M are molecular weight mark Quasi- protein).

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

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

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

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

2. the incomplete fluid nutrient medium of BMGY is added by fermenter volume 60% (V/V) in the preparation of seedling albumen, press simultaneously Defoaming agent is added in culture medium 0.1% (V/V), is passed through high-temp steam sterilizing 30 minutes, is down to 32 DEG C to culture medium temperature, YNB is added And biotin, it is inoculated with production Pichia pastoris X33-PEDV-S1 or X33- TGEV-S1 or X33- PDCoV-S1 secondary seed Liquid, fermentor parameter setting are respectively mixing speed 800r/min, 30 DEG C of temperature, maintain D0 value (dissolved oxygen amount) 20%.Culture 24 Bacterium solution after hour adds methanol, and the speed of adding of methanol is the culture of 2ml/h/L inducing expression, according to the above ferment control parameter And technique inducing expression 120 hours；Fermented and cultured bacterium solution through tube centrifuge 10000r/min be centrifuged 30 minutes, harvest it is upper Clearly, after ammonium sulfate precipitated protein being added, 30 minutes harvest precipitatings is centrifuged by 12000r/min, suitable physiological saline solution is added Albumen precipitation.

3. protein liquid is placed in inactivation bottle by inactivation, metered 10% formalin is mixed them thoroughly with adding with shaking, The ultimate density of formalin is 0.1%.It is poured into another inactivation bottle after adding formalin, the disease nearby adhered to avoid bottleneck Poison fails to contact inactivator.37 DEG C inactivation 16 hours after take out, set 2-8 DEG C of preservation.

4. the inspection of semifinished product

4.1 steriling tests carry out steriling test by existing " Chinese veterinary pharmacopoeia " annex.

4.2 determining the protein quantity detect protein content by Bradford method.

4.3 inactivations, which are examined, takes a small amount of inoculation YPD solid medium for the protein liquid after inactivation, sets 30 DEG C and continues culture 72 Hour.Sterile length of being born is observed, inactivation is sentenced and examines qualification.

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

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

The Arlacel-85 of 0.5% volume is dispersed in the squalene phase of 5% volume by the preparation of 1 adjuvant, the tween-with 0.5% volume 80 are mixed into the 10mM sodium citrate buffer solution of 94% volume, and then mixed at high speed obtains thick lotion.Then by this thick lotion High pressure homogenizer (1200bar, 6-8 circulations) is iterated through, the lotion with uniform droplet size is generated；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.

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

3 emulsifications take 1 part of oily phase to be put in 50L-200L high-speed shearing machine, start the stirring of four leaf paddle slow rotations, while Xu Xu is added 1 part of water phase, with 300-500r/min, emulsifies 5 minutes.After emulsification, 10mL vaccine is taken to be centrifuged 15 points with 3000r/min Clock, the water phase that tube bottom is precipitated should be no more than 0.5ml.

4 vaccine product inspections

4.1 character

Appearance vaccine should be milky emulsion, free from admixture and qualification is answered in outer packing.

Dosage form 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 shaking gently It is dispersed in water.

Stability is drawn vaccine 10ml and is added in centrifuge tube, and with 3000r/min centrifugation 15 minutes, the water phase that tube bottom is precipitated was answered No more than 0.5ml.

Viscosity is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.

4.2 loading quantity inspections are carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.

4.3 steriling tests are carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.

With 3-5 age in days piglet 10, (corresponding sow should be later stage of pregnancy detection pig epidemic diarrhea, passes 4.4 safety verifications 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 simultaneously, is raised, is observed continuously 14 at identical conditions, the feeding of record test pig, drinking-water and clinical setting. Any locally and systemically adverse reaction as caused by vaccine should not occur.

4.5 efficacy test

4.5.1 the immune piglet serum neutralizing antibody of inspection is inoculated with the piglet of 1 times of immunizing dose, uses within 2 weeks after immune and neutralizes Antibody test method examines the serum for Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig fourth type coronavirus disease Neutralizing antibody, 10 piglets at least should turn sun by 8 piglets, for three kinds of viral neutralize antibody titers >=1:32, can reach Good immune effect；

4.5.2 piglet immunological agent 15 that Immunization test 1 multiple dose of inoculation is done to piglet, are divided into for 3 weeks after immune 3 groups (5/group), together with the piglet of the same age 15 (being also classified into 3 groups, 5/group) of non-vaccine inoculation, with 10^5.0 TCID₅₀/ ml pig Epidemic diarrhea virus, transmissible gastro-enteritis virus and the virulent oral challenge respectively of pig fourth type coronavirus disease, exempt from after attacking poison Epidemic disease piglet should have 4/5 protection respectively, and the morbidity of control pig 5/5 or immune piglet 5/5 are protected, and control piglet 4/5 falls ill, and be judged to exempt from Epidemic disease is qualified.

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

Pig epidemic diarrhea, the transmissible gastroenteritis of swine, pig fourth type coronavirus disease three manufactured experimently in embodiment 4, embodiment 3 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 subunits of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus in 1 materials Example 3 Vaccine preproduction；Transmissible gastroenteritis of swine, pig epidemic diarrhea, porcine rotavirus (G5 type) trigeminal live vaccine (the weak magnificent strain of poison+ CV777 plants+NX plants of weak poison), Jilin decent job biological products limited liability company produces (lot number 2016006)；Pig transmissible stomach and intestine Scorching, pig epidemic diarrhea bivalent inactivated vaccine, Jilin decent job biological products limited liability company produce (lot number 2016003).

2 methods

The design of 2.1 animal experiments

With 3-5 age 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 setting control 15 Only, raise, be observed continuously 14 at identical conditions, the feeding of record test pig, drinking-water and clinical setting, after 14d respectively into Row blood sampling, and strong virus attack was used in the blood sampling same day.15 of negative control group are not immunized, and are divided into 3 groups, are respectively used to three kinds of diseases Poison attacks the negative control of poison.Three vaccine groups are respectively equipped with one group of (5) blank control group, only immune without attacking poison.Specifically Grouping 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 referring to the 4.5.1 item method in embodiment 3, is taken a blood sample respectively to immune latter 14 days piglets, point From serum, measurement is directed to the neutralization of Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig fourth type coronavirus respectively Antibody titer.

2.2.2 Immunization is protected

Efficacy test is carried out referring to the 4.5.2 item method applied in example 3, immune latter 14 days piglets are used respectively corresponding strong Poison is attacked.It is observed continuously 10, records the incidence of each group piglet.

3 results

3.1 antibody determination results

With sick three subunit vaccines of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus in embodiment 3 Preproduction, transmissible gastroenteritis of swine, pig epidemic diarrhea, porcine rotavirus (G5 type) trigeminal live vaccine (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 pig fourth type are directed to after three subunit vaccines are immune in piglet serum 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 immune The neutralize antibody titers of piglet are not higher than 1:16.Therefore, generated after three subunit vaccines are immune 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 and commercially available three attenuated live vaccines, commercially available bivalent inactivated vaccine are immune Neutralize antibody titers compare

3.2 Immunizations protect result

With sick three subunit vaccines of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus in embodiment 3 Preproduction, transmissible gastroenteritis of swine, pig epidemic diarrhea, porcine rotavirus (G5 type) trigeminal live vaccine (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, after 14d respectively with it is corresponding it is virulent attacked, continuously Observation 10 days, that attacks poison protection the results are shown in Table 3.

After 3 three subunit vaccine preproduction of table and commercially available three attenuated live vaccines, commercially available bivalent inactivated vaccine are immune Malicious protecting effect is attacked to compare

The result shows that pig epidemic diarrhea of the invention, transmissible gastroenteritis of swine, pig fourth type coronavirus disease three are sub- single Position vaccine safety is high, and immunogenicity is good, it is immune after antibody generate quickly and hold time length compared with antibody titers, vaccine saves Phase is long, and immunizing dose is small, and the adjuvant of selection is easy to inject, can a needle prevent and treat three kinds of main diarrhea related diseases viral diseases, raising The survival rate of piglet.

Sequence table

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<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 (4)

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

The amino acid sequence of the Porcine epidemic diarrhea virus S1 albumen is SEQ ID NO:1；

The amino acid sequence of 1 albumen of pig infectious gastroenteritis virus S is SEQ ID NO:2；

The amino acid sequence of the pig fourth type coronavirus S1 albumen is SEQ ID NO:3；

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

The Porcine epidemic diarrhea virus S1 albumen is the pichia pastoris yeast for being CGMCC NO:14372 by deposit number Bacterium expression preparation；1 albumen of pig infectious gastroenteritis virus S is the Bath for being CGMCC NO:14371 by deposit number The expression preparation of moral Pichia yeast；It is CGMCC NO:14370 that the pig fourth type coronavirus S1 albumen, which is by deposit number, Pichia Pastoris expression preparation.

2. pig epidemic diarrhea according to claim 1, transmissible gastroenteritis of swine, pig fourth type coronavirus disease three are sub- single Position vaccine, which is characterized in that the vaccine adjuvant be aluminium hydroxide gel, mineral oil, Gel 01, propolis, ISA206, ISA760VG or MF59.

3. pig epidemic diarrhea according to claim 1, transmissible gastroenteritis of swine, pig fourth type coronavirus disease three are sub- single Position vaccine, which is characterized in that the vaccine adjuvant is MF59.

4. pig epidemic diarrhea according to claim 1, transmissible gastroenteritis of swine, pig fourth type coronavirus disease three are sub- single Position vaccine, which is characterized in that Porcine epidemic diarrhea virus S1 protein content >=150 μ g/ml, pig in vaccine described in every part The S1 protein content of infectious gastroenteritis virus >=150 μ g/ml, S1 protein content >=150 μ g/ of pig fourth type coronavirus Ml, 50%(V/V) MF59.