CN106511991B - Application of the Ii gene order in Porcine epidemic diarrhea virus gene vaccine - Google Patents

Abstract

The present invention relates to gene vaccine technical field, in particular to application of a kind of Ii gene order in Porcine epidemic diarrhea virus gene vaccine, Ii gene base sequence is shown in sequence 8 in sequence table.Ii genophore and S1(m) genophore is mixed to or will be used for after the connection of Ii gene and S1(m) gene the preparation of Porcine epidemic diarrhea virus gene vaccine, S1(m) gene base sequence is shown in sequence 10 in sequence table.On the basis of the S1 gene through codon optimization, successfully construct eukaryon expression plasmid pVAX-S1(m) and pVAX-Ii-S1(m), the effect that the immunogenicity and Ii of mouse immuning test verifying S1 albumen play in improving humoral and cellular immune response as molecule adjuvant, head exempts from rear 63d and specific antibody occurs, it is significantly raised that head exempts from rear 84d antibody, new thinking is proposed to develop PEDV vaccine, is laid a good foundation for the test of pig body.

Description

Application of the Ii gene order in Porcine epidemic diarrhea virus gene vaccine

Technical field

The present invention relates to gene vaccine technical field, in particular to a kind of Ii gene order is in Porcine epidemic diarrhea virus base Because of the application in vaccine.

Background technique

Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus A kind of acute intestinal communicable disease caused by (Porcine epidemic diarrhea virus, PEDV), clinical symptoms with Diarrhea, vomiting, dehydration are main feature.PEDV belongs to coronavirus, is the single strand plus RNA virus for having cyst membrane.PEDV coding 4 A structural proteins S protein (spike protein), E protein (small member protein), M albumen (member ) and N protein (nucleoprotein) protein.S protein is in receptor combination, induction neutralizing antibody and virus and cell is promoted to melt Close etc. plays an important role.

From 2010, PED in south China broke out by province, causes up to a million pigs dead in short 1 year and spreads to The whole nation causes heavy losses to the pig breeding industry in China.In addition, South Korea, Japan, Vietnam, even PED occur sudden and violent in the U.S. for Thailand Hair, leads to piglet extensive death.PED, which is broken out, damages the most serious piglet in especially birth 7d to piglet, presents and propagates The features such as range is wide, infection rate and the death rate are high.But Adult Pig infection PEDV only shows slight clinical symptoms.Due to the course of disease compared with It is short, have little time to generate active immunity response in piglet body, the immunization method mainly taken at present is immune sow, is generated containing anti- The colostrum of body, piglet permit colostrum acquisition passivity immune by inhaling.Commercialized vaccine is mainly attenuated vaccine, including China at present CV777, the KPEDV-9 and DR13 of South Korea, Japan P-5V, but the immune effect of these vaccines is undesirable.In recent years, state The various forms of vaccines of inside and outside scholar's research and probe, including Transgenic Plant Vaccines, recombinant adenovirus vaccine and subunit's epidemic disease Seedling etc., but not can induce effective immune response and immunoprotection.Researcher confirms that PEDV prevalence strain morphs, Especially immunogenicity preferable S protein variation is maximum, this may is that previous vaccine cannot effectively prevention and control current PED the reason of.

DNA vaccination is also referred to as nucleic acid vaccine, is will have protective antigen gene to be cloned into eukaryon table by recombinant DNA technology It up to carrier, is imported in host through certain approach, pass through host cell expression foreign protein and induces generation immune response.Mesh The preceding DNA vaccination by U.S. FDA approval into clinical test includes the DNA epidemic disease such as AIDS, tumour, Ebola virus and malaria Seedling.

DNA vaccination can correctly express exogenous proteins after being inoculated with host, through the Transcription/Translation System of host cell Native conformation can induce comprehensive immune response in target organ and cell continuous expression antigen.In addition, DNA vaccination system Standby simplicity, cost is relatively low, storage is convenient, and no virulence returns strong or remaining danger, can be avoided immune evasion phenomenon.But It is that there are biosafety issues for DNA vaccination, such as a possibility that exogenous DNA and host genome integration and immune tolerance；Separately Outside, plasmid imports the low efficiency of host and foreign gene low results under DNA vaccination immune effect host cell expression is horizontal Drop.How to improve the antibody level of DNA vaccination induction is the emphasis of current research DNA vaccination.

Mhc class ii molecule constant chain (Invariant chain, Ii) is important point in the synthesis of mhc class ii molecular biosciences Sub- companion belongs to II type transmembrane protein, is in non-polypeptide.Chaperone of the Ii as mhc class ii molecule, in mhc class ii molecule It plays an important role in the formation of compound, sorting and internalization, while Ii can block the antigen binding position of mhc class ii molecule Point, prevents it in conjunction with endogenous antigen, plays protection prematurity mhc class ii molecular action.Significantly Ii can promote , T cell differentiation mature into B cell and enhancement antigen submission, have been used for a kind of immune carrier.

So far the phase applied in Porcine epidemic diarrhea virus gene vaccine without the constant chain fusion protein of mhc class ii molecule It closes and records.

Summary of the invention

In order to solve the above immune effect of DNA vaccination in the prior art it is low, so far without mhc class ii molecule constant chain merge Related the problem of recording that albumen is applied in Porcine epidemic diarrhea virus gene vaccine, this application provides a kind of Ii gene sequences The application being listed in Porcine epidemic diarrhea virus gene vaccine.

What the present invention was obtained through the following steps:

Application of the Ii gene order in Porcine epidemic diarrhea virus gene vaccine, the Ii gene base sequence are shown in sequence Sequence 8 in table.

Ii genophore and Porcine epidemic diarrhea virus S1 (m) genophore are preferably mixed or are incited somebody to action by the application Ii gene is used for the preparation of Porcine epidemic diarrhea virus gene vaccine, S1 after connecting with Porcine epidemic diarrhea virus S1 (m) gene (m) gene base sequence is shown in sequence 10 in sequence table.

The application, preferably Ii gene are connect with S1 (m) gene by 2A gene, and Ii-2A gene base sequence is shown in sequence Sequence 9 in list.

The body fluid that the application, preferably Ii genes amplification Porcine epidemic diarrhea virus S1 (m) gene protein induction generate Immune and cellullar immunologic response improves ELISA antibody level, and the T cell of secretion IL-4 is promoted to generate.

The application, preferred operations are as follows:

(1) primer for expanding Ii gene is designed and synthesized, sequence 6 and 7 in sequence table is seen, designs and synthesizes for expanding The primer for increasing Porcine epidemic diarrhea virus S1 (m) gene, is shown in sequence 4 and 5 in sequence table；

(2) pig spleen RNA is extracted, the cDNA obtained using reverse transcription is template, with sequence 6 and 7 in sequence table through PCR amplification Ii gene is obtained, base sequence is shown in sequence 8 in sequence table；Using S1 (m) gene of optimum synthesis as template, with sequence in sequence table 4 and 5 obtain S1 (m) gene through PCR amplification, and base sequence is shown in sequence 10 in sequence table；

(3) Ii gene and S1 (m) gene are inserted into plasmid pVAX respectively and obtain recombinant plasmid pVAX-Ii and pVAX-S1 (m), it is used after two kinds of recombinant plasmids being mixed.

The application, preferred operations are as follows:

(1) primer for expanding Ii gene is designed and synthesized, sequence 6 and 7 in sequence table is seen, designs and synthesizes for expanding The primer for increasing the Ii gene Ii-2A of aftosa 2A gene, is shown in sequence 1,2 and 3 in sequence table, designs and synthesizes for expanding pig stream The primer of row diarrhea virus S1 (m) gene is shown in sequence 4 and 5 in sequence table；

(2) pig spleen RNA is extracted, the cDNA obtained using reverse transcription is template, with sequence 6 and 7 in sequence table through PCR amplification Ii gene is obtained, base sequence is shown in sequence 8 in sequence table；Using obtained Ii gene as template, respectively with 1 He of sequence in sequence table 2, sequence 1 and 3 gradually expands through PCR and obtains Ii-2A gene in sequence table, and base sequence is shown in sequence 9 in sequence table；It is closed with optimizing At S1 (m) gene be template, obtain S1 (m) gene through PCR amplification with sequence 4 and 5 in sequence table, base sequence is shown in sequence table Middle sequence 10；

(3) Ii-2A gene is connect with S1 (m) gene, and connects and obtains recombinant plasmid VAX-Ii-2A- into plasmid pVAX S1(m)。

The application, preferably PCR system are 2 × primerSTAR Mix, 12.5 μ l, F primer, 1.0 μ l, R primer, 1.0 μ L, template 0.2 μ l, ddH₂11.3 μ l of O amounts to 25.0 μ l.

The application, preferably PCR response procedures are 98 DEG C of 1s；98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 2min 30s carry out 35 A circulation, 72 DEG C of 5min.

In order to improve the immune effect of DNA vaccination, a variety of methods have been attempted in this experiment, such as inclined according to mammalian codons Preferendum has carried out S1 gene modification；Molecule adjuvant Ii is added and improves the protein induced immune response of S1；In mouse leg before immune The efficiency of intramuscular injection lidocaine hydrochloride raising cellular uptake eucaryon plasmid.Molecule adjuvant Ii raising S1 albumen is wherein added to lure The immune response effect led is more satisfactory.

Beneficial effects of the present invention:

1) this test successfully constructs eukaryon expression plasmid pVAX-S1 on the basis of the S1 gene through codon optimization (m) it is being improved with pVAX-Ii-2A-S1 (m), the immunogenicity and Ii of mouse immuning test verifying S1 albumen as molecule adjuvant The effect played in humoral and cellular immune response, head exempt from rear 63d and specific antibody occur, and head exempts from rear 84d antibody apparent increase,

2) eukaryotic expression recombinant plasmid of the S1 albumen and Ii albumen of the codon optimization of this experimental construction amalgamation and expression, through small The immunological characteristic of mouse immunity test validating DNA vaccine, proposes new thinking to develop PEDV vaccine, has established base for the test of pig body Plinth.

Detailed description of the invention

Fig. 1 is the PCR amplification of purpose gene, A:M:1Kb DNA Marker；1:Negative control；2:S1(m)； B:M:DL2000DNA Marker；1:Negative control；2:Ii；C:M:DL2000DNA Marker；1:Negative control；2:Ii-2A；

Fig. 2 is that the digestion of recombinant vector is identified, A:M:DL5000DNA Marker；1:pVAX-Ii(BamHI/EcoRV)； 2:pVAX(BamHI/EcoRV)；B:M:DL5000DNA Marker；1:pVAX-S1(m)(EcoRV/XhoI)；2:pVAX (EcoRV/XhoI)；C:M:DL5000DNA Marker；1:pVAX-Ii-2A-S1(m)(BamHI/EcoRV/XhoI)；2:pVAX (BamHI/EcoRV/XhoI)；

Fig. 3 is that IFA identifies protein expression after Transfected Recombinant Plasmid HEK-293A cell；

Fig. 4 is that Western blot identifies Transfected Recombinant Plasmid HEK-293A expression of cellular proteins, A:1:pVAX-Ii-2A- S1(m)；2:pVAX-S1(m)；3:HEK-293A.B:1:pVAX-Ii-2A-S1(m)；2:pVAX-Ii；3:HEK-293A；Fig. 5 is ELISA antibody test；

Fig. 6 is neutralizing antibody detection；

Fig. 7 is the special lymphopoiesis response of PEDV；

Fig. 8 is cell factor IL-4 content in spleen lymphocyte；

Fig. 9 is cell factor IFN-γ content in spleen lymphocyte.

Specific embodiment

Invention is further explained combined with specific embodiments below:

Embodiment 1

1 materials and methods

1.1 strains, plasmid and cell

E.coil DH5 α, PEDV low virulent strain are saved by this laboratory.PVAX1 plasmid is saved by this laboratory.HEK-293A Cell, Vero cell are saved by this laboratory.S1 gene (S1 (m)) through codon optimization is closed by Shanghai JaRa biotech firm At.

1.2 reagents and experimental animal

Trizol Plus、Oligod(T)₁₈, dNTPs (10mM), PrimerSTAR high fidelity enzyme, T4DNA ligase, limit Property restriction endonuclease processed is purchased from Takara company.Reverse transcription reagent box, transfection reagent Lipofectamine 2000 are purchased from Invitrogen.Rabbit-anti S1 protein polyclone antibody is prepared by this laboratory and is saved.Rabbit-anti CD74 antibody is purchased from Santa Cruz Biotech.DNA gel QIAquick Gel Extraction Kit, Plasmid Miniprep Kit are purchased from BIOMEGA company.The goat-anti rabbit of HRP label The goat anti-rabbit igg of IgG and FITC label is purchased from Wuhan doctor moral company.Isopropanol and dehydrated alcohol are tried purchased from Chinese medicines group chemistry Agent Co., Ltd.Chloroform is purchased from Shanghai Ling Feng chemical reagent Co., Ltd.Human peripheral lymphocyte separating liquid is purchased from day Saliva Hao sun biological products Co., Ltd.

Balb/c mouse is purchased from Military Medical Science Institute's Experimental Animal Center.

The amplification of 1.3 target gene

1.3.1 primer

It is designed and synthesized according to CD74 gene (Ii) sequence (GenBank:AK394321) that GenBank is issued for expanding The primer of Ii gene and the Ii gene (Ii-2A) containing aftosa 2A gene, introduced respectively in upstream and downstream primer BamHI and EcoRV；With the PEDV S gene order (GenBank:KF453516) of GenBank publication for standard, Shanghai JaRa biotech firm Pass through codon optimization and synthesizes S1 (1-2379bp) gene, i.e. S1 (m) gene；It designs and synthesizes for expanding S1 (m) gene Primer, introduce EcoRV and XhoI respectively in upstream and downstream primer.Primer sequence is as follows:

Table is used to expand the primer and its sequence of different S1 and Ii segments

1.3.2 PCR system and program

The fresh spleen of pig is taken, total serum IgE is extracted, utilizes Oligod (T)₁₈Reverse transcription is cDNA；Using cDNA as template, primer Ii-F/Ii-R is through PCR amplification Ii gene；Using PCR product Ii gene as template, Ii-F/Ii-R-1, Ii-F/Ii-R-2 through PCR by Step amplification Ii-2A gene；Using S1 (m) gene of optimum synthesis as template, primer S1 (m)-F/S1 (m)-R is through PCR amplification S1 (m) Gene.

PCR system and program are as follows: 2 × primerSTAR Mix, 12.5 1.0 1.0 μ l μ l of μ l μ l, R of μ l μ l, F, template 0.2 μ l μ l, ddH₂11.3 μ l μ l of O amounts to 25.0 μ l μ l.

PCR response procedures are 98 DEG C of 1s；98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 2min 30s carry out 35 and recycle, and 72 DEG C 5min.PCR product is separated through 1.0% agarose gel electrophoresis, and PCR product S1 (m), Ii and Ii-2A are recycled through DNA fragmentation glue Kits.Recycling segment is saved backup in -20 DEG C.

Using S1 (m) gene of codon optimization as template, amplification obtains S1 (m) segment (Fig. 1 of size about 2379bp Middle A)；Spleen total serum IgE is extracted, reverse transcription obtains cDNA and as template, and amplification obtains Ii segment (Fig. 1 of size about 663bp Middle B)；Using Ii segment as template, the Ii segment containing aftosa 2A gene is obtained by twice PCR, i.e. Ii-2A, size is about 702bp (C in Fig. 1).

The building and identification of 1.4 recombinant plasmids

1.4.1 the building of recombinant plasmid pVAX-Ii and pVAX-Ii-2A

1.4.1.1 Ii, Ii-2A segment and pVAX plasmid enzyme restriction and recycling

Digestion pVAX plasmid and segment Ii, Ii-2A are distinguished using restriction enzyme BamHI and EcoRV, and digestion system is such as Under: each 5.0 μ l μ l of 1.0 μ l μ l, 10 × K buffer of restriction enzyme BamHI and EcoRV, segment or each 15ug of plasmid and 8ug, moisturizing to 50 μ l μ l.37 DEG C of effect 3h in water-bath, 1% agarose gel electrophoresis separation, cut purpose band through DNA Gel reclaims kit recycling.

1.4.1.2 target fragment connect and converts with carrier

Connection reaction total volume is 10 μ l μ l.System is as follows: 10 × Ligase Buffer, 1.0 μ l, T4DNA ligase 0.5 μ l, 7.5 1.0 μ l of μ l, pVAX of segment.Overnight in 16 DEG C of connections on small-sized connection instrument.E.coil DH5 α impression is prepared simultaneously State cell.Connection product was converted into DH5 α competent cell in second day, coating contains 100ug/ml kanamycins (K⁺) LB it is flat Plate, 37 DEG C of culture 14-16h, picking single bacterium colony is to containing 100ug/ml K⁺In LB liquid medium, in shaking table 200rpm 37 DEG C culture 4-6h, draw 1 μ l bacterium solution do template, according to the method described above PCR amplification carry out Preliminary Identification.

1.4.1.3 a small amount of extractions and identification of recombinant plasmid

Plasmid is extracted using plasmid extraction kit.Recombinant plasmid is identified in digestion, and digestion system is as follows: restriction enzyme BamHI and EcoRV each 1.0 μ l of 0.5 μ l, 10 × K buffer, 2.0 μ l of recombinant plasmid, moisturizing to 10 μ l.Digestion pVAX simultaneously Plasmid compares.37 DEG C of effect 3h, 1% agarose gel electrophoresis separate and observe result in water-bath.The positive will be accredited as Two kinds of plasmids be respectively designated as pVAX-Ii and pVAX-Ii-2A, and send to Invitrogen company be sequenced.

1.4.2 the building of recombinant vector pVAX-S1 (m) and pVAX-Ii-S1 (m)

1.4.2.1 S1 (m) segment, pVAX and pVAX-Ii-2A plasmid enzyme restriction and recycling

Digestion S1 (m) segment, pVAX and pVAX-Ii-2A plasmid, enzyme are distinguished using restriction enzyme EcoRV and XhoI It is as follows to cut system: each 5.0 μ l of 1.0 μ l, 10 × H buffer of restriction enzyme EcoRV and XhoI, segment or each 15ug of plasmid And 8ug, moisturizing to 50 μ l.37 DEG C of effect 3h in water-bath, 1% agarose gel electrophoresis separation, cut purpose band through DNA Gel reclaims kit recycling.

1.4.2.2 target fragment connect and converts with carrier

Connection reaction total volume is 10 μ l.System is as follows: 10 × Ligase Buffer, 1.0 μ l, T4DNA ligase, 0.5 μ 3.5 μ l of l, S1 (m) segment, 1.0 μ l of plasmid, moisturizing to 10 μ l.Overnight in 16 DEG C of connections on small-sized connection instrument.It prepares simultaneously E.coil DH5 α competent cell.Second day by connection product convert DH5 α competent cell, coating containing 100ug/ml card that Mycin (K⁺) LB plate, 37 DEG C of culture 14-16h, picking single bacterium colony is to containing 100ug/ml K⁺In LB liquid medium, in 37 DEG C of culture 4-6h of shaking table 200rpm draw 1 μ l bacterium solution and do template, and PCR amplification carries out Preliminary Identification according to the method described above. 1.4.2.3 a small amount of extractions and identification of recombinant plasmid

Plasmid is extracted using plasmid extraction kit.Recombinant plasmid is identified in digestion, and digestion system is as follows: restriction enzyme EcoRV and XhoI each 1.0 μ l of 0.5 μ l, 10 × H buffer, 2.0 μ l of recombinant plasmid, moisturizing to 10 μ l.Digestion pVAX matter simultaneously Grain compares.37 DEG C of effect 3h, 1% agarose gel electrophoresis separate and observe result in water-bath.The positive will be accredited as Two kinds of plasmids are respectively designated as pVAX-S1 (m) and pVAX-Ii-2A-S1 (m) (referred to as pVAX-Ii-S1 (m)), and send to The sequencing of Invitrogen company.

Using BamHI/EcoRV digestion recombinant plasmid pAVX-Ii, the band (Fig. 2 being consistent with the expected size of Ii gene is obtained Middle A)；Using EcoRV/XhoI digestion recombinant plasmid pAVX-S1 (m), the band being consistent with the expected size of S1 (m) gene is obtained (B in Fig. 2)；Using BamHI/EcoRV/XhoI digestion recombinant plasmid pAVX-Ii-S1 (m), obtain and S1 (m) gene and Ii-2A Two bands (C in Fig. 2) that the expected size of gene is consistent.Recombinant plasmid is sent to Invitrogen and is sequenced, sequencing result is shown Gene is all correct.

The eukaryotic expression of 1.5 recombinant plasmids is identified

1.5.1 transfection

By four kinds of plasmid pVAX, pVAX-Ii, pVAX-S1 (m) and pVAX-Ii-S1 (m) according to Lipofectamine 2000 operational manuals transfect HEK-293A cell.Specific step is as follows: the day before transfection, and HEK-293A cell is uniformly laid on 24 holes and 96 porocyte plates are added after complete medium in 37 DEG C of cell incubator cultures；Reach 90- to cell density within second day When 95%, complete medium is discarded, the complete medium for not containing antibiotic is added.By 1ug plasmid and 3 μ l Lipofectamine2000 is diluted in respectively in culture medium of the 100 μ l without containing antibiotic and serum, is mixed gently, room temperature is incubated Educate 5min；Above two liquid is gently mixed uniformly, 20min is stored at room temperature；Culture medium in 24 orifice plates is discarded, mixing is added Liquid is placed in 37 DEG C of cell incubators and is incubated for 6h；Mixed liquor is discarded, 1ml complete medium is added, is placed in 37 DEG C of cell incubators Cultivate 72h.In addition, it is thin 96 holes will to be added after 0.3ug plasmid and 0.8 μ l Lipofectamine immixture according to above-mentioned steps In born of the same parents' plate, it is placed in 37 DEG C of cell incubator culture 72h.

1.5.2 IFA

96 porocyte plates for taking out 72h after transfecting, discard cell culture medium, and PBS is washed 2 times；PBS is discarded, every hole is added Methanol: 100 μ l of propyl alcohol (1:1) is placed in -20 DEG C of standing 20min；Discard methanol: propyl alcohol, PBS are washed 3 times；Rabbit-anti PEDV is added 100 hole μ l/ of S1 positive serum (1:20) or rabbit-anti CD74 polyclonal antibody (1:100), 37 DEG C of incubation 60min；Antibody is discarded, PBST is washed 6 times, every minor tick 5min；50 hole μ l/ FITC- goat anti-rabbit igg (1:100), 37 DEG C of incubation 45min are added；It discards Antibody, PBST are washed 3 times, every minor tick 5min；In fluorescence microscopy microscopic observation result.Normal HEK-293A is arranged simultaneously to make For control.

72h after recombinant plasmid transfected cell carries out IFA verification experimental verification recombinant plasmid pVAX- using rabbit-anti S1 positive serum The expression of S1 (m) and pVAX-Ii-S1 (m) S1 albumen in HEK-293A cell, as a result, it has been found that pVAX-S1 (m) and pVAX-Ii- There is fluorescence in HEK-293A cell after S1 (m) transfection, and HEK-293A cell and normal HEK- after pVAX transfection 293A cell does not have fluorescence；Utilize the how anti-progress IFA verification experimental verification recombinant plasmid pVAX-Ii and pVAX-Ii-S1 of rabbit-anti CD74 (m) in HEK-293A cell Ii albumen expression, as a result, it has been found that pVAX-Ii and pVAX-Ii-S1 (m) transfection after HEK- There is fluorescence in 293A cell, and the HEK-293A cell and normal HEK-293A cell after pVAX transfection do not have fluorescence (figure 3)。

1.5.3 Western blot

24 porocyte plates for taking out 72h after transfecting, discard cell culture medium, and PBS is washed 2 times；Cell pyrolysis liquid 100 is added The hole μ l/, is placed in and acts on 30min on ice；Cell is collected, 5 × Loading Buffer is added and boils 5min in 100 DEG C；It utilizes 10% protein adhesive carries out SDS-PAGE, protein isolate.Processing HEK-293A cell compares in the same way simultaneously.SDS- After PAGE, glue is cut into suitable size, half-dried transfer printing is transferred, and transfer condition is 20V, 35min.Transfer terminates Afterwards, NC film is placed in the PBST containing 10% skimmed milk, room temperature acts on 3-5h；PBST is discarded, rabbit-anti PEDV S protein is added Positive serum (1:200) or rabbit-anti CD74 polyclonal antibody (1:1000), antibody are dilute using the PBST containing 5% skimmed milk It releases, is stood overnight in 4 DEG C.Next day, antibody is discarded, PBST washs 4-6h；The addition diluted goat anti-rabbit igg-HRP of PBST (1: 15000), room temperature jog 30min；Antibody is discarded, PBST washs 1-2h；It is developed the color with chemical luminescence reagent kit, carries out X-ray in darkroom Piece exposure observes result after developed and fixing.

72h after Transfected Recombinant Plasmid HEK-293A cell harvests cell and separates through 10%SDS-PAGE.Utilize rabbit-anti S1 Positive serum carries out Western blot analysis, and discovery pVAX-S1 (m) and pVAX-Ii-S1 (m) swimming lane are equal at correspondingly sized place There is band, and HEK-293A swimming lane does not have band；Utilize the how anti-progress Western blot analysis of rabbit-anti CD74, discovery There is band at correspondingly sized place in pVAX-Ii and pVAX-Ii-S1 (m) swimming lane, and HEK-293A swimming lane does not have band (Fig. 4).

1.6 mouse immuning test

1.6.1 a large amount of extractions of plasmid

200 μ L strains are inoculated in 6ml and contain 100ug/ml kanamycins (K⁺) LB liquid medium in, 37 DEG C 200rpm shaken cultivation is stayed overnight.6ml recombinant bacterium is inoculated in 500ml and contains 100ug/ml K by next day⁺LB liquid medium in, 37 DEG C of 200rpm shaken cultivation 16-20h.7500rpm 10min is centrifuged bacterium solution, the STE for collecting thallus and being pre-chilled with 4 DEG C of 300ml (0.1M NaCl, 10mM Tris-HCl, 1mM EDTA, pH 8.0) is washed twice.7500rpm 10min is centrifuged bacterium solution, collects Thallus and the solution I (50mM glucose, 10mM EDTA, 25mM Tris-Cl, PH 8.0) that 4 DEG C of 6ml pre-coolings are added are resuspended thin Bacterium；Solution II (the 0.2M NaOH that 6ml is newly prepared is added；1%SDS), it gently turns upside down centrifuge tube for several times, is placed in and makees on ice Use 5min；The solution III (glacial acetic acid 11.5mL, 5M potassium acetate 60mL, water 28.5mL) of 4 DEG C of 8ml pre-coolings is added, gently up and down Reverse centrifuge tube is generated to white precipitate for several times, is placed in and is acted on 5min on ice；4 DEG C of 12000rpm are centrifuged 15min, and supernatant is moved to In new centrifuge tube and be added 0.6 times of volume isopropanol, be placed in -20 DEG C of effect 60min；4 DEG C of 12000rpm are centrifuged 15min, It discards supernatant and that 70% ethanol washing of 15ml is added is primary；4 DEG C of 12000rpm are centrifuged 10min, discard supernatant, after drying at room temperature 4.5ml ddH is added₂O dissolution；500 μ l RNase A (10mg/mL), 37 DEG C of effect 60min are added；Isometric phenol is added: Chloroform, mixing are placed on room temperature effect 5min；4 DEG C of 12000rpm are centrifuged 10min, transfer supernatant to new centrifuge tube, addition etc. The chloroform of volume, mixing are placed on room temperature effect 5min；4 DEG C of 12000rpm centrifugation 10min, transfer supernatant to new centrifuge tube, Isometric dehydrated alcohol is added, mixing is placed on room temperature effect 25min；4 DEG C of 12000rpm are centrifuged 15min, discard supernatant simultaneously It is primary that 70% ethanol washing of 10ml is added；4 DEG C of 12000rpm are centrifuged 10min, discard supernatant, 3ml is added after drying at room temperature ddH₂O dissolution.Spectrophotometric determination plasmid concentration and purity are placed in -20 DEG C of preservations.

1.6.2 mice group and immune

60 cleaning grade mouse are randomly divided into 6 groups, every group 10,1,2 and 3 group is immunized pVAX-Ii+pVAX-S1 respectively (m), pVAX-Ii-S1 (m) and pVAX-S1 (m)；4, pVAX-Ii, pVAX and PBS are immunized respectively for 5 and 6 groups, as a control group.Exempt from Only, the previous day is immunized in 100 μ g/ of epidemic disease dosage, and leg muscle injects 2% lidocaine hydrochloride, and 50 μ μ l/ are only.Immunization route is leg Intramuscular injection, immunizing dose are 100ug/.Head exempts from rear 21d, 42d and 63d with identical approach and dosage booster immunization.

After head exempts from rear 63d and 84d, every group takes 5 mouse at random, and eyeball takes a blood sample and separates serum and examines for ELISA antibody It surveys and neutralizing antibody detects；Aseptic collection mouse spleen, separation lymphocyte are tested for lymphocyte proliferation.

1.6.3 ELISA antibody test

With the PEDV coated elisa plate purified through ultracentrifugation, protein concentration is adjusted to 10 μ g/ml with antigen coat liquid, 100 μ μ l are added in every hole, and 37 DEG C of placement 1h, 4 DEG C overnight, and PBST is washed 3 times, and each 5min is patted dry；200 μ μ l5% are added in every hole Skimmed milk, 37 DEG C of placement 3h, PBST are washed 3 times, are patted dry；2 doubling dilution blood serum sample, 37 DEG C of placements since 1:25 with PBST 1h discards sample, and PBST is washed 3 times, and each 5min is drained；Every hole be added with the diluted HRP- goat anti-mouse igg of PBST (1: 10000), 37 DEG C of placement 30min discard sample, and PBST is washed 3 times, and each 5min is drained；In light protected environment, TMB is developed the color Liquid A and B are mixed in equal volume, and 100 μ μ l, 37 DEG C of effect 10-15min are added in every hole；50 μ l 2M H are added in every hole₂SO4 terminates anti- It answers, OD450 value is read in microplate reader；Sample S/N value is calculated, is the serum with the serum greatest dilution of value >=2.1 S/N Potency.

ELISA antibody test result is shown: head exempts from rear 63d, and 1,2 and 3 immune groups can detect specific antibody, but each Antibody level difference is unobvious (P > 0.05) between group；Head exempts from rear 84d, and each immune group antibody level increases, and 1,2 and 3 is immune Group antibody level is apparently higher than other three groups (P < 0.05), the obvious (P of 1 and 2 immune group antibody levels difference compared with 3 immune groups < 0.05), antibody level difference is unobvious (P > 0.05) (Fig. 5) between 1 and 2 immune groups.

1.6.4 neutralizing antibody detects

PEDV virus titer is measured before virus neutralization tests.Vero cell is uniformly laid in 96 porocyte plates, to cell Single layer is covered with to carry out connecing poison.Virus is made into 10 times of doubling dilutions with maintaining liquid, is inoculated in cell hole, 100 holes μ l/ are each dilute Degree of releasing does 3 repetitions, in 37 DEG C of cell incubator culture 3-5d.Setting does not connect poison cell and does negative control simultaneously.Observe CPE simultaneously The CPE hole count for recording each dilution calculates PEDV TCID according to Reed-Muench method₅₀。

Vero cell is uniformly laid in 96 porocyte plates by 1d before virus neutralization tests.Serum to be checked is inactivated through 56 DEG C 30min, 1200rpm are centrifuged 5min, collect supernatant；250 μ μ l of blood serum sample is taken to be added to the maintenance containing 2%FBS of 250 μ l In liquid, serum is done into 1:2 dilution, 250 μ l are therefrom drawn after mixing and are added in the new maintaining liquid of 250 μ μ l, it is dilute to be 1:4 It releases ..., such successively doubling dilution；PEDV is diluted to 200TCID with maintaining liquid₅₀/ 100 μ μ l, and will be after itself and dilution Serum mixes in equal volume, in 37 DEG C of effect 1h；Culture medium in 96 porocyte plates is discarded, virus and serum mixed liquor, 100 μ μ are added The hole l/, each serum dilution do 4 repetitions, in 37 DEG C of cell incubator culture 4-5d.Negative serum control, sky are set up simultaneously White control, virus-negative serum control.When there is not CPE in negative serum control and blank control, and virus-negative serum When CPE occurs in control, test sets up and records the CPE hole count of each serum dilution.To completely inhibit the maximum of the serum of CPE Dilution is the neutralization titer of the serum.

Neutralizing antibody testing result is shown: head exempts from rear 63d, unobvious (the P > of neutralizing antibody level difference between each immune group 0.05)；Head exempts from rear 84d, and 1,2 and 3 immune group neutralizing antibody levels are apparently higher than other three groups (P < 0.05), but this 1,2 and 3 Neutralizing antibody level difference between immune group is unobvious (P > 0.05) (Fig. 6).

1.6.5 lymphocyte proliferation assay

1.6.5.1 the preparation of splenic lymphocytes

Mouse is extractd into eyeball bloodletting and cervical dislocation is put to death, impregnates 5min in 75% alcohol；It is sterile to cut off mouse abdomen Portion's skin opens peritonaeum and carefully takes out spleen；Spleen is put into the pouch of 200 mesh copper mesh sewing, with No. 4 syringe needles in spleen Upper bundle pin hole；Spleen is gently scraped with dress curved needle head on the injector, overflows spleen cell, a little PBS is added and with suction Pipe piping and druming prepares single cell suspension for several times；Splenocyte suspension is gently added to containing human peripheral lymphocyte separating liquid from In heart pipe, 2000rpm is centrifuged 15min；White cloud and mist layer in centrifuge tube is drawn with suction pipe, is moved to containing 5ml erythrocyte cracked liquid Centrifuge tube in, 2000rpm be centrifuged 10min；It discards supernatant, 1640 culture medium of 5ml is added and washs cell, 2000rpm centrifugation 10min；It discards supernatant, with the 1640 resuspension cells containing 10%FBS and adjusts cell concentration to 5 × 10⁶A/ml is added 96 Porocyte plates, 100 holes μ μ l/.

1.6.5.2 lymphocyte proliferation assay

Using the PEDV purified through ultracentrifugation as stimulator antigen, ConA is positive control, final concentration of 10ug/ml；37 66h is cultivated in DEG C cell incubator, draws part supernatant, and 20 μ l μ l MTT (5mg/ml), 37 DEG C of cell incubators relayings are added Continuous culture 6h；Culture medium is discarded, 100 μ l μ l DMSO are added in every hole, and crystallization is melted in oscillation, read OD570 numerical value in microplate reader； Calculate stimulus index: SI=stimulation hole OD value/do not stimulate hole OD value.

Distinguish 63d and 84d separating mouse splenic lymphocytes after the first exemption, carries out the examination of PEDV specific lymphoproliferation It tests.As the result is shown: head exempts from rear 63d, and specific lymphocyte increment occur in 1,2 and 3 immune groups, but poor compared with other three groups Different unobvious (P > 0.05)；Head exempts from rear 84d, and the stimulus index of 1,2 and 3 immune groups increases, and is significantly higher than other three groups of (P < 0.05) (Fig. 7).

1.6.5.3 cytokine assay

Head is collected to exempt from rear 63d and 84d mouse spleen lymphocyte proliferation assay on the cell of each group stimulation hole stimulation 66h Clearly, IFN-γ and IL-4 content, concrete operation step in supernatant is detected to be carried out according to kit specification.

IL-4 content detection is as the result is shown in mouse spleen lymphocyte culture supernatant, and: head exempts from rear 63d, between each immune group IL-4 content difference is unobvious (P > 0.05)；Head exempts from rear 84d, and the IL-4 content of 1,2 and 3 immune groups is significantly raised and significantly high In other three groups (P < 0.05), 1 and 2 immune group IL-4 contents are significantly higher than 3 immune groups (P < 0.05), between 1 and 2 immune groups IL-4 content difference is unobvious (P > 0.05), (Fig. 8).

IFN-γ content detection is as the result is shown in mouse spleen lymphocyte culture supernatant, and: head exempts from rear 63d, between each immune group IFN-γ content difference is unobvious (P > 0.05)；Head exempts from rear 84d, and the IFN-γ content of 1,2 and 3 immune groups is significantly raised, and aobvious It writes and is higher than other three groups (P < 0.05), the IFN-γ content difference between 1,2 and 3 immune groups is unobvious (P > 0.05) (Fig. 9). 1.6.5.4 data statistic analysis

Using SPSS software, statistical analysis of data carries out ANOVA and LSD multiple analysis, compares each group difference, P < 0.05 indicates that significant difference, P < 0.01 indicate that difference is extremely significant.

In the present invention, we construct Ii and S1 fusion, and the 2A gene of intermediate aftosa is connected, Western Blot is the result shows that recombinant plasmid can correctly express albumen, wherein verifying recombinant plasmid pVAX- using rabbit-anti S positive serum When Ii-S1 (m) protein expression, there are size two bands of about 110kD and 85kD, be Ii-S1 (m) fusion protein and S1 egg respectively White size；Occur size two bands of about 110kD and 25kD when verifying how anti-using rabbit-anti Ii, is Ii-S1 (m) fusion respectively The size of albumen and Ii albumen, the reason of causing this phenomenon is that self-cleavage occurs for 2A albumen, so that Ii-S1 (m) merges egg The cracking of white hair first portion.The immunogenicity of mouse validating DNA vaccine is immunized using recombinant plasmid.The result shows that head exempts from rear 84d, 1, 2 and 3 immune groups can detect specific antibody and neutralizing antibody, and be apparently higher than other three groups (P < 0.01), in addition 1 It is significantly higher than 3 immune groups (P < 0.05) with 2 immune group specific antibody levels.It is protein induced that this illustrates that Ii significantly improves S1 Humoral immune response.Cytokine content testing result is shown in splenic lymphocytes culture supernatant, and head exempts from rear 84d, 1,2 and 3 IL-4 and IFN-γ content are significantly higher than other three groups (P < 0.01) in immune group mouse spleen lymphocyte culture supernatant, and 1 It is significantly higher than 3 immune groups (P < 0.05) with 2 immune group IL-4 contents.This illustrates that Ii promotes the T cell generation of secretion IL-4. Mouse immuning test confirm expression of recombinant plasmid S1 albumen can effective induction body fluid be immune and cellullar immunologic response, and Ii significantly enhances humoral and cellular immune response response.

In conclusion eukaryon expression plasmid can correctly express Ii, S1 and Ii-2A-S1 (m) fusion protein；Mouse immune Test confirms that Ii can enhance the humoral immunity and cellullar immunologic response of the protein induced generation of S1, provides for prevention and control PEDV new Method.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by the limit of embodiment System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, substitution, simplification should be Equivalence replacement mode, is included within the scope of the present invention.

<110>Agricultural University Of Nanjing

<120>application of the Ii gene order in Porcine epidemic diarrhea virus gene vaccine

<160> 11

<210> 1

<211> 33

<212> DNA

<213>artificial synthesized

<400> 1

TATAGGATCCGCCACCATGGAGGACCAGCGCGA 33

<210> 2

<211> 47

<212> DNA

<213>artificial synthesized

<400> 2

GACGTCGCCGGCCAACTTGAGAAGGTCAAAGTTCAGGATGACTTGGC 47

<210> 3

<211> 48

<212> DNA

<213>artificial synthesized

<400> 3

GCAGATATCGGGCCCTGGGTTGGACTCGACGTCGCCGGCCAACTTGAG 48

<210> 4

<211> 30

<212> DNA

<213>artificial synthesized

<400> 4

ATAGATATCGCCACCATGGAGAGCCTCACA 30

<210> 5

<211> 30

<212> DNA

<213>artificial synthesized

<400> 5

AGTCTCGAGTCAGATGCTCATGGAAAAGTT 30

<210> 6

<211> 33

<212> DNA

<213>artificial synthesized

<400> 6

TATAGGATCCGCCACCATGGAGGACCAGCGCGA 33

<210> 7

<211> 30

<212> DNA

<213>artificial synthesized

<400>7

GCAGATATCTTACAGGATGACTTGGCCGAG 30

<210> 8

<211> 645

<212> DNA

<213> CDS

<400> 8

atggaggacc agcgcgacct catctccaac catgagcagc tgcccatgct gggccagcgc 60

cccggggccc ccgagagcaa gtgcagccgt ggagctctgt acacaggctt ttctgtcctg 120

gtggctctgc tcctggctgg ccaggccacc accgcctact tcctgtacca gcagcagggc 180

cggctggaca agctgacggt cacctctcag aacttgcagc tggagagcct gcggatgaag 240

cttcccaagc cctccaagcc tttgagcaag atgcgggttt ccgcccccat gctgatgcag 300

gccctgccca tggaaggccc ggagcctatg cgcaacgcca ccaagtacgg caacatgacc 360

caggaccacg tgatgcacct gctcctgaag tctgaccccc tgggagtgta cccgaagctg 420

aaggggagcc tcccagaaaa tctgaagcac ctcaagaaca ccatggacgg tgtgaactgg 480

aagctctttg agaactggct gcgtcagtgg ctcttgtttg aaatgagcaa gaactcgctg 540

gaggagacac cctttgaggt tccgccaaaa gacccactgg agacggagga cctgtcgtcc 600

gggctgggcg tgaccaagca ggatctcggc caagtcatcc tgtaa 645

<210> 9

<211> 702

<212> DNA

<213> CDS

<400> 9

atggaggacc agcgcgacct catctccaac catgagcagc tgcccatgct gggccagcgc 60

cccggggccc cggagagcaa gtgcagccgt ggagctctgt acacaggctt ttctgtcctg 120

gtggctctgc tcctggctgg ccaggccacc accgcctact tcctgtacca gcagcagggc 180

cggctggaca agctgacggt cacctctcag aacttgcagc tggagagcct gcggatgaag 240

cttcccaagc cctccaagcc tttgagcaag atgcgggttt ccgcccccat gctgatgcag 300

gccctgccca tggaaggccc ggagcctatg cgcaacgcca ccaagtacgg caacatgacc 360

caggaccacg tgatgcacct gctcctgaag tctgaccccc tgggagtgta cccgaagctg 420

aaggggagcc tcccagaaaa cctgaagcac ctcaagaaca ccatggacgg tgtgaactgg 480

aagctctttg agaactggct gcgtcagtgg ctcttgtttg aaatgagcaa gaactcgctg 540

gaggagacac cctttgaggt tccgccaaaa gacccactgg agacggagga cctgtcgtcc 600

gggctgggcg tgaccaagca ggatctcggc caagtcatcc tgaactttga ccttctcaag 660

ttggccggcg acgtcgagtc caacccaggg cccgaattct gc 702

<210> 10

<211> 2379

<212> DNA

<213> mat_peptide

<400>10

atggagagcc tcacatactt ctggctcctg ctgcccgtgc tcagcacact cagcctgcca 60

caggatgtca cacgctgctc cgccaagacc aatttccgcc ggttcttcag caagtttaac 120

gtccaagctc ccgccgtggt ggtcctgggg ggctacctgc caatcgggga gaaccagggc 180

gtgaatagca cctggtactg cgccggacaa caccctaccg ccagcggggt ccatgggatt 240

ttcgtcagcc acattcgcgg aggccacggg ttcgaaatcg gcatcagcca ggagcctttc 300

gacccctccg ggtatcagct ctatctccac aaagccacaa acgggaacac caacgccacc 360

gcccggctgc gcatctgcca gttccccagc atcaagaccc tggggcccac agccaataac 420

gacgtgacca ccgggcgcaa ttgtctcttc aacaaggcca tcccagccca catgtccgag 480

cattccgtcg tggggatcac atgggacaat gaccgggtga ccgtgttcag cgataaaatc 540

tattatttca atttcaaaaa tgactggagc cgcgtcgcca ccaaatgcta caattccggc 600

gggtgcgcta tgcaatatgt gtatgagcct acctattaca tgctgaatgt gaccagcgcc 660

ggggaggacg ggatctccta ccagccctgc acagctaatt gcattgggta cgccgccaat 720

gtgtttgcca ccgaacccaa cggacacatc cccgaggggt tttccttcaa taactggttc 780

ctgctctcca acgacagcac cctcgtgcat ggcaaggtgg tgtccaatca acccctgctg 840

gtgaactgcc tcctcgctat tcctaagatt tatggactcg gccaattttt ctcctttaac 900

caaaccattg atggggtctg caatggcgcc gctgtccaac gcgctcccga ggccctgcgg 960

ttcaacatta acgataccag cgtgatcctc gccgagggct ccatcgtgct ccacacagct 1020

ctgggcacaa atttcagctt cgtctgctcc aactccagcg acccccatct cgctacattt 1080

gctatccccc tgggcgctat ccaggtcccc tactactgtt ttctcaaggt cgacacctac 1140

aatagcaccg tgtacaagtt tctggccgtc ctgcccccta cagtgcgcga gatcgtcatt 1200

accaagtacg gcgacgtgta tgtcaacgga ttcgggtacc tccacctcgg gctgctcgac 1260

gccgtgacaa ttaactttac cgggcacggc acagacgacg acgtctccgg cttttggaca 1320

atcgctagca caaacttcgt ggacgctctc atcgaattcc aaggaaccgc catccagcgc 1380

atcctctact gcgacgaccc tgtgagccaa ctgaagtgct cccaggtcag ctttgatctg 1440

gacgacggct tctaccctat cagcagcacc aacctgctgt cccacgagca acccacctcc 1500

tttgtgaccc tgcccagctt caacgaccac tccttcgtga acattacagt gagcgctgcc 1560

ttcggcggac atagcggggc caacctcatc gcctccgaca caaccatcaa cgggtttagc 1620

agcttttgcg tcgatacacg ccagtttacc atctccctct tctataatgt caccaatagc 1680

tacgggtacg tgagcaattc ccaggattcc aattgcccct tcacactcca gagcgtgaac 1740

gactacctga gcttctccaa gttctgcgtc agcacctccc tgctcgcttc cgcctgcacc 1800

atcgacctct ttggctaccc cgagttcggc agcggagtca aatttacaag cctgtacttt 1860

cagttcatta agggcgaact catcaccggc accccaaaac cactcgaagg cgtgaccgat 1920

gtgagcttta tgacactcga tgtctgtaca aagtatacaa tctatggctt caaaggggaa 1980

ggcattatta ccctgattaa ttccagcttt ctcgctgggg tgtactacac aagcgatagc 2040

ggccagctgc tggccttcaa gaacgtcaca tccggggctg tgtactccgt caccccctgt 2100

tccttttccg aacaagccgc ctacgtggat gacgacatcg tcggcgtgat ttccagcctc 2160

agcaacagca catttaactc cacccgcgag ctgcctgggt tcttttacca cagcaatgac 2220

ggaagcaact gcaccgagcc tgtgctggtc tactccaaca tcggggtgtg caagagcggc 2280

agcatcggct atgtgccctc ccaaagcggg caggtgaaga tcgccccaac agtcacagga 2340

aacatctcca tccctaccaa cttttccatg agcatctga 2379

<210> 11

<211> 3093

<220>

<221>CDS

<222>(1) ... (702)

<220>

<221>mat_peptide

<222>(715) ... (3093)

<400>11

atggaggacc agcgcgacct catctccaac catgagcagc tgcccatgct gggccagcgc 60

cccggggccc cggagagcaa gtgcagccgt ggagctctgt acacaggctt ttctgtcctg 120

gtggctctgc tcctggctgg ccaggccacc accgcctact tcctgtacca gcagcagggc 180

cggctggaca agctgacggt cacctctcag aacttgcagc tggagagcct gcggatgaag 240

cttcccaagc cctccaagcc tttgagcaag atgcgggttt ccgcccccat gctgatgcag 300

gccctgccca tggaaggccc ggagcctatg cgcaacgcca ccaagtacgg caacatgacc 360

caggaccacg tgatgcacct gctcctgaag tctgaccccc tgggagtgta cccgaagctg 420

aaggggagcc tcccagaaaa cctgaagcac ctcaagaaca ccatggacgg tgtgaactgg 480

aagctctttg agaactggct gcgtcagtgg ctcttgtttg aaatgagcaa gaactcgctg 540

gaggagacac cctttgaggt tccgccaaaa gacccactgg agacggagga cctgtcgtcc 600

gggctgggcg tgaccaagca ggatctcggc caagtcatcc tgaactttga ccttctcaag 660

ttggccggcg acgtcgagtc caacccaggg cccgaattct gcgatatcgc caccatggag 720

agcctcacat acttctggct cctgctgccc gtgctcagca cactcagcct gccacaggat 780

gtcacacgct gctccgccaa gaccaatttc cgccggttct tcagcaagtt taacgtccaa 840

gctcccgccg tggtggtcct ggggggctac ctgccaatcg gggagaacca gggcgtgaat 900

agcacctggt actgcgccgg acaacaccct accgccagcg gggtccatgg gattttcgtc 960

agccacattc gcggaggcca cgggttcgaa atcggcatca gccaggagcc tttcgacccc 1020

tccgggtatc agctctatct ccacaaagcc acaaacggga acaccaacgc caccgcccgg 1080

ctgcgcatct gccagttccc cagcatcaag accctggggc ccacagccaa taacgacgtg 1140

accaccgggc gcaattgtct cttcaacaag gccatcccag cccacatgtc cgagcattcc 1200

gtcgtgggga tcacatggga caatgaccgg gtgaccgtgt tcagcgataa aatctattat 1260

ttcaatttca aaaatgactg gagccgcgtc gccaccaaat gctacaattc cggcgggtgc 1320

gctatgcaat atgtgtatga gcctacctat tacatgctga atgtgaccag cgccggggag 1380

gacgggatct cctaccagcc ctgcacagct aattgcattg ggtacgccgc caatgtgttt 1440

gccaccgaac ccaacggaca catccccgag gggttttcct tcaataactg gttcctgctc 1500

tccaacgaca gcaccctcgt gcatggcaag gtggtgtcca atcaacccct gctggtgaac 1560

tgcctcctcg ctattcctaa gatttatgga ctcggccaat ttttctcctt taaccaaacc 1620

attgatgggg tctgcaatgg cgccgctgtc caacgcgctc ccgaggccct gcggttcaac 1680

attaacgata ccagcgtgat cctcgccgag ggctccatcg tgctccacac agctctgggc 1740

acaaatttca gcttcgtctg ctccaactcc agcgaccccc atctcgctac atttgctatc 1800

cccctgggcg ctatccaggt cccctactac tgttttctca aggtcgacac ctacaatagc 1860

accgtgtaca agtttctggc cgtcctgccc cctacagtgc gcgagatcgt cattaccaag 1920

tacggcgacg tgtatgtcaa cggattcggg tacctccacc tcgggctgct cgacgccgtg 1980

acaattaact ttaccgggca cggcacagac gacgacgtct ccggcttttg gacaatcgct 2040

agcacaaact tcgtggacgc tctcatcgaa ttccaaggaa ccgccatcca gcgcatcctc 2100

tactgcgacg accctgtgag ccaactgaag tgctcccagg tcagctttga tctggacgac 2160

ggcttctacc ctatcagcag caccaacctg ctgtcccacg agcaacccac ctcctttgtg 2220

accctgccca gcttcaacga ccactccttc gtgaacatta cagtgagcgc tgccttcggc 2280

ggacatagcg gggccaacct catcgcctcc gacacaacca tcaacgggtt tagcagcttt 2340

tgcgtcgata cacgccagtt taccatctcc ctcttctata atgtcaccaa tagctacggg 2400

tacgtgagca attcccagga ttccaattgc cccttcacac tccagagcgt gaacgactac 2460

ctgagcttct ccaagttctg cgtcagcacc tccctgctcg cttccgcctg caccatcgac 2520

ctctttggct accccgagtt cggcagcgga gtcaaattta caagcctgta ctttcagttc 2580

attaagggcg aactcatcac cggcacccca aaaccactcg aaggcgtgac cgatgtgagc 2640

tttatgacac tcgatgtctg tacaaagtat acaatctatg gcttcaaagg ggaaggcatt 2700

attaccctga ttaattccag ctttctcgct ggggtgtact acacaagcga tagcggccag 2760

ctgctggcct tcaagaacgt cacatccggg gctgtgtact ccgtcacccc ctgttccttt 2820

tccgaacaag ccgcctacgt ggatgacgac atcgtcggcg tgatttccag cctcagcaac 2880

agcacattta actccacccg cgagctgcct gggttctttt accacagcaa tgacggaagc 2940

aactgcaccg agcctgtgct ggtctactcc aacatcgggg tgtgcaagag cggcagcatc 3000

ggctatgtgc cctcccaaag cgggcaggtg aagatcgccc caacagtcac aggaaacatc 3060

tccatcccta ccaacttttc catgagcatc tga 3093

Claims (3)

1. Application of the 1.Ii gene order in preparation Porcine epidemic diarrhea virus gene vaccine, by Ii gene and pig epidemic diarrhea Viral S1(m) preparation of Porcine epidemic diarrhea virus gene vaccine is used for after gene connection, the Ii gene base sequence is shown in sequence Sequence 8, S1(m in list) gene base sequence is shown in sequence 10 in sequence table；

   It is specifically shown in following operation:

   (1) primer for expanding Ii gene is designed and synthesized, sequence 6 and 7 in sequence table is seen, designs and synthesizes for expanding mouth The primer of the Ii gene Ii-2A of fever aphthous 2A gene, is shown in sequence 1,2 and 3 in sequence table, designs and synthesizes for expanding pig popularity Diarrhea virus S1(m) gene primer, see sequence 4 and 5 in sequence table；

   (2) pig spleen RNA is extracted, the cDNA obtained using reverse transcription is obtained with sequence 6 and 7 in sequence table through PCR amplification as template Ii gene, base sequence are shown in sequence 8 in sequence table；Using obtained Ii gene as template, respectively with sequence 1 and 2, sequence in sequence table Sequence 1 and 3 gradually expands through PCR and obtains Ii-2A gene in list, and base sequence is shown in sequence 9 in sequence table；With optimum synthesis S1(m) gene is template, obtains S1(m through PCR amplification with sequence 4 and 5 in sequence table) gene, base sequence is shown in sequence in sequence table Column 10；

   (3) Ii-2A gene and S1(m) gene is connected, and connects and obtains recombinant plasmid pVAX-Ii-2A- S1 into plasmid pVAX (m).
2. 2. application according to claim 1, it is characterised in that Ii gene and S1(m) gene connected by 2A gene, Ii-2A Gene base sequence is shown in sequence 9 in sequence table.
3. 3. application according to claim 1, it is characterised in that Ii genes amplification Porcine epidemic diarrhea virus S1(m) gene egg The humoral immunity and cellullar immunologic response that white induction generates, improve ELISA antibody level, and the T cell of secretion IL-4 is promoted to generate.