CN106520817A

CN106520817A - Preparation method and applications of porcine circovirus type II recombinant lactobacillus plantarum oral vaccine

Info

Publication number: CN106520817A
Application number: CN201610980068.XA
Authority: CN
Inventors: 赖强; 倪能能; 钟泽民; 华国良; 刘德辉
Original assignee: Guangzhou Welltech Biotechnology Co Ltd
Current assignee: Guangzhou Welltech Biotechnology Co Ltd
Priority date: 2016-11-08
Filing date: 2016-11-08
Publication date: 2017-03-22

Abstract

The invention provides a preparation method of porcine circovirus type II recombinant lactobacillus plantarum. The preparation method comprises following steps: separated Cap sequence is connected with expression vector pPSCPSP so as to construct expression plasmid pPSCPSP-Cap; expression plasmid pPSCPSP-Cap is inserted into lactobacillus plantarum LP-1 so as to obtain porcine circovirus recombinant lactobacillus plantarum pPSCPSP-Cap-LP-1; a cultured product of pPSCPSP-Cap-LP-1 can be applied directly as an oral vaccine to stimulate bodies to generate antibodies effectively, and reduce the morbidity of porcine circovirus type II obviously. Application of the oral vaccine is beneficial for avoiding of retarded growth, emaciation, sow reproduction disorder, dyspnea, occasionally induced jaundice, diarrhea, extensive pathological changes of visceral organs and skin caused by serious infection of porcine circovirus in livestock breeding, improving of pig herd immunity and health conditions, and increasing of the economic benefit of breeding.

Description

A kind of preparation method of porcine circovirus 2 type recombinant plant lactobacillus oral vaccine and Using

Technical field

The present invention relates to vaccine preparation technology field, more particularly, to a kind of porcine circovirus 2 type recombinant plant breast bar The preparation method and application of fungus oral vaccine.

Background technology

Pig circular ring virus（Porcine circovirus, PCV）Belong to PCV-II section（Circovridae）PCV-II Category（Circovirus）, serotype is PCV1 and PCV2, clinically causes the mainly PCV2 of disease.Circovirus infection is main Cause postweaning multisystemic asthenia syndrome（PMWS）, the disease is also closely related with other various syndromes, and such as pig is congenital Tremble（CT）, dermatitis nephrotic syndrome（PNDS）, respiratory diseases in pigs syndrome, the disease such as sow breeding difficulty and enteritis. PCV2 Jing are often viral with pig breeding and RDS（PRRSV）Or pig parvoviral（PPV）Disease is sent out or secondary sense Dye bacterium, makes ill pig aggravation, causes great economic loss to global pig industry.PCV2 full-length genomes 1766bp～1768bp, 3 reading frames of main code ORF1, ORF2 and ORF3, the capsid egg of wherein ORF2 coding viruses In vain, it is main structural proteins, and PCV2 major antigen albumen, antibody can be produced as antigenic stimulus body, hence it is evident that reduce The incidence of disease of pig.

From 1991, the disease is reported in Canada first, subsequently broken out in Asia, South and North America and Europe in succession.China In 2000, after reported first, there is the infection of PCV2, the disease infects the just pig industry for having given China in rising trend always Cause serious economic loss.Mainly by vaccinating, the vaccine on market is mainly totivirus for the sick prevention and control at present Inactivated vaccine and gene engineering vaccine.The main strain of inactivated vaccine is divided into：SH strains, LG strains, WH strains, ZJ/c strains；Inactivated vaccine is using process In repeatedly need to be inoculated with, the time produced required for immunoprotection is long, and cannot be used for urgent immunization campaign.The gene work of commercialization Cheng Miao is mainly the vaccine of the Bo Linge of import, and protected effect is notable but expensive.

Lactic acid bacteria is a kind of nonpathogenic Gram positive bacteria, is generally acknowledged probio, the stable state in maintenance intestines and stomach, The normal flora of regulating intestinal canal, suppresses the breeding of spoilage organisms, suppresses cholesterol absorption, reducing blood lipid, step-down, immunological regulation to resist swollen The aspects such as knurl, cancer prevention play an important role.In past more than 20 years, lactic acid bacteria is used as a kind of transmission prevention and treatment The carrier of molecule has caused the concern of researcher.As carrier：Lactic acid bacteria safety, no endotoxin express foreign protein Purifying is needed not move through, can be taken together with thalline.The capacity of foreign gene is big in addition, inducing cellular immune, produces a large amount of Cell factor, be preferable recombinant vaccine carrier, particular for oral vaccine.Furthermore, lactic acid bacteria can be deposited in enteron aisle Field planting living, after oral genetic engineering lactic acid bacteria, the albumen of expression constantly in enteron aisle can be produced and be played and act on accordingly, Therefore, lactic acid bacteria can be used as the pathogen recognition carrier of preferable food-grade.

Compared with vaccinating, oral vaccine has irreplaceable advantage：Mucosa-immune saving work, saving time, Animal stress is reduced, can enhancing effect and specificity when chronic disease is treated.Used as oral vaccine, lactic acid bacteria can be in the gastrointestinal tract Survival, is played a role by mucosa-immune, produces cellular immunity and humoral immunity.Therefore, oral vaccine is in the research and development of medicine Have broad application prospects.

The content of the invention

The technical problem solved required for of the invention is to overcome the defect for vaccinating presence, there is provided a kind of pig circular ring virus The preparation method of 2 type recombinant plant lactobacillus oral vaccines.

Second object of the present invention is to provide the structure of pig circular ring virus recombinant lactobacilli.

Third object of the present invention is to provide the oral vaccine of pig circular ring virus recombinant lactobacilli.

Fourth object of the present invention is to provide the preparation method of the oral vaccine of pig circular ring virus recombinant lactobacilli.

5th purpose of the present invention is to provide the application of the oral vaccine of pig circular ring virus recombinant lactobacilli.

The purpose of the present invention is achieved by following technical scheme：

A kind of preparation method of porcine circovirus 2 type recombinant plant lactobacillus, comprises the following steps：

S1. amplification is separated, the capsid protein Cap sequences of PCV2 strains, the Cap sequences such as SEQ ID NO is obtained：Shown in 8；

S2. Cap sequences are connected with expression vector pPSCPSP, construction expression plasmid pPSCPSP-Cap；

S3. expression plasmid pPSCPSP-Cap is proceeded in Lactobacillus plantarum LP-1, that is, obtains pig circular ring virus recombinant plant breast Bacillus pPSCPSP-Cap-LP-1.

Preferably, the PCV2 strains directly can be obtained from the lymph node of the pathological material of disease of pig farm collection.

The PCV strains being separated to from pathological material of disease in this research belong to PCV2 type strains, are clinically to cause pig circular ring virus disease The strain of disease, this research also carries out sequencing identification to the strain, with NCBI gene pools in carry out gene BLAST and compare, and absolutely greatly The homology of most PCV2 types strains is up to 99%.Expression Cap protein is expanded with the strain there is clinical meaning.

The sequence of PCV2 strains such as SEQ ID NO in this research：7.For expanding capsid protein Cap sequences（ORF2, sequence Such as SEQ ID NO：8）Primer P5 and P6 be being designed according to the ORF2 gene orders in PCV2 genes.

Preferably, the plasmid pPSCPSP-Cap is that the method that electricity consumption is converted is proceeded in Lactobacillus plantarum LP-1.

The pPSCPSP carriers selected in the present invention do not need abduction delivering, and expression is high, and expression is stable, expression The function of the immunoprotection that the Cap protein of PCV2 can be brought into normal play.

In addition, the present invention is also by designing specific primer, it is final expand in capsid protein Cap sequences, therefore, the present invention It is P5 and P6, sequence such as SEQ ID NO also to protect for the primer for expanding Cap sequences described in S1：1 and SEQ ID NO：Shown in 2.

Preferably, carrier pPSCPSP described in S2 is with pIA β as skeleton, comprising super promoter SCP and M6 precursor protein The sequence construct of signal peptide and obtain.

The present invention also provides the porcine circovirus 2 type recombinant plant lactobacillus pPSCPSP-Cap-LP- that methods described is obtained 1；PPSCPSP-Cap-LP-1 can be colonized in pig body, and biologically active is high.

The present invention also provides the preparation containing the pPSCPSP-Cap-LP-1.

The present invention also provides biologies of the pPSCPSP-Cap-LP-1 prevention and treatment Infection of Porcine circovirus is prepared Application in product.

The present invention also provides vaccines of the pPSCPSP-Cap-LP-1 prevention and treatment Infection of Porcine circovirus is prepared In application.

The present invention also provide it is a kind of prevention and treatment circovirus infection oral vaccine preparation method, be by Culture during pPSCPSP-Cap-LP-1 is inoculated in culture medium obtains full culture, by the full culture directly as oral epidemic disease Seedling.

Compared with prior art, the invention has the advantages that：

The present invention provides a kind of preparation method of porcine circovirus 2 type recombinant plant lactobacillus, by by isolated Cap sequences Row are connected with expression vector pPSCPSP, construction expression plasmid pPSCPSP-Cap；And expression plasmid pPSCPSP-Cap is proceeded to into plant In thing lactobacillus LP-1, pig circular ring virus recombinant plant lactobacillus pPSCPSP-Cap-LP-1 is obtained, Cap sequences are made in plant breast Successful expression in bacterium, all active in vivo and in vitro, pPSCPSP-Cap-LP-1 can be colonized in pig body, and biologically active is high； The culture of pPSCPSP-Cap-LP-1 can be used directly as oral vaccine so that work is saved in production, the time is saved, is subtracted Few Animal stress, used as oral vaccine, lactic acid bacteria can be survived in the gastrointestinal tract, be played a role by mucosa-immune, produce cell Immunity and humoral immunity；Oral vaccine can effectively stimulate body to produce antibody, can significantly decrease pig gyrate virus II type The incidence of disease, contribute to solving retarded growth caused by the institute of pig circular ring virus severe infections in livestock-raising, become thin, it is female Situations such as pig breeding dysfunction, expiratory dyspnea, even presentation extensive pathological change with jaundice, diarrhoea, internal organs and skin, improve The immune level of swinery, so as to improve the general level of the health of swinery, increases the brought economic benefit of cultivation.

Description of the drawings

Fig. 1 is the double digestion identification of restructuring Lactobacillus plantarum expression vector pPSCPSP-Cap, wherein, M：DNA maker 2000；1：PPSCPSP-Cap plasmids；2：PPSCPSP-Cap single endonuclease digestions；3：PPSCPSP-Cap double digestions.

Fig. 2 is the PCR identifications of restructuring Lactobacillus plantarum pPSCPSP-Cap-LP-1；Wherein, M：DNA maker 2000； 1：Negative control；2：PPSCPSP-Cap-DH5 α positive controls；3～10：PCR identification groups.

Fig. 3 is SDS-PAGE after restructuring Lactobacillus plantarum expression supernatant process；Wherein, M is albumen Marker；1 is sky It is white to compare；2 is pPSCPSP-Cap-LP-1.

Fig. 4 is the Westernblot that restructuring Lactobacillus plantarum expresses supernatant, wherein, M is albumen Marker；1 is the positive Control；2 is the Sample supernatants of pPSCPSP-Cap-LP-1.

Fig. 5 is the zoopery of restructuring Lactobacillus plantarum oral vaccine, wherein being set to blank group（PBS groups）, A groups （Control group）, B groups（Vaccine group）, C groups（10 times of bacterial concentration gavages）, D groups（100 times of bacterial concentration gavages）, E（Original bacteria liquid is dense Degree drinking-water）, F（10 times of bacterial concentration drinking-water）.

Fig. 6 be Mouse oral recombinant plant lactobacillus oral vaccine for a period of time after, Lactobacillus plantarum is default in intestines and stomach Plant situation；Wherein, it is cloudy：Negative control, sun：Oral recombinant plant lactobacillus pPSCPSP-Cap-LP-1, M：DNA maker 2000,1～10：The detached Lactobacillus plantarum single bacterium colony from excrement.

Specific embodiment

Present disclosure is further illustrated with reference to Figure of description and specific embodiment, but be should not be construed as to this The restriction of invention.Without departing from the spirit and substance of the case in the present invention, that what is the inventive method, step or condition made is simple Modification is replaced, and belongs to the scope of the present invention；If not specializing, in embodiment, technological means used is art technology Conventional meanses known to personnel.

Constructed Lactobacillus plantarum expression vector pPSCPSP in this patent, is with carrier pIA β 8 as skeleton, to originate InL.caseiCETC5276 super promoter SCP, M6 precursor protein sequence builds what is obtained.

The clone of 1 PCV-II of embodiment

DNA virus extracts kit is used from the pathological material of disease of pig farm collection, genome is extracted, two pairs of primers is designed, by the side of PCR Method, detects positive sample, then the sequence that the totivirus of PCV2 is amplified by P3, P4 from the pathological material of disease for being gathered with primer P1, P2 Row：

Primer P1：AATGGCATCTTCAACACCC

Primer P2：TTAAGGGTTAAGTGGGGGGTC

Primer P3：CCCAAGCTTCCGCGGGCTGGCTGATTTGAAAGT

Primer P4：GGGGTACCCCGCGGAAATTTCTGACAAACGTTAC

Detailed process is as follows：

S1. the genome for being extracted with pathological material of disease carries out the detection of virus-specific, the program of PCR as template with primer P1, P2 For：Denaturation, 94 DEG C of 5min；Denaturation, 94 DEG C of 45s；Annealing, 55 DEG C of 30s；Extend, 72 DEG C of 30s；72 DEG C after 30 circulations Extend 10min eventually, after electrophoresis, the sample that can amplify the band of 460bp or so is defined as into positive.

S2. with the positive sample that detects as template, the detection of virus-specific, the journey of PCR are carried out with primer P3, P4 Sequence is：Denaturation, 94 DEG C of 5min；Denaturation, 94 DEG C of 45s；Annealing, 54 DEG C of 30s；Extend, 72 DEG C of 2min；72 after 30 circulations The band of 1700bp or so is amplified after DEG C eventually extending 10min electroresis appraisals.

S3. with after 1% agarose gel electrophoresis, glue reclaim is cloned into pMD19-T to the pcr amplification product in S2 after purification vector.Convert to E. coli competent DH5 α competence, the M13 primers carried with carrier T, carry out the identification of bacterium solution PCR, Positive bacterium solution, with antibacterial agents box extract plasmid, send to Shanghai Sheng Gong Biological Co., Ltd. sequencing, by sequencing result with Sequence alignment in Gene bank, correct plasmid is named：pMD19-PCV2.

The structure of 2 Lactobacillus plantarum expression vector of embodiment

Design pair of primers（P6 and P5）, for expanding ORF2 reading frames（Cap protein）Sequence

P5：CCCTCGAGATGATGACGTATCCAAGGAGGCGTTTC

P6：CCGAGCTCTTACTTAGGGTTAAGTGGGGGGTCTTTAAG

The structure of recombinant lactobacilli expression vector：（1）With plasmid pMD19-PCV2 as template, P5 and P6 is primer, uses high-fidelity Enzymatic amplification goes out the sequence of the Cap protein of PCV2, and the program of PCR is：Denaturation, 94 DEG C of 5min；Denaturation, 94 DEG C of 45s；Annealing, 54℃ 30s；Extend, 72 DEG C of 2min；Amplify 700bp's or so after 72 DEG C of whole extension 10min electroresis appraisals after 30 circulations Band.

（2）By the band of 700bp or so, after purifying is reclaimed, it is connected with pPSCPSP carriers：Product after PCR amplifications, Reinstate with the expression vector one of Lactobacillus plantarum after being reclaimed with 1% agarose gel electrophoresisXhoIWithSacIDouble digestion reacts.Glue PCV2 Cap proteins sequence and pSCPSP purpose fragments are reclaimed, with ligase, the reaction overnight in 16 DEG C.Connection product is converted To DH5 α competence, according to the resistant gene on pPSCPSP carriers, carried out with the resistance LB flat board of chloramphenicol and ampicillin Resistance screening, enters performing PCR identification with bacterium solution, shakes bacterium upgrading grain, useXhoIWithSacICarry out digestion identification（Fig. 1）, single endonuclease digestion Band differs markedly from the purpose fragment of the plasmid of ring-type, the visible 700bp of double digestion group or so.Show recombinant expression carrier Successfully build, the bacterium for being accredited as the positive is named as pPSCPSP-Cap-DH5 α.

The plasmid of the plasmid pPSCPSP-Cap for having identified is spent into the sequencing of Shanghai Sheng Gong Biological Co., Ltd., gene is discharged Mutation.The expression vector pPSCPSP-Cap electricity consumptions for successfully building are converted to Lactobacillus plantarum LP-1, clicking on parameter is： The μ F of 1.7kv, 2000 Ω, 25,4.0ms, 2nm.The MRS fluid nutrient mediums of the bacterium solution precooling after electricity turn are resuspended, 37 DEG C of incubation 2h. Bacterium solution is taken after centrifugation and is uniformly coated on chloramphenicol（25μg/ml）Resistant panel on 37 DEG C culture.Single bacterium colony, enters performing PCR identification （Fig. 2）, primer is P5, P6.Identify that correct bacterium is named as pPSCPSP-Cap- LP-1.

The expression of 3 albumen of embodiment and identification

First, the SDS-PAGE identifications of expression and expression product of the genes of interest in Lactobacillus plantarum：By the positive weight after conversion Group bacterium pPSCPSP-Cap-LP-1 and empty carrier pPSCPSP-LP-1 is connected to overnight incubation in MRS fluid nutrient mediums, overnight will train Bacterium solution after supporting is inoculated in appropriate MRS liquid in 2% ratio, and 37 DEG C of culture 28h or so take supernatant, is sunk with 80% ammonium sulfate Form sediment, albumen PBS renaturation, after concentration with 12% isolated protein electrophoresis, with the detection of SDS-PAGE protein electrophoresises accordingly Protein band（Fig. 3）, Fig. 3 results show empty plasmid group there is no purpose band, recombinant plant lactobacillus group has the mesh of 28kDa or so Band.

2nd, the Westernblot identifications of expression and expression product of the genes of interest in Lactobacillus plantarum：Albumen supernatant After being processed with the method for step one, after carrying out SDS-PAGE protein electrophoresises, go on pvdf membrane, 200mA, 30min, with 5% it is de- After fat milk powder room temperature closing 2h is processed, with the monoclonal antibody of PCV2（1：200 are diluted with 3%BSA）4 DEG C of night incubations, after washing film Resisted with HRP is marked two（1：2000 are diluted with 3%BSA）Incubation at room temperature 1h, stays figure with albumen visualizer（Fig. 4）, Fig. 4 results show Show purpose band visible in 28kDa or so.

The zoopery of 4 recombinant plant lactobacillus pPSCPSP-Cap-LP-1 oral immunity mouse of embodiment

6～7 week old female mices are randomly divided into into 8 groups, 8 per group；It is set to blank group（PBS groups）, A groups（Control group）, B Group（Vaccine group）, C groups（10 times of bacterial concentration gavages）, D groups（100 times of bacterial concentration gavages）, E（Original bacteria liquid concentration is drunk water）, F （10 times of bacterial concentration drinking-water）.

Oral group, persistently drinks water daily, lactic acid bacteria is added in water, with 10⁸CFU/mL, 10⁹The bacteria concentration drinking-water of CFU/mL （Original bacteria liquid concentration is counted：10⁸CFU/mL）.Vaccine group leg muscle inject 100ul inactivated vaccines, head exempt from 14 days after booster immunization one It is secondary.14 days after head exempts from, 28 days, take 4 at random, extract eyeball blood sampling and collect serum, do the antibody test of ELISA.Whole In individual experimentation, lactic acid bacteria is processed：By the bacterium of 37 DEG C of incubated overnights, washed after three times with aseptic PBS, it is resuspended to concentration to be 10¹⁰CFU/mL（With reference to the Chinese literature of lactic acid bacteria oral vaccine）, entirely the packet of experiment is as shown in table 1.

Carry out the 14th day of zoopery, 28 days, blood sampling in 42 days was clear, after serum collection, was first placed on 37 DEG C of standing 2h, 3000r, is centrifuged 10min, separates out serum.Serum Antibody content, experimental result are detected with mouse PCV2 antibody assay kits As shown in table 2, multigroup of the experimental data that antibody test is obtained uses single factor analysis, compares and checked using t between group, P< 0.05 thinks with statistical significance.

Mouse PCV2 antibody content detection data interpretations of result：

Statistical analysis are carried out to all data of experiment detection using SPSS softwares, 14 days after exempting in experimentation one, 28 My god, two exempt from 14 days afterwards, and the antibody content of 28 days each groups carries out single factor test method analysis, compares and use t inspections, immunity between group Afterwards, blank group, A groups are extremely notable with other group differences（P<0.01）, blank group is notable with A group differences（P>0.05）, One exempts from 14 days B groups and F group othernesses are not notable（P>0.05）, C groups, D groups, E group differences are notable（P<0.05）；One exempts from 28 My god, E groups and F group differences it is not notable（P>0.05）, C groups, D groups, E group differences are notable（P<0.05）；Two exempt from 14 days, B Organize and D groups, B groups and F groups, D groups and E group differences be not notable（P>0.05）；Two exempt from 28 days, and E groups and F group differences do not show Write（P>0.05）.Test result indicate that, oral vaccine can stimulate body to produce corresponding antibody, this reality by oral and gavage The recombinant plant lactobacillus pPSCPSP-Cap-LP-1 for testing middle structure can stimulate body to produce the antibody of PCV2, and activity is good （Fig. 5）.

Recombinant plant lactobacillus oral immunity mouse 20 days or so, gathers fresh excrement, uses specific primer P5, P6 to enter Row bacterium solution PCR identifies that can detection Lactobacillus plantarum be colonized in intestines and stomach, experimental result such as Fig. 6.

SEQUENCE LISTING

<110>Guangzhou Ward Bioisystech Co., Ltd

<120>A kind of preparation method and application of porcine circovirus 2 type recombinant plant lactobacillus oral vaccine

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Claims

1. a kind of preparation method of porcine circovirus 2 type recombinant plant lactobacillus, it is characterised in that comprise the following steps：

2. the preparation method of porcine circovirus 2 type recombinant plant lactobacillus according to claim 1, it is characterised in that use In amplification S1 described in Cap sequences primer be P5 and P6, sequence such as SEQ ID NO：1 and SEQ ID NO：Shown in 2.

3. the preparation method of porcine circovirus 2 type recombinant plant lactobacillus according to claim 1, it is characterised in that S2 The carrier pPSCPSP be with pIA β as skeleton, the sequence construct comprising super promoter SCP and M6 precursor protein signal peptide and .

4. the porcine circovirus 2 type recombinant plant lactobacillus pPSCPSP- that claims 1 to 3 any one methods described is obtained Cap-LP-1。

5. the preparation containing pPSCPSP-Cap-LP-1 described in claim 4.

6. pPSCPSP-Cap-LP-1 described in claim 5 is in the biological products for preparing prevention and treatment Infection of Porcine circovirus Application.

7. pPSCPSP-Cap-LP-1 described in claim 5 prepare prevention and treatment Infection of Porcine circovirus vaccine in should With.

8. it is a kind of prevention and treatment circovirus infection oral vaccine preparation method, it is characterised in that by pPSCPSP- Culture during Cap-LP-1 is inoculated in culture medium obtains full culture, by the full culture directly as oral vaccine.