CN107158371A - A kind of gene engineered subunit bigeminy oral vaccine - Google Patents

Abstract

The present invention provides a kind of gene engineered subunit bigeminy oral vaccine, it is Porcine epidemic diarrhea virus and transmissible gastro-enteritis virus gene engineered subunit bigeminy oral vaccine, is that use can recombinantly express Porcine epidemic diarrhea virus S protein and prepared by the Lactococcus lactis of pig infectious gastroenteritis virus S LN albumen；Wherein Porcine epidemic diarrhea virus S protein, its amino acid sequence is SEQ ID NO:1；The nucleotides sequence of its encoding gene is classified as SEQ ID NO:2；Described SLN albumen, is to connect A, D antigen site gene of transmissible gastro-enteritis virus and N321 antigen site genes.Inactivated vaccine prepared by the present invention is safe and reliable; energy offer is effectively homologous to attack malicious protection; stronger immunity can be produced after immune; the morbidity and mortality of inoculation swinery are significantly reduced; the prevalence of pig epidemic diarrhea and transmissible gastroenteritis of swine can effectively be prevented and propagated; the economic loss that this disease is caused to pig industry is reduced, is had broad application prospects.

Description

A kind of gene engineered subunit bigeminy oral vaccine

Technical field

The invention belongs to vaccine preparation technology field, and in particular to a kind of gene engineered subunit bigeminy oral vaccine.

Background technology

Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) is by many viraleses of Buddhist nun (Nidovirales) Porcine epidemic diarrhea virus (the Porcine epidemic of coronaviridae (Coronaviridae) Diarrhea virus, PEDV) it is caused to suffer from diarrhoea, vomit, be dehydrated and to the high fatal rate of suckling pig as the one of principal character Kind of winter multiple high degree in contact enteric infectious disease.The pig of all ages and classes and different cultivars is susceptible, suckling pig, feeder pig Or the incidence of disease of growing and fattening pigs, up to 100%, the especially harm to suckling pig is the most serious, case fatality rate average out to 50%, provisions Pig industry brings serious harm.

Transmissible gastroenteritis of swine (Transmissible Gastroenteritis, TGE) is by many mesh of Buddhist nun, coronavirus One kind caused by the transmissible gastro-enteritis virus (Transmissible Gastroenteritis Virus, TGEV) of section with The high degree in contact enteric infectious disease that diarrhoea, vomiting, dehydration and the high fatal rate of piglet are characterized.The disease has highly infectious, This disease is once broken out on pig farm, and causes piglet intimate 100% within 2 week old of birth dead, loses extremely serious, gives pig industry band Carry out high risks.

Pig epidemic diarrhea and transmissible gastroenteritis of swine are often concurrent in piglet in cold season, cause grice diarrhoea, take off Water, death, infectiousness are strong, it is difficult to cure, therefore develop both viral bigeminy vaccines to prevention diarrhea of pigs and gastroenteritis disease Outburst, reduce the pig death rate, reduce pig farm economic loss be extremely important.

Vaccine immunization is to prevent both sick major measures, but currently available vaccines, existing vaccines generally requires injection, conventional Vaccinate can produce stress and vaccine the problem of absorb.Research was found in the past, and the secreting type produced by Intestinal Mucosal Immunization resists Body (sIgA) is resistant to the potent antibodies of PEDV and TGEV infection, and such as IgG, IgM immune protective effect are unsatisfactory.Therefore select A kind of bacterium that can be settled down in intestinal mucosa is selected as development of the Host Strains for pig epidemic diarrhea and transmissible gastroenteritis of swine vaccine It is extremely important.

Lactococcus lactis is a kind of easily by the normal in bacterium of mucosal absorption, because of its for a long time should in food industry every field With being proved without pathogenic, to be acknowledged as safe level microbe.Using some Lactococcus lactis in intestines and stomach, uropoiesis, life Grow in system or mucosa adhesion survival and it is not pathogenic the features such as, carried out extensively viable bacteria it is oral after in vivo field planting and epidemic disease The research of seedling.

At present, pig epidemic diarrhea and transmissible gastroenteritis of swine incidence are high, more sophisticated the reason for morbidity, and Specific treatment medicine is had no, general symptomatic treatment effect is not good, so preferably vaccine inoculation prevents from breaking out.However, business on the market The vaccine of product is mainly inactivated vaccine and Attenuate vaccine.Although inactivated vaccine safety and stability, heavy dose of inoculation or application concentration are needed Antigen；Duration of immunity is short, need to often strengthen inoculation；Producing complete immunity needs 2 weeks, is unfavorable for urgent immunization campaign and reduction vaccine Expense and inactivated vaccine can only can not obtain sIgA by injecting immune, piglet, protecting effect is not good.And Attenuate vaccine is due to existing Cost is high, easy reversion, have the defects such as latent infection danger, it is difficult to popularization and application in practice.Traditional vaccine is popular in preventing and treating pig Property diarrhoea and transmissible gastroenteritis of swine in the problem of expose more and more, complete protection can not be provided, developed a kind of new Reliable vaccine is extremely urgent.In the immunologic mechanism of pig epidemic diarrhea and transmissible gastroenteritis of swine, mucosa-immune has very Important effect.The inactivated vaccine of traditional non-bowel input does not produce sIgA antibody, and cell mediated immune response is weak and ties up Hold the time short.The advantage that oral immunity is protruded is can effectively to stimulate enteron aisle local immunity cell to produce secretory IgA, and this is especially Be adapted to intestinal mucosa infectious disease, and avoid caused by regular injection stress and vaccine absorb problem.

The content of the invention

The present invention provides a kind of gene engineered subunit bigeminy oral vaccine, i.e., a kind of Porcine epidemic diarrhea virus and pig pass Metachromia marcy agent gene engineered subunit bigeminy oral vaccine, the advantage that oral immunity is protruded is can effectively to stimulate enteron aisle Local immunity cell produces secretory IgA, and this is specially adapted to intestinal mucosa infectious disease, and avoids caused by regular injection Stress and vaccine absorb problem, solve that vaccine immunity irritability is strong, immunogenicity is not strong, virose problem.

Porcine epidemic diarrhea virus and transmissible gastro-enteritis virus gene engineered subunit bigeminy provided by the present invention Oral vaccine, is that use can recombinantly express Porcine epidemic diarrhea virus S protein and pig infectious gastroenteritis virus S LN albumen Prepared by Lactococcus lactis；

Wherein Porcine epidemic diarrhea virus S protein, its amino acid sequence is SEQ ID NO:1；The nucleosides of its encoding gene Acid sequence is SEQ ID NO:2；

Described SLN albumen, is by A, D antigen site gene of transmissible gastro-enteritis virus and N321 antigen site bases Because what is connected,

Preferably, the order of connection of described SLN protein coding genes is D antigen sites gene, N321 antigen sites Gene, Staphylococal Protein A locus gene；

Described D antigen sites gene, N321 antigen sites gene, Staphylococal Protein A locus gene are connected by Linker Come,

Recorded as one kind of embodiment is specific, the amino acid sequence of SLN albumen is SEQ ID NO:3；Its encoding gene For SEQ ID NO:4.

Inactivated vaccine prepared by the present invention is safe and reliable, can provide it is effectively homologous attack malicious protection, it is immune after can produce Stronger immunity, the morbidity and mortality of inoculation swinery are significantly reduced, and can effectively prevent pig epidemic diarrhea and pig to pass The prevalence of metachromia gastroenteritis and propagation, reduce the economic loss that this disease is caused to pig industry, have broad application prospects.

Brief description of the drawings

Fig. 1：The PCR qualification result figures of two kinds of recombinant bacteriums；

Fig. 2：The double digestion qualification result figure of two kinds of recombinant bacteriums；

Fig. 3：The western-blotting result figures of two kinds of recombinant bacteriums.

Embodiment

The present invention is described in detail with reference to embodiment.

Embodiment 1：The preparation of oral vaccine

First, synthesis and amplifying target genes

According to totivirus RNA sequence, after confrontation point in situ is optimized, from two flexible Linker ((GGGGS) 3) TGEV A, D antigen site gene and N321 antigen site genes are connected and (be sequentially：D antigen sites gene- Linker-N321 antigen site gene-Linker-A antigen sites gene), it is SLN the sequence designations, and be sent to biological public affairs Department's synthesis.

Design specific primer extracts RNA from the PEDV variants obtained respectively, and PEDV S1 bases are expanded after reverse transcription Cause and TGEV SLN genes simultaneously connect pMD18-T carriers, and conversion chooses positive monoclonal into DH5 α impressions, bacterium solution PCR identifications, Extract plasmid enzyme restriction identification and sequencing identification is correct.

1st, expression vector establishment

To identify correct S1 genes and SLN genes respectively with pMG36e expression vector restriction enzyme Xba I and Hind III double digestions, and carry out glue reclaim purifying.By both 16 DEG C of connections overnight, culture 16 in e. coli jm109 is transformed into small When.It is correct that picking monoclonal bacterium colony enters performing PCR identification, the identification of plasmid double digestion and sequencing identification.

3rd, Recombinant Lactococcus lactis is built

After identifying that correct two kinds of positive plasmids are softly mixed with competent cell MG1363 respectively, place on ice 5min, is transferred in 2mm precooling electricity conversion cup, rapid electric shock, shock parameters are voltage 2.5kV, the electric shock time is 5ms, The SGM17 recovery medias of 900 μ l ice precoolings are added after electric shock, mixes, bacterium solution is transferred in 1.5ml centrifuge tubes, put on ice 10min, 28 DEG C of recovery culture 2h are put, take appropriate bacterium solution to be coated on the GM17 agar mediums containing 5 μ g/ml erythromycin, 28 DEG C Anaerobic culturel 2-3d.In picking single bacterium colony on flat board, the GM17 Liquid Cultures containing 5 μ g/ml erythromycin are inoculated in respectively In base, after 28 DEG C of Anaerobic culturels are stayed overnight, extract plasmid respectively from Lactococcus lactis, and PCR identifications are carried out to plasmid respectively, it is double Digestion identification and sequencing identification are correct.Two kinds of Recombinant Lactococcus lactis kinds are respectively designated as：MG1363/pMG36e-S1 and MG1363/pMG36e-SLN。

4th, the induced expression of recombinant protein

Take 50 μ L two kinds of strains to be inoculated in 5mL respectively and contain newly matching somebody with somebody in GM17 fluid nutrient mediums for erythromycin, in 28 DEG C Incubated overnight, when reaching 1.0 to OD600, adds nisin to final concentration of 10ng/ml, continues to cultivate, in termination after 10 hours Induction.

Then SDS-PAGE Protein Detection experiments are carried out：As a result show expressed destination protein be about 86KD and 15KD；Western identification recombinant proteins have many anti-abilities reacted with anti-PED and anti-TGE, it was demonstrated that recombinant bacterium is expressed Foreign protein there is good reactionogenicity.

5th, recombinant bacterium is frozen

100 microlitres are taken respectively from the nutrient solution of two kinds of recombinant protein bacterial strains of expression, and 50 containing erythromycin are inoculated in respectively In milliliter GM17 culture mediums.When 28 DEG C of non-oscillating cultures are to OD600=1.0, two kinds of bacterium solutions are mixed, and contain 3% with 100 milliliters 10% skim milk mixed liquor of sucrose is well mixed, and is packed as 2 milliliters and is often managed, and is freezed.

6th, the stability test of bacterium is frozen

(1) fungi preservation temperature：By the lyophilized culture of two kinds of strain mixtures respectively at -20 DEG C, 4 DEG C and 25 DEG C guarantors Deposit 3 months, then respectively take 5 to be recovered with the above method and count, influence of the relatively more different storage temperatures to bacterial number.As a result show Show saved as with -20 DEG C it is best.

Influence of the different storage temperatures of table 1 to the trimestral mixed reorganization strain bacterium number of preservation

(2) the fungi preservation time：The lyophilized culture of two kinds of strain mixtures is frozen 3,6,12, respectively take 5 within 18 months, Recovery is counted in the same way, influence of the relatively more different holding times to bacterial number.As a result show strain in -20 DEG C of conditions Lower to preserve the 18 months influence difference to strain quantity not notable by lyophilized, shows to freeze strain very stable.

Influence of the different holding times of table 2 to mixed reorganization strain bacterium number

(3) PCR is identified：Picking freezes 3,6,12, after being recovered on MRS solid mediums of the strain of 18 months, take MRS Single bacterium colony on solid plate, plus 100 μ l sterilizing distilled waters, after being heated 5 minutes in boiling water bath, with the PCR for providing two genes for oneself Primer amplification S1 and SLN genes show two positive purpose bands, show to freeze bacterium stabilization.

7th, the preparation of vaccine

The bacterium powder freezed is taken, 1ml physiological saline is added, is well mixed, it is directly oral.

Embodiment 2：

(1) potency test of oral vaccine

1 material

1.1 medicine

Restructuring oral vaccine prepared by Qingdao Agricultural University laboratory.

1.2 test sites experimental animal in one's power

Test site is Qingdao of Shandong province Chengyang District Hai Wei pig farms, and experimental animal is the piglet of 20 first 30 ages in days.

2 test methods

2.1 immune vaccines simultaneously detect antibody

The piglet of 20 first 30 ages in days is equally divided into control group and immune group.Every piglet of immune group gavages the oral epidemic disease of combination 1 part of seedling, control group gavages isodose sterile saline.7d, 14d, 21d, 28d couple after immune preceding and immune Every pig is taken a blood sample, and surveys antibodies in blood content.

Expression and Fluctuation of the recombinant bacterium in pig body after 2.2 vaccine immunities

Oral immunity group is coated on 5 μ g/ after gathering rectum cotton swab, sample treatment respectively immune 7 days afterwards, 14 days, 28 days The MRS flat boards of ml erythromycin, through 28 DEG C of culture 24h, every group of random 10 bacterium colonies of picking are carried after expanding culture with alkaline lysis Plasmid is taken, and carries out restriction analysis, digestion products observe digestion post-fragment size, checking excrement inspection bacterium through agarose gel electrophoresis Whether it is recombinant bacterium MG1363 (pMG36e).

3 results and analysis

Antibody level result after 3.1 oral immunities

Control group and immune group are all higher to S1 and SLN sIgA maternal antibodies level.Immune group head exempted from after 14 days, due to The decay of maternal antibody and the possible partial neutralisation of vaccine, antibody titer has declined, but still is significantly higher than control group；Exempt from After epidemic disease 28 days, control group continues to decline for the antibody level of above-mentioned antigen, and the antibody level of immune group then continues rise, extremely 60 days after immune, remain to detect higher level antibody.It is as shown in the table：

S1 and SLN antigentic specificity sIgA antibody levels are directed in Swine serum

Expression and Fluctuation result of 3.2 recombinant bacteriums in pig body

Recombinant bacterium oral immunity group is 14 days after immune, collection rectum cotton swab is detected respectively within 28 days, is not detected by Recombinant plasmid；Recombinant bacterium oil emulsion adjuvant emulsion seedling intramuscular injection group also fails to detect recombinant plasmid in the above-mentioned period.According to antibody Testing result, recombinant bacterium oil-emulsion inactivated vaccine stimulates body to produce anti-mainly by the slow release of its immune protective antigen Body；Compared with control group, it is immune after 14,28 days antibody levels significantly raise；And recombinant bacterium viable bacteria oral immunity group is in above-mentioned rank The level that section produces antibody is relatively low, it is also oral with viable bacteria after in vivo time-to-live shorter testing result match.

4 conclusions

It can be seen that recombinant bacterium has good immunogenicity, energy by the tracking of the antibody level produced to immune rear pig body Enough body is stimulated to produce the specific sIgA antibody for PEDV S1 and TGEV SLN, and can be long lasting for higher level.Knot Fruit shows that the vaccine immunity effect of this laboratory research is good.

Embodiment 3：The safety testing of oral vaccine

1 material

1.1 medicine

Restructuring oral vaccine prepared by Qingdao Agricultural University laboratory.

1.2 test sites experimental animal in one's power

Test site is Qingdao of Shandong province Chengyang District Hai Wei pig farms, and experimental animal is the piglet of 20 first 30 ages in days.

2 test methods

2.1st, single-dose safety is tested

30 age in days piglets 80, are divided into 2 groups, respectively test group and control group, every group of 40 pigs.Test group every is gavaged Recombinant lactic acid bacteria 1ml, control group such as gavages at the dose aseptic physiological saline, after gavaging once, the continuous 14 days spiritual shapes of observation piglet State, whether there is the ill symptomses such as diarrhoea, feed intake decline and occurs, and the production performances such as survival rate, weightening, efficiency of feed utilization are carried out Statistics.

2.2nd, single dose repeats safety testing

30 age in days piglets 80, are divided into 2 groups, respectively test group and saline control group, every group of 40 pigs.Test group Every gavages recombinant lactic acid bacteria 1ml, the dose aseptic physiological saline such as control group is gavaged, and continuously gavages seven days, continuous 14 after medication Its observation piglet state of mind, whether there is the ill symptomses such as diarrhoea, feed intake decline and occurs, and to survival rate, weightening, feed conversion The production performances such as rate are counted.

2.3rd, overdose safety testing

30 age in days piglets 80, are divided into 2 groups, respectively test group and saline control group, every group of 40 pigs.Test group Every gavages recombinant lactic acid bacteria 2ml, the dose aseptic physiological saline such as control group is gavaged, and gavages after 1 time, continuous 14 days observation piglets The state of mind, whether there is the ill symptomses such as diarrhoea, feed intake decline and occurs, and to productivitys such as survival rate, weightening, efficiencies of feed utilization It can be counted.

3rd, result of the test

The safety testing result that vaccine is used 30 age in days piglet single doses

The safety testing result that vaccine is reused to 30 age in days piglet single doses

The safety testing result that vaccine is used 30 age in days piglet overdoses

Each test group piglet is after vaccine is gavaged, and the reaction of continuous 14 days observation piglets, piglet declines without diarrhoea, feed intake Deng ill symptomses, the health survival of all experiment piglets, increased weight.After off-test it is random select 5 and cut open kill, observe cut open inspection Change, tissue and organ and the basic indifference of saline control group of each group piglet cut open inspection.

Gene engineered subunit oral vaccine prepared by the present invention is safe and reliable, effectively Intestinal Mucosal Immunization can be caused anti- Should, stronger immunity can be produced after being immunized, the morbidity and mortality of immune piglet are significantly reduced, and can effectively prevent pig Epidemic diarrhea disease and transmissible gastroenteritis of swine, reduce the economic loss that this disease is caused to pig industry, have before wide application Scape.

SEQUENCE LISTING

<110>Qingdao Agricultural University

<120>A kind of gene engineered subunit bigeminy oral vaccine

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Claims (6)

1. a kind of Porcine epidemic diarrhea virus and transmissible gastro-enteritis virus gene engineered subunit bigeminy oral vaccine, it is special Levy and be, described oral vaccine is that use can recombinantly express Porcine epidemic diarrhea virus S protein and transmissible gastroenteritis of swine Prepared by the Lactococcus lactis of viral SLN albumen；

Described Porcine epidemic diarrhea virus S protein, its amino acid sequence is SEQ ID NO:1；

Described SLN albumen, is by A, D antigen site gene of transmissible gastro-enteritis virus and N321 antigen sites gene company Pick up what is come.

2. oral vaccine as claimed in claim 1, it is characterised in that described SLN albumen, the order of connection of its encoding gene For D antigen sites gene, N321 antigen sites gene, Staphylococal Protein A locus gene.

3. oral vaccine as claimed in claim 1, it is characterised in that described D antigen sites gene, N321 antigen site bases Cause, Staphylococal Protein A locus gene are connected by Linker.

4. oral vaccine as claimed in claim 1 or 2, it is characterised in that the amino acid sequence of described SLN albumen is SEQ ID NO:3。

5. oral vaccine as claimed in claim 1, it is characterised in that described Porcine epidemic diarrhea virus S protein, it is encoded The nucleotides sequence of gene is classified as SEQ ID NO:2.

6. oral vaccine as claimed in claim 1 or 2, it is characterised in that described SLN albumen, its encoding gene is SEQ ID NO:4。