CN107961373A - A kind of strain of gene engineered subunit oral vaccine and its construction method and purposes for being used to prevent pig epidemic diarrhea - Google Patents

A kind of strain of gene engineered subunit oral vaccine and its construction method and purposes for being used to prevent pig epidemic diarrhea Download PDF

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CN107961373A
CN107961373A CN201711125034.3A CN201711125034A CN107961373A CN 107961373 A CN107961373 A CN 107961373A CN 201711125034 A CN201711125034 A CN 201711125034A CN 107961373 A CN107961373 A CN 107961373A
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coe
dcpep
ppg
targeting
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徐义刚
王丽
马孙婷
唐丽杰
李经
李一经
姜艳平
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东北农业大学
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
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    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/54Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541Mucosal route
    • A61K2039/542Mucosal route oral/gastrointestinal
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/55Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552Veterinary vaccine
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
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    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA Viruses positive-sense
    • C12N2770/00011MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA Viruses positive-sense ssRNA Viruses positive-sense
    • C12N2770/20011Coronaviridae
    • C12N2770/20022New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA Viruses positive-sense
    • C12N2770/00011MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA Viruses positive-sense ssRNA Viruses positive-sense
    • C12N2770/20011Coronaviridae
    • C12N2770/20034Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention discloses a kind of strain of gene engineered subunit oral vaccine and its construction method and purposes for being used to prevent pig epidemic diarrhea.The present invention is used as the transmission live vector of vaccine antigen by the use of the indigenous lactic bacteria strain being located away from chitling road, select in PEDV S proteins and epitopic core epitope area COE is as immunogene, it is the transmission speed for effectively improving vaccine antigen at the same time, shorten the immune response time, introduce antigen delivery cell M cell-targeting Peptide C ol and antigen processing, presenting cells Dendritic Cells targeting peptides DCpep, the recombination lactic acid bacteria preparation in expression PEDV S proteins with epitopic core epitope area COE and M cell-targeting Peptide C ol and Dendritic Cells targeting peptides DCpep is built using constitutive expression carrier, so that vaccine antigen is secreted with the breeding of engineering strain, the drawbacks of avoiding induction type recombinant lactic acid bacteria.The it is proposed of the present invention provides effective technological means to prevent the development of the gene engineered subunit oral vaccine of pig epidemic diarrhea.

Description

It is a kind of be used for prevent pig epidemic diarrhea the strain of gene engineered subunit oral vaccine and Its construction method and purposes

Technical field

It is more particularly to a kind of to be used to prevent pig epidemic diarrhea the present invention relates to a kind of vaccine strain and its construction method The strain of gene engineered subunit oral vaccine and its construction method, further relate to the vaccine strain and are taken orally in preparation prevention pig epidemic diarrhea Application in vaccine.The invention belongs to veterinary drug technical field.

Background technology

Pig epidemic diarrhea (porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV) causes a kind of acute highly contagious disease of pig, is harm One of Important Infectious Diseases that China's pig breeding industry develops in a healthy way, the disease is using watery diarrhea, vomiting and dehydration as main feature, in the world Property distribution, harm is serious, and heavy economic losses is brought to pig breeding industry.Vaccine inoculation is to prevent the sick major measure, at present Using it is more be TGEV-PEDV dyad inactivated vaccines and weak poison by Harbin Veterinary Medicine Inst., China Academy of Agriculture's exploitation Seedling.

Pig epidemic diarrhea inactivated vaccine immunizing dose is big, and it is of high cost to prepare inactivated vaccine.Pig epidemic diarrhea is weak Although malicious seedling immunizing dose reduces many, expense is relatively low, and generation immunity is very fast, is adapted to the urgent immunity inoculation of swinery, The virulence of attenuated vaccine strain virus is easily returned by force, has potential virulence enhancing risk, to the dangerous property of some pole susceptible animals.In recent years Come, traditional commodities vaccine (inactivated vaccine and attenuated vaccine) Vaccine effectiveness is gradually reduced, and causes immune often failure, and pig is popular Property diarrhea incidence rise year by year, the reason is that many, as attenuated vaccine strain virus vitro recombination virulence returns strong, PEDV Appearance of variant etc..PEDV can infect the pig of all ages and classes, different lines, wherein it is the most serious with suckling pig harm, extremely The rate of dying is up to 90%.The major source of infection that PED occurs is morbid pig and with malicious pig, and major transmission path is typical excrement-mouth way Infect in footpath.Research shows that piglet is mainly obtained source of parents sIgA antibody and produced by colostrum exempts from the passive of coronavirus property diarrhea Epidemic disease is protected.Therefore, mucosa-immune is the important channel for protecting piglet adaptive immune, is produced by body after sow vaccine inoculation Newborn source sIgA antibody so to suckling pig provide protection, or immune piglet make its own produce mucosal immune response, Cai Nengwei Body provides enough protections to viral infection resisting.However, tradition PED inactivated vaccines and Attenuate vaccine mainly using injecting pathway as It is main, it is impossible to which that stimulating animal body mucosal system produces the sIgA antibody of sufficient amount, and then causes protecting effect bad.Based on PEDV Can through intestinal mucosa infect and cause a disease the characteristics of, with reference to PEDV can induce produce neutralizing antibody neutralizing epitope area, and based on The time of enhancing vaccine immunity antigen transmission efficiency and then shortening body generation immune response develops oral vaccine, is that science is prevented Control the desirable route of PED.

Lactic acid bacteria has probiotic, tolerance digestive tract environment stress ability, enteron aisle colonization ability etc., is to transmit oral vaccine The preferable live vector of antigenic substance, the most-often used lactic acid bacteria strains of the research field are Lactobacillus casei at present, and are made With being naturally occurring in animal intestinal tract, probiotic its effect of preferably indigenous lactic bacteria strain transmission vaccine antigen will be more preferable. In addition, selected marker when usually using antibiotic resistance at present as structure lactic acid bacteria expression system, with this genoid work The application of journey lactobacillus preparation in practice, the transfer of resistance factor will bring serious biological safety consequence.In addition, this grinds Study carefully the recombinant lactic acid bacteria system built in field based on induction type, i.e. the expression of vaccine antigen needs derivant to induce, and In the environment for lacking derivant, recombinant lactic acid bacteria will stop the secretion of vaccine antigen, be unfavorable for large-scale production, further restrict The popularization and application of the recombination lactic acid bacteria preparation.

Therefore, it is necessary to the infection that more safe and effective, cheap, the convenient vaccine of inoculation carrys out prevention and control PEDV.

The content of the invention

The technical problems to be solved by the invention be to provide it is a kind of based on pig source lactobacillus reuteri structure be used for prevent The gene engineered subunit oral vaccine strain of pig epidemic diarrhea and its construction method and purposes.

In order to achieve the above object, present invention employs following technological means:

The present invention is used as the transmission of vaccine antigen by the use of the indigenous lactic bacteria strain-lactobacillus reuteri being located away from chitling road Live vector, selects in PEDV S proteins and epitopic core epitope area COE (499-638aa) is used as immunogene, which can Effectively induction body produces neutralizing antibody and then suppresses PEDV infection;At the same time to effectively improve the transmission speed of vaccine antigen, contract The short immune response time, present invention introduces antigen delivery cell M cell-targeting Peptide C ol and antigen processing, presenting cells tree Prominent shape cell-targeting Peptide D Cpep, to build the double targeted delivery antigen systems of genetic engineering lactic acid bacteria;Use a kind of constitutive expression Targeted in vector construction expression PEDV S proteins with epitopic core epitope area COE and M cell-targeting Peptide C ol and Dendritic Cells The recombination lactic acid bacteria preparation of Peptide D Cpep so that vaccine antigen is secreted with the breeding of engineering strain, avoids induction type weight The drawbacks of group lactic acid bacteria.By above-mentioned design, final obtain of the present invention complies with current cultivation industry emphasis green, safety and environmental protection hair A kind of gene engineered subunit oral vaccine strain for being used to prevent pig epidemic diarrhea of exhibition theory.

On the basis of the studies above, it is sub- single that the present invention proposes a kind of genetic engineering for being used to prevent pig epidemic diarrhea Position oral vaccine strain, the vaccine strain transmit live vector using the lactobacillus reuteri for being located away from chitling road as vaccine antigen, Wherein containing in constitutive expression PEDV S proteins and epitopic core epitope area COE and targeting M cells and DC cells targeting peptides The recombinant expression carrier of fusion protein, the order of connection of fusion protein are:COE- targets targeting peptides-targeting DC cells of M cells Targeting peptides, COE sequences, target M cells targeting peptides and target DC cells targeting peptides between connected with flexible sequence Connect

Wherein, it is preferred that the pig source lactobacillus reuteri is named as lactobacillus reuteri LJ-15, is deposited in China Type Tissue Collection, in Wuhan University, its culture presevation numbering is CCTCC NO.M 2017570 for address.Experiment card Bright, this plant of the probiotic of lactobacillus reuteri LJ-15 is significantly better than common bacterial strain-Lactobacillus casei at present.

Wherein, it is preferred that marked containing Flag at 5 ' ends in the PEDV S proteins with epitopic core epitope area COE genes Sign sequence, the nucleotide sequence of the COE genes containing Flag sequence labels as shown in SEQ ID NO.5, coding targeting M cells and The targeting peptides of DC cells are respectively as shown in SEQ ID NO.11 and shown in SEQ ID NO.12.

Wherein, it is preferred that the recombinant expression carrier is prepared by following steps:

(1) using the geneome RNA and reverse transcription of isolation kit method extraction Porcine epidemic diarrhea virus into cDNA, with CDNA obtains COE genes as template through PCR;Preferably, Flag sequence labels are contained at the 5 ' ends for expanding the COE genes of acquisition, The nucleotide sequence of COE genes containing Flag sequence labels is as shown in SEQ ID NO.5;

(2) the COE fragments obtained using step (1) is templates, by M cell-targetings peptide (Co1) and DC cell-targeting peptides (DCpep) encoding gene is building up to the downstream of COE genes by fusion DNA vaccine method, obtains fusion COE-Co1-DCpep, Then COE-Co1-DCpep fusions are connected into pMD19-T simple carriers, COE genes, M cell-targetings peptide coding base Connected between cause, DC cell-targeting DNA encoding peptides by flexible sequence, obtained recombinant vector is named as pMD19-T-COE- Co1-DCpep;

Preferably, upstream and downstream primer is respectively SEQ ID NO.6 and SEQ ID NO.10 institutes used by fusion DNA vaccine Show;

(3) structure of lactic acid bacteria constitutive expression plasmid pPG-T7g10-PPT

Transformed on the basis of plasmid pPG612, double digestion is carried out to plasmid pPG612 using XbaI and XhoI, with HCE-T7g10-PgsA anchor-MCS-rrnBT1T2 sequences connect, and construct constitutive and secretive expression lactic acid bacteria vector, order Entitled pPG-T7g10-PPT, wherein HCE are bacillus constitutive and secretive expression promoter, its nucleotide sequence such as SEQ ID Shown in NO.1;T7g10 is translational enhancer sequence, its nucleotide sequence is as shown in SEQ ID NO.2, the core of PgsA anchor Nucleotide sequence is as shown in SEQ ID NO.3;MCS is polyclone enzyme enzyme site;RrnBT1T2 is Escherichia coli transcription terminator sequence Row, its nucleotide sequence is as shown in SEQ ID NO.4;

(4) using restriction endonuclease to plasmid pMD19-T-COE-Co1-DCpep and Expression Plasmid in LAB PPG-T7g10-PPT carries out double digestion processing, target gene fragment is purified through DNA plastic recovery kits, by recovery purifying PMD19-T-COE-Co1-DCpep genetic fragments are connected with expression vector pPG-T7g10-PPT purpose fragments, structure recombination expression Plasmid is in the constitutive expression PEDV S proteins and epitopic core epitope area COE and targeting M cells and DC cells The recombinant expression carrier of peptide fusion protein is targeted, is named as pPG-PPT-COE-Col-DCpep.

Wherein, it is preferred that in the constitutive expression PEDV S proteins and epitopic core epitope area COE and targeting M is thin The recombinant expression carrier of the targeting peptide fusion protein of born of the same parents and DC cells is transferred to the impression of lactobacillus reuteri by electric method for transformation In state cell.

Further, the invention also provides a kind of method of the gene engineered subunit oral vaccine strain described in structure, Comprise the following steps:

(1) using the geneome RNA and reverse transcription of isolation kit method extraction Porcine epidemic diarrhea virus into cDNA, with CDNA obtains COE genes as template through PCR;Preferably, Flag sequence labels are contained at the 5 ' ends for expanding the COE genes of acquisition, The nucleotide sequence of COE genes containing Flag sequence labels is as shown in SEQ ID NO.5;

(2) the COE fragments obtained using step (1) is templates, by M cell-targetings peptide (Co1) and DC cell-targeting peptides (DCpep) encoding gene is building up to the downstream of COE genes by fusion DNA vaccine method, obtains fusion COE-Co1-DCpep, Then COE-Co1-DCpep fusions are connected into pMD19-T simple carriers, COE genes, M cell-targetings peptide coding base Connected between cause, DC cell-targeting DNA encoding peptides by flexible sequence, obtained recombinant vector is named as pMD19-T-COE- Co1-DCpep;

Preferably, upstream and downstream primer is respectively SEQ ID NO.6 and SEQ ID NO.10 institutes used by fusion DNA vaccine Show;

(3) structure of lactic acid bacteria constitutive expression plasmid pPG-T7g10-PPT

Transformed on the basis of plasmid pPG612, double digestion is carried out to plasmid pPG612 using XbaI and XhoI, with HCE-T7g10-PgsA anchor-MCS-rrnBT1T2 sequences connect, and construct constitutive and secretive expression lactic acid bacteria vector, order Entitled pPG-T7g10-PPT, wherein HCE are bacillus constitutive and secretive expression promoter, its nucleotide sequence such as SEQ ID Shown in NO.1;T7g10 is translational enhancer sequence, its nucleotide sequence is as shown in SEQ ID NO.2, the core of PgsA anchor Nucleotide sequence is as shown in SEQ ID NO.3;MCS is polyclone enzyme enzyme site;RrnBT1T2 is Escherichia coli transcription terminator sequence Row, its nucleotide sequence is as shown in SEQ ID NO.4;

(4) using restriction endonuclease to plasmid pMD19-T-COE-Co1-DCpep and Expression Plasmid in LAB PPG-T7g10-PPT carries out double digestion processing, target gene fragment is purified through DNA plastic recovery kits, by recovery purifying PMD19-T-COE-Co1-DCpep genetic fragments are connected with expression vector pPG-T7g10-PPT purpose fragments, structure recombination expression Plasmid is in the constitutive expression PEDV S proteins and epitopic core epitope area COE and targeting M cells and DC cells The recombinant expression carrier of peptide fusion protein is targeted, is named as pPG-PPT-COE-Col-DCpep;

(5) preparative separation is in the competent cell of the lactobacillus reuteri in chitling road, through electric transformation technology by the weight of structure Group plasmid pPG-PPT-COE-Col-DCpep is transferred to lactobacillus reuteri competent cell, screens recombinant lactic acid bacteria transformant, obtains Positive restructuring lactic acid bacteria is obtained, is as used for the gene engineered subunit oral vaccine strain for preventing pig epidemic diarrhea.

In the described method, it is preferred that the lactobacillus reuteri is named as lactobacillus reuteri LJ-15, preservation In China typical culture collection center, in Wuhan University, its culture presevation numbering is CCTCC NO.M 2017570 for address.

Further, the invention also provides the gene engineered subunit oral vaccine strain is preparing prevention or controlling Treat the purposes in the medicine of prevention pig epidemic diarrhea.

A kind of gene engineered subunit oral vaccine for preventing pig epidemic diarrhea, it contains gene work of the present invention The oral vaccine strain of journey subunit.

In addition, the present invention is located away from lactobacillus reuteri in chitling road also within protection scope of the present invention, it is described Lactobacillus reuteri, be named as lactobacillus reuteri LJ-15 (Lactobacillus reuteri LJ-15), Classification And Nomenclature For lactobacillus reuteri LJ-15 (Lactobacillus reuteri LJ-15), it is deposited in China typical culture collection The heart, address is in Wuhan University, its culture presevation numbering is CCTCC NO.M 2017570, and the preservation time is on October 9th, 2017.

The present invention mainly passes through the characteristics of intestinal mucosa Infective of pig according to PEDV, green from mucosa-immune angle design Colour circle is protected, safe and efficient, probiotic good genetic engineering lactic acid bacteria oral vaccine, is of great significance to prevention and control PED.

Brief description of the drawings

Fig. 1 is the excrement characterization for feeding different lactic acid bacteria piglets;

Fig. 2 is index and spleen index;

Fig. 3 is thymus index;

Fig. 4 is the content of IgG in serum;

Fig. 5 is the content of sIgA in intestines mucus;

Fig. 6 is IL-1 contents in serum;

Fig. 7 is Serum IL-2;

Fig. 8 is piglet intestinal segment pathology section examination;

Fig. 9 is the Vector map of pPG-T7g10-PPT;

Figure 10 is recombinant expression carrier collection of illustrative plates;

(a):pPG-PPT-COE;(b):pPG-PPT-COE-DCpep;(c):pPG-PPT-COE-Col;(d)pPG-PPT- COE-Col-DCpep;

Figure 11 is restructuring plasmid identification result;

(a) Sac I and Apa I double digestions pPG-PPT-COE;(b) Sac I and Apa I double digestions pPG-PPT-COE- Col;(c) Sac I and Apa I double digestions pPG-PPT-COE-DCpep;(d) Sac I and Apa I double digestions pPG-PPT-COE- Col-DCpep;(e) recombinant plasmid PCR results;

Figure 12 is purpose protein expression qualification result;

M:Protein molecular weight standard;1:pPG-COE/LJ-15;2:pPG-COE-Col/LJ-15;3:pPG-COE- DCpep/LJ-15;4:pPG-COE-Col-DCpep/LJ-15;5:Empty carrier recombinant lactic acid bacteria

Figure 13 is the laser co-focusing result of purpose protein expression positioning;

A、B、C:Empty carrier bacterium pPG/LJ-15;D、E、F:pPG-COE/LJ-15;G、H、I:pPG-COE-Col/LJ-15; J、K、L:pPG-COE-DCpep/LJ-15;M、N、O:pPG-COE-Col-DCpep/LJ-15;

Figure 14 is specific IgG level in immune serum;

Figure 15 is horizontal for mucosal antibodies sIgA;

(a):SIgA is horizontal in immunized mice excrement;(b):SIgA is horizontal in the outer genital tract of immunized mice;(c):Immunized mice intestines stick SIgA is horizontal in liquid;

Figure 16 is immunized mice lymphopoiesis index;

Figure 17 is cytokines measurement result.

A:PBS groups;B:PPG/LJ-15 groups;C:PPG-COE/LJ-15 groups;D:PPG-COE-Col/LJ-15 groups;E:pPG- COE-DCpep/LJ-15 groups;F:PPG-COE-Col-DCpep/LJ-15 groups.

Culture presevation information:

Strain name:Lactobacillus reuteri LJ-15

Lactobacillus reuteri LJ-15

Classification And Nomenclature:Lactobacillus reuteri LJ-15

Lactobacillus reuteri LJ-15

Depositary institution:China typical culture collection center

Address:Chinese Wuhan Wuhan Universitys

Culture presevation is numbered:CCTCC NO.M 2017570

The preservation time:On October 09th, 2017

Embodiment

The present invention is further described with reference to specific embodiments and the drawings, the advantages and features of the present invention will be with Description and it is apparent.But embodiment is only exemplary, does not form any restrictions to the scope of the present invention.Art technology Personnel should be understood that without departing from the spirit and scope of the invention can be to the details and form of technical solution of the present invention Modify or replace, but these modifications and replacement are each fallen within protection scope of the present invention.

The probiotic comparison of LJ-15 plants of 1 lactobacillus reuteri of embodiment and Lactobacillus casei

Inventor is separated to one plant of lactobacillus reuteri from the enteron aisle of pig, is named as lactobacillus reuteri LJ- 15 plants, China typical culture collection center is deposited in, its culture presevation numbering is CCTCC NO.M2017570.In order to utilize LJ-15 plants of lactobacillus reuteri probiotic is commonly used its as the carriers for transmitting oral vaccine immunity antigen with the field at present Lactobacillus casei carried out systemic comparative analysis.

1st, diarrhea of weaned piglets situation is suppressed

3 groups of weanling pig point, every group 10, experiment I groups piglet feeds LJ-15 containing lactobacillus reuteri plants of daily ration, real Daily ration of the II groups piglet feeding containing Lactobacillus casei is tested, experiment III groups piglet feeds normal daily ration, the piglet of timing observation daily Because ablactation stress caused by diarrhea situation, continuous observation 30 days.Statistical result showed (is shown in Table 1), and Luo Yishi breasts are added in daily ration LJ-15 plants of bacillus can significantly improve piglet because ablactation stress caused by diarrhea situation, significantly reduce Incidence of Diarrhea, and raise The fecal consistency of lactobacillus reuteri LJ-15 piglets discharge is fed higher than feeding Lactobacillus casei group (see figure 1), is shown LJ-15 plants of lactobacillus reuteri can be effectively improved piglet gastrointestinal tract environment, improve body resistance stress ability.

1 delactational piglets diarrhea rate of table

2nd, to the influence of normal immune vaccine antibody level

During evaluation suppresses diarrhea of weaned piglets effect, piglet normal immunological, according to standard immunization protocol respectively in piglet 1 Pseudorabies attenuated vaccine and swine fever attenuated vaccine immunity are inoculated with when age in days and 30 age in days, continues to feed 21 days (piglets about 50 days Age) after, immune piglet internal antibody level is detected.The results show that resist in feeding lactobacillus reuteri LJ-15 piglet bodies The antibody level of pseudorabies (table 2) and anti-swine fever (table 3) is higher than Lactobacillus casei group and normal diet group, shows in daily ration The vaccine antibody that addition lactobacillus reuteri LJ-15 can effectively improve delactational piglets is horizontal, its effect is better than Lactobacillus casei.

2 hog cholera antibody testing result of table

Blocking rate=(OD negative control-OD samples)/OD feminine gender × 100%;If blocking rate >=40%, for the positive;If≤ 30%, for feminine gender.

3 pseudorabies antibody testing result of table

Blocking rate=OD samples/OD feminine gender × 100%;If blocking rate≤60%, for the positive;If > 70%, for feminine gender.

3rd, to the influence of intestinal microflora

After feeding piglet lactobacillus reuteri LJ-15 and Lactobacillus casei, with PCR-DGGE methods in intestine of young pigs The structure of flora is analyzed, influence of the two kinds of lactic acid bacterias of evaluation to microbial population of animal intestinal tract.Gathered respectively in different time points young Swine excrement, the STb gene of faecal microbiota, PCR amplification 16S rDNA V3 areas are extracted with DNA extraction kit, and carry out denatured gradient Gel electrophoresis, to analyze biodiversity index (H), richness (S) and the uniformity (E).The results show (is shown in Table 4), with feeding Feed normal daily ration to compare with feeding Lactobacillus casei group intestine of young pigs micropopulation structure, feeding piglet lactobacillus reuteri LJ- 15 can preferably improve intestine of young pigs Bacterial community, maintain intestine of young pigs microecological balance.

Intestine of young pigs Bacterial community changes after table 4 feeds lactic acid bacteria

A. lactobacillus reuteri group;B. Lactobacillus casei group;C. normal diet group

4th, to the influence of intestine of young pigs and immune organ

After piglet lactobacillus reuteri LJ-15 and Lactobacillus casei 30d is fed, piglet is slaughtered, takes out whole intestinal segments, chest Gland and spleen, measure intestinal segment length and weigh, count every group of piglet immunological shoot formation.Statistic analysis result shows that feeding is young Pig lactobacillus reuteri LJ-15 can promote intestine of young pigs development (the results are shown in Table 5) and improve piglet spleen to a certain extent Dirty index (see Fig. 2) and thymus index (see Fig. 3), its probiotic effect are better than Lactobacillus casei.

5 small intestinal length of table and its weight

5th, total IgG and the total sIgA antibody levels of casing slime in piglet serum

Different time points gather serum and casing slime sample after feeding piglet lactobacillus reuteri LJ-15 and Lactobacillus casei This, detects IgG antibody level and total sIgA antibody levels in casing slime in piglet serum, to evaluate the non-of lactic acid bacteria induction body Specific immunity characteristic.Testing result shows that lactobacillus reuteri LJ-15 is added in feed can significantly improve piglet serum and intestines Non-specific immunoglobulin level (the result is shown in Fig. 4 and Fig. 5), strengthens its active immunity, and lactobacillus reuteri in mucus The ability of LJ-15 induction body nospecific immunities is better than Lactobacillus casei.

6th, IL-1 and IL-2 measure in piglet serum

IL-1 and IL-2 is a kind of important cytokine that body produces, and participates in the immune regulation of body.Testing result is shown Show, lactobacillus reuteri LJ-15 is added in feed can significantly improve the secretion level of cell factor IL-1 and IL-2, with enhancing The resistance of body, and lactobacillus reuteri LJ-15 promotes cell factor IL-1 and IL-2 secretion capacity to be better than cheese breast bar Bacterium.

7th, to the protecting effect of intestine of young pigs

Piglet after lactobacillus reuteri LJ-15 and Lactobacillus casei 14 days is fed respectively, and gavage mode attacks production by oral administration Enterotoxigenic escherichia coli, collection piglet duodenum, jejunum, ileum and colon sample, prepares the paraffin section of each intestinal segment simultaneously Observation Histopathologic changes are dyed through HE, to evaluate the guarantor of lactobacillus reuteri LJ-15 and Lactobacillus casei to intestine of young pigs Protect effect.Testing result is shown (see Fig. 8):Lactobacillus reuteri LJ-15 group piglet intestinal villus structures are fed than more complete, enteraden For structure than more visible, lamina propria has a small amount of hyperemia;Lactobacillus casei group piglet intestinal segment fine hair partial exfoliation is fed, is scattered in intestines Chamber, it is congested under mucous membrane, there is inflammatory cell infiltration;The intestinal segment intestinal villus for feeding normal diet group piglet comes off seriously, and lamina propria fills Blood.As it can be seen that lactobacillus reuteri LJ-15 is better than Lactobacillus casei to the protecting effect of intestine of young pigs.

In conclusion the present invention separates the probiotic of lactobacillus reuteri LJ-15 obtained from chitling road and is significantly better than Lactobacillus casei, is the carrier bacterium of the oral vaccine antigen of ideal transmission.

The gene engineered subunit oral vaccine of prevention pig epidemic diarrhea of the embodiment 2 based on pig source lactobacillus reuteri The structure of strain

1st, the structure of lactic acid bacteria constitutive expression plasmid pPG-T7g10-PPT

Transformed on the basis of plasmid pPG612, double digestion is carried out to plasmid pPG612 using XbaI and XhoI, will The carrier segments obtained after glue reclaim are connected with HCE-T7g10-PgsA anchor-MCS-rrnBT1T2 sequences, construct composition Type secreting, expressing lactic acid bacteria vector pPG-T7g10-PPT, wherein bacillus Geobacillus toebii composing types secrete table Up to promoter HCE nucleotide sequence as shown in SEQ ID NO.1, the nucleotide sequence such as SEQ ID of T7g10 translational enhancers Shown in NO.2, the nucleotide sequence of PgsAanchor is as shown in SEQ ID NO.3, Escherichia coli rrnBT1T2 transcription terminator sequences For row as shown in SEQ ID NO.4, MCS is polyclone enzyme enzyme site.Vector map is as shown in Figure 9.

Specific construction method refers to document, and (comparison of the structure and expression effect of composing type lactic acid bacteria expression vectors, brings up Jade for asking rain strain, Northeast Agricultural University's master thesis) method progress.And carrier pPG-T7g10-PPT has been documented in document (fusion Express the non-resistance mark recombinant lactobacillus casei expression systems of TGEV 6Ds and PEDV ps420, Wang Yusai, Northeast Agricultural University Master thesis) in, preserved by Northeast Agricultural University and provided.

2nd, the structure of pPG-PPT-COE-Col-DCpep carriers

The PEDV epidemic strains that the present invention uses were isolated from the pig of Heilongjiang Province's outburst pig epidemic diarrhea epidemic situation in 2016 , using isolation kit method extraction virus genome RNA and reverse transcription is into cDNA, using cDNA as template, with reference to GenBank In the sequence delivered, a pair of of specific primer C-F and C-R are designed according to PEDV S gene orders, 5 ' ends of primer introduce SacI restriction enzyme sites and Flag sequence labels, 3 ' ends introduce ApaI restriction enzyme sites, contain Flag sequence labels to expand 5 ' ends COE genetic fragments, its nucleotide sequence such as SEQ ID NO.5;Using COE fragments as template, using C-F as sense primer, respectively Using C-Co1, C-DCpep, C-Co1-DCpep as anti-sense primer (end of primer 3 ' introduces ApaI restriction enzyme sites), amplification respectively includes The COE-Co1 fragments of the M cell list targeting modifications of Flag sequence labels, the Dendritic Cells list targeting comprising Flag sequence labels The COE-Co1- of the COE-DCpep fragments of modification, the double targeting modifications of M cells and Dendritic Cells comprising Flag sequence labels DCpep fragments.Primer sequence is shown in Table 6.

6 primer sequence of table

aRestriction enzyme site;bFlag labels;cTerminator codon;dDCpep(SEQ ID NO.12);fCo1(SEQ ID NO.11);gFlexible linker.

COE-Co1, COE-DCpep, COE-Col-DCpep fusion are connected into pMD19-T simple carriers successively, Construct recombinant plasmid pMD19-T-COE-Col, pMD19-T-COE-DCpep and pMD19-T-COE-Col-DCpep.Utilize Restriction endonuclease Sac I and Apa I carries out double digestion to above-mentioned plasmid and Expression Plasmid in LAB pPG-T7g10-PPT Processing, target gene fragment is purified through DNA plastic recovery kits.By COE-Co1, COE-DCpep, COE-Col- of recovery purifying DCpep genetic fragments are connected with expression vector pPG-T7g10-PPT purpose fragments respectively, structure recombinant expression plasmid pPG- T7g10-PPT-COE-Col, pPG-T7g10-PPT-COE-DCpep and pPG-T7g10-PPT-COE-Col-DCpep, letter Claim pPG-PPT-COE-Col, pPG-PPT-COE-DCpep and pPG-PPT-COE-Co1-DCpep, Vector map such as Figure 10 b- Shown in d.

Structure does not contain the recombinant expression plasmid of targeting peptides at the same time, is named as pPG-PPT-COE, Vector map such as Figure 10 a It is shown.

3rd, the structure of the pig source lactobacillus reuteri of PEDV COE albumen is expressed

The competent cell of pig source lactobacillus reuteri LJ-15 is prepared, through electric transformation technology by the recombinant plasmid of structure PPG-PPT-COE-Co1-DCpep and pPG-PPT-COE, pPG-PPT-COE-Col, pPG-PPT-COE-DCpep are transferred to pig source Lactobacillus reuteri LJ-15 competent cells, recombinant lactic acid bacteria is screened in the Selective agar medium tablet containing chlorampenicol resistant Transformant, positive restructuring lactic acid bacteria are respectively designated as pPG-COE-Col-DCpep/LJ-15, pPG-COE/LJ-15, pPG-COE- Col/LJ-15,pPG-COE-DCpep/LJ-15.Positive recombinant plasmid digestion and PCR qualification results are shown in Figure 11.

4. the expression and identification of albumen

4.1Western blot qualification results

Static gas wave refrigerator restructuring lactobacillus reuteri pPG-COE-Col-DCpep/LJ-15, pPG-COE/LJ-15, pPG- COE-Col/LJ-15 and pPG-COE-DCpep/LJ-15, is collected by centrifugation thalline, at 2 × SDS cellular lysate liquid after cultivating 16h The use of the anti-COE protein antibodies in mouse source is primary antibody after reason, using the expression of Western blot method testing goal albumen, detection Shown in the result is shown in Figure 12, destination protein is expressed.

The laser co-focusing detection of 4.2 destination proteins positioning

The cellular localization of restructuring lactobacillus reuteri express express target protein, detection knot are detected using laser confocal microscope Fruit as shown in Figure 13, recombinates lactobacillus reuteri pPG-COE/LJ-15, pPG-COE-Col-DCpep/LJ-15, pPG-COE- There is obvious yellow-green fluorescence in the phage surface of Col/LJ-15 and pPG-COE-DCpep/LJ-15, and control group pPG/ LJ-15 does not have found green fluorescence, the results showed that destination protein is expressed and showed in phage surface.

5. recombinate lactobacillus reuteri Evaluation of Immunogenicity

The present invention have rated the restructuring lactobacillus reuteri of structure using 4-6 week old BALB/c mouse as animal model system Immunogenicity.Immune programme is:Gavage approach inoculation recombinant lactic acid bacteria pPG-COE/LJ-15, pPG-COE-Col/ by oral administration LJ-15, pPG-COE-DCpep/LJ-15 and pPG-COE-Col-DCpep/LJ-15,200 μ L concentration of inoculation are every mouse every time 109The bacterium amount of CFU/mL, continuous immunity three days, after being immunized 1 time, 2 weeks daily with identical dosage booster immunization once.With oral PBS is inoculated with as control, using ELISA method detection immunized mice excrement, outer genital tract, in intestinal mucosa sIgA antibody levels and IgG antibody is horizontal in serum.Concrete outcome is as follows:

The 5.1 horizontal testing results of serum antibody IgG

0d, 10d, 17d, 27d, 34d and 41d prepare immune mouse serum after just exempting from, and serum is detected by ELISA IgG antibody is horizontal, the result is shown in Figure 14.Testing result is shown, can detect within the 10th day after first immunisation restructuring lactobacillus reuteri Go out IgG antibody, antibody level is gradually increasing, and reaches peak value within the 34th day after exempt from first, with control group (pPG/LJ-15 groups and PBS groups) compared to difference extremely significantly (p < 0.01), control group has no difference in immune front and rear IgG antibody level.In addition, targeting is repaiied Decorations restructuring lactobacillus reuteri pPG-COE-Col/LJ-15, pPG-COE-DCpep/LJ-15 and pPG-COE-Col-DCpep/ LJ-15 immune groups are exempted to can induce body generation after two weeks significantly in non-targeted modification restructuring lactobacillus reuteri pPG- in head The antibody level (p < 0.05) of COE/LJ-15 immune groups.Double targeting modification restructuring lactobacillus reuteri pPG-COE-Col- The antibody level that DCpep/LJ-15 induction bodies produce is higher than single targeting modification group.

5.2 mucous membrane sIgA antibody test results

In immunized mice excrement shown in sIgA testing results as Figure 15 a, respectively at just exempt from after the 0th, 4,6,8,10,12,14, 16th, 20,22,24,26,28,30,32,34,36d takes stool in mice, and it is horizontal to detect sIgA in excrement by ELISA, non-targeted SIgA antibody level of the modification restructuring lactobacillus reuteri pPG-COE/LJ-15 immune groups in immune rear 6d immunized mice excrement Significantly raised, peaking during 10d, begins to decline, after secondary immunity, antibody level quickly raises, and reaches in 26d afterwards Peak value, and antibody level is significantly higher than head and exempts from rear peak value.Targeting modification recombinant bacterium pPG-COE-Col/LJ-15, pPG-COE- DCpep/LJ-15 and pPG-COE-Col-DCpep/LJ-15 immune groups 4d antibody levels after immune start to raise, to the Peaking during 10d, and the antibody level that produces of targeting modification restructuring lactobacillus reuteri immune group induction be significantly higher than it is non-targeted Modification restructuring lactobacillus reuteri immune group (p < 0.05).Double targeting modification restructuring lactobacillus reuteri immune group induction bodies The antibody level of generation is higher than single targeting modification and recombinates lactobacillus reuteri group, but the not notable (p of difference>0.05).

The outer reproduction mucous membrane sIgA antibody levels testing result of immunized mice is as shown in Figure 15 b, oral immunity restructuring Luo Yishi Lactobacillus pPG-COE/LJ-15, pPG-COE-Col/LJ-15, pPG-COE-DCpep/LJ-15 and pPG-COE-Col-DCpep/ LJ-15 groups mouse 10d after immune can detect sIgA antibody outside immunized mice in genital tract, the difference compared with control group Extremely significantly (p<0.01), and 34d reaches peak value after head exempts from.Immune group comparison among groups is the results show that just exempt from rear antibody level Gradually rise, just exempt from before rear 10d between group antibody level difference not significantly (p>0.05);17d after just exempting from, targeting modification restructuring Bacterium pPG-COE-Col-DCpep/LJ-15, pPG-COE-Col/LJ-15 and pPG-COE-DCpep/LJ-15 immune group antibody water It is flat to be significantly higher than non-targeted modification recombinant bacterium pPG-COE/LJ-15 immune groups (p<0.05);Double targeting modification recombinant bacterium immune groups The antibody level that body produces is induced to be higher than single targeting modification recombinant bacterium immune group, but the not notable (p of difference>0.05);Empty carrier The sIgA antibody levels of bacterium pPG/LJ-15 control groups and PBS control group mouse in its immune front and rear outer genital tract have no difference.

Immunized mice intestinal mucosa sIgA antibody levels testing result is as shown in Figure 15 c, and each group is after initial immunity 40d gathers mouse intestines mucus sample detection sIgA antibody levels, and compared with before being immunized, control group sIgA antibody levels have no difference (p >0.05), immune group antibody level difference extremely significantly (p<0.01), double targeting modification recombinant bacterium immune group antibody levels are higher than Single targeting modification recombinant bacterium immune group.

5.3 spleen lymphocyte proliferation results

Shown in the result is shown in Figure 16, non-targeted modification recombinant bacterium pPG-COE/LJ-15 immune groups are in 0.5 μ g/mL and 5 μ g/mL Under antigen concentration stimulates, mouse lymphocyte propagation stimulus index and PBS groups and the thorn of empty carrier bacterium control group under the same terms Sharp index is compared, difference extremely significantly (p<0.01);Targeting modification restructuring lactobacillus reuteri pPG-COE-Col/LJ-15, pPG- COE-DCpep/LJ-15 and pPG-COE-Col-DCpep/LJ-15 immune groups are significantly in non-targeted recombinant bacterium immune group (p< 0.05);Double targeting modifications recombinate lactobacillus reuteri pPG-COE-Col-DCpep/LJ-15 immune groups in 0.5 μ g/mL and 5 μ g/ Under mL antigen concentrations stimulate, mouse lymphocyte propagation stimulus index is significantly higher than single targeting modification restructuring lactobacillus reuteri PPG-COE-Col/LJ-15 groups and pPG-COE-DCpep/LJ-15 groups (p<0.05).

5.4 cytokines measurement results

40d after head exempts from, it is sterile to take mouse spleen, splenocyte suspension is made, adjustment cell concentration is 5 × 106A/mL, is adopted Stimulated with two antigen concentrations, cell factor IFN-γ in cells and supernatant is detected using BioSource ELISA kits Horizontal with IL-4, as shown in Figure 17, each immune group cell factor IL-4 (Figure 17 b), IFN-γ (Figure 17 a) are horizontal for testing result It is significantly higher than control group (p<0.05), wherein pPG-COE-Col-DCpep/LJ-15, pPG-COE-Col/LJ-15, pPG-COE- The value of DCpep/LJ-15, pPG-COE/LJ-15 immune group Th2/Th1 are respectively 1.24,1.18,1.08,1.053, it is seen that targeting The immune response of peptide modified antigen induction is based on Th2 type cell immune responses.

To sum up, the present invention is using PEDV neutralizing epitopes area COE as immunogene, and merges M cells and DC cell-targeting peptides, profit It is to transmit carrier with constitutive expression carrier pPG-PPT and pig source lactobacillus reuteri, successfully constructs expression COE fusion targetings The genetic engineering lactobacillus reuteri of peptide, oral immunity confirm that the restructuring lactobacillus reuteri can effectively induce local mucous membrane Immune response, and the humoral immune response of body generation system can be stimulated, it is shown that good immune effect.

Sequence table

<110>Northeast Agricultural University

<120>A kind of strain of gene engineered subunit oral vaccine and its construction method and use for being used to prevent pig epidemic diarrhea On the way

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<170> PatentIn version 3.3

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gagctcgatt ataaggatga cgatgacaag aagcttgtta ctttgccatc gtttaatgat 60

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attgcatctg acactactat caatgggttt acttctttct gtgttgacac tagacaattt 180

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Claims (10)

  1. A kind of 1. gene engineered subunit oral vaccine strain for being used to prevent pig epidemic diarrhea, it is characterised in that the epidemic disease Seedling strain transmits live vector using the lactobacillus reuteri for being located away from chitling road as vaccine antigen, wherein containing constitutive expression PEDV In S protein and epitopic core epitope area COE and targeting M cells and DC cells targeting peptide fusion protein recombinant expression carrier, The order of connection of fusion protein is:The targeting peptides of targeting peptides-targeting DC cells of COE- targeting M cells, COE sequences, targeting M are thin It is attached between the targeting peptides of born of the same parents and the targeting peptides of targeting DC cells with flexible sequence.
  2. 2. gene engineered subunit mouth vaccine strain as claimed in claim 1, it is characterised in that the described chitling road that is located away from Lactobacillus reuteri is named as lactobacillus reuteri LJ-15, is deposited in China typical culture collection center, and address is big in Wuhan Learn, its culture presevation numbering is CCTCC NO.M 2017570.
  3. 3. gene engineered subunit mouth vaccine strain as claimed in claim 1, it is characterised in that the PEDV S proteins neutralize Contain Flag sequence labels, the nucleotide of the COE genes containing Flag sequence labels in 5 ' ends of epitopic core epitope area COE genes Sequence as shown in SEQ ID NO.5, coding targeting M cells and DC cells targeting peptides respectively as shown in SEQ ID NO.11 and Shown in SEQ ID NO.12.
  4. 4. gene engineered subunit mouth vaccine strain as claimed in claim 1, it is characterised in that the recombinant expression carrier leads to Following steps are crossed to be prepared:
    (1) made using the geneome RNA and reverse transcription of isolation kit method extraction Porcine epidemic diarrhea virus into cDNA with cDNA For template, COE genes are obtained through PCR;Preferably, Flag sequence labels are contained at the 5 ' ends for expanding the COE genes of acquisition, contain The nucleotide sequence of the COE genes of Flag sequence labels is as shown in SEQ ID NO.5;
    (2) the COE fragments obtained using step (1) compile M cell-targetings peptide (Co1) and DC cell-targetings peptide (DCpep) as template Code gene is building up to the downstream of COE genes by fusion DNA vaccine method, fusion COE-Co1-DCpep is obtained, then by COE- Co1-DCpep fusions are connected into pMD19-T simple carriers, COE genes, M cell-targetings DNA encoding peptide, DC cells Connected between targeting DNA encoding peptide by flexible sequence, obtained recombinant vector is named as pMD19-T-COE-Co1-DCpep;
    Preferably, upstream and downstream primer is respectively shown in SEQ ID NO.6 and SEQ ID NO.10 used by fusion DNA vaccine;
    (3) structure of lactic acid bacteria constitutive expression plasmid pPG-T7g10-PPT
    Transformed on the basis of plasmid pPG612, double digestion is carried out to plasmid pPG612 using XbaI and XhoI, with HCE- T7g10-PgsA anchor-MCS-rrnBT1T2 sequences connect, and construct constitutive and secretive expression lactic acid bacteria vector, are named as PPG-T7g10-PPT, wherein HCE are bacillus constitutive and secretive expression promoter, its nucleotide sequence such as SEQ ID NO.1 It is shown;T7g10 is translational enhancer sequence, its nucleotide sequence is as shown in SEQ ID NO.2, the nucleotide of PgsA anchor Sequence is as shown in SEQ ID NO.3;MCS is polyclone enzyme enzyme site;RrnBT1T2 is Escherichia coli transcription terminator sequences, its Nucleotide sequence is as shown in SEQ ID NO.4;
    (4) using restriction endonuclease to plasmid pMD19-T-COE-Co1-DCpep and Expression Plasmid in LAB pPG- T7g10-PPT carries out double digestion processing, target gene fragment is purified through DNA plastic recovery kits, by the pMD19- of recovery purifying T-COE-Co1-DCpep genetic fragments are connected with expression vector pPG-T7g10-PPT purpose fragments, build recombinant expression plasmid, In the as described constitutive expression PEDV S proteins and epitopic core epitope area COE and targeting M cells and DC cells targeting The recombinant expression carrier of peptide fusion protein, is named as pPG-PPT-COE-Col-DCpep.
  5. 5. genetic engineering lactic acid bacteria oral vaccine strain as claimed in claim 1, it is characterised in that the constitutive expression In PEDV S proteins and epitopic core epitope area COE and targeting M cells and DC cells targeting peptide fusion protein recombination expression Carrier is transferred to by electric method for transformation in the competent cell of pig source lactobacillus reuteri.
  6. 6. a kind of method for building claim 1-5 any one of them gene engineered subunit oral vaccine strains, its feature exist In comprising the following steps:
    (1) made using the geneome RNA and reverse transcription of isolation kit method extraction Porcine epidemic diarrhea virus into cDNA with cDNA For template, COE genes are obtained through PCR;Preferably, Flag sequence labels are contained at the 5 ' ends for expanding the COE genes of acquisition, contain The nucleotide sequence of the COE genes of Flag sequence labels is as shown in SEQ ID NO.5;
    (2) the COE fragments obtained using step (1) compile M cell-targetings peptide (Co1) and DC cell-targetings peptide (DCpep) as template Code gene is building up to the downstream of COE genes by fusion DNA vaccine method, fusion COE-Co1-DCpep is obtained, then by COE- Co1-DCpep fusions are connected into pMD19-T simple carriers, COE genes, M cell-targetings DNA encoding peptide, DC cells Connected between targeting DNA encoding peptide by flexible sequence, obtained recombinant vector is named as pMD19-T-COE-Co1-DCpep;
    Preferably, upstream and downstream primer is respectively shown in SEQ ID NO.6 and SEQ ID NO.10 used by fusion DNA vaccine;
    (3) structure of lactic acid bacteria constitutive expression plasmid pPG-T7g10-PPT
    Transformed on the basis of plasmid pPG612, double digestion is carried out to plasmid pPG612 using XbaI and XhoI, with HCE- T7g10-PgsA anchor-MCS-rrnBT1T2 sequences connect, and construct constitutive and secretive expression lactic acid bacteria vector, are named as PPG-T7g10-PPT, wherein HCE are bacillus constitutive and secretive expression promoter, its nucleotide sequence such as SEQ ID NO.1 It is shown;T7g10 is translational enhancer sequence, its nucleotide sequence is as shown in SEQ ID NO.2, the nucleotide of PgsA anchor Sequence is as shown in SEQ ID NO.3;MCS is polyclone enzyme enzyme site;RrnBT1T2 is Escherichia coli transcription terminator sequences, its Nucleotide sequence is as shown in SEQ ID NO.4;
    (4) using restriction endonuclease to plasmid pMD19-T-COE-Co1-DCpep and Expression Plasmid in LAB pPG- T7g10-PPT carries out double digestion processing, target gene fragment is purified through DNA plastic recovery kits, by the pMD19- of recovery purifying T-COE-Co1-DCpep genetic fragments are connected with expression vector pPG-T7g10-PPT purpose fragments, build recombinant expression plasmid, In the as described constitutive expression PEDV S proteins and epitopic core epitope area COE and targeting M cells and DC cells targeting The recombinant expression carrier of peptide fusion protein, is named as pPG-PPT-COE-Col-DCpep;
    (5) preparative separation is in the competent cell of the lactobacillus reuteri in chitling road, through electric transformation technology by the restructuring matter of structure Grain pPG-PPT-COE-Col-DCpep is transferred to lactobacillus reuteri competent cell, screens recombinant lactic acid bacteria transformant, obtains sun Property recombinant lactic acid bacteria, be as used to prevent the gene engineered subunit oral vaccine strain of pig epidemic diarrhea.
  7. 7. method as claimed in claim 6, it is characterised in that the lactobacillus reuteri for being located away from chitling road is named as Lactobacillus reuteri LJ-15, is deposited in China typical culture collection center, address is in Wuhan University, its culture presevation numbering For CCTCC NO.M 2017570.
  8. 8. claim 1-5 any one of them gene engineered subunit oral vaccine strain is preparing prevention or Prevention pig stream Purposes in the medicine of row diarrhea.
  9. 9. a kind of gene engineered subunit oral vaccine for preventing pig epidemic diarrhea, it is characterised in that contain claim 1-5 Any one of them gene engineered subunit oral vaccine strain.
  10. 10. one plant of lactobacillus reuteri being located away from chitling road, is named as lactobacillus reuteri LJ-15, Chinese allusion quotation is deposited in Type culture collection, in Wuhan University, its culture presevation numbering is CCTCC NO.M 2017570 for address, and the preservation time is On October 9th, 2017.
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