CN104988107B - A kind of recombinant Lactobacillus of high efficient expression foot-and-mouth disease virus antigen gene and its preparation method and application - Google Patents
A kind of recombinant Lactobacillus of high efficient expression foot-and-mouth disease virus antigen gene and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of recombinant Lactobacillus of high efficient expression foot-and-mouth disease virus antigen gene and its preparation method and application, belong to biotechnology.The recombinant Lactobacillus of the present invention, which contains, to be introduced the Bacillus acidi lactici signal peptide gene of transformation in N-terminal and carries out the VP 1 Gene of Foot-and-Mouth Disease virus sequence of codon optimization, nucleotide sequence such as SEQ ID NO：Shown in 1.The present invention is transformed the structure of Bacillus acidi lactici signal peptide, a connection small peptide GSGGSGG is borrowed to be merged with the VP 1 Gene of Foot-and-Mouth Disease virus after codon optimization the Bacillus acidi lactici signal peptide gene of transformation, gene order after fusion is inserted into Escherichia coli lactic acid bacteria shuttle expression vector, construction recombination plasmid pSIP/opti FMDV A/VP1, by pSIP/opti FMDV A/VP1 recombinant plasmid transformeds to Bacillus acidi lactici.The present invention establishes appropriate reconstruction signal peptide structure to improve the new approaches of heterologous protein secretion efficiency and new method, theoretical foundation and technical support is provided further to research and develop novel mucosa-immune vaccine for recombinant Lactobacillus expression system.
Description
Technical field
The present invention relates to a kind of recombinant Lactobacillus and its preparation method and application, more particularly to a kind of high efficient expression mouth hoof
Recombinant Lactobacillus of epidemic disease viral antigen genes and its preparation method and application, belongs to biotechnology.
Background technology
Aftosa (Foot and mouth disease, FMD) is by foot and mouth disease virus (Foot and mouth
Disease virus, FMDV) caused by a kind of infectious disease that infects of acute, hot, high degree in contact artiodactylous, to propagate
Rapidly, incidence is high and famous.It is classified as one of animal epidemic of 93 kinds of statutory reports by World Organization for Animal Health (OIE).
Foot and mouth disease virus belongs to Picornaviridae Hostis, and viral genome is single-stranded positive RNA, about 8.5kb, there is 7 blood
Clear type, without cross protection between type.Its open reading frame is by L genes, P1 structural protein genes (including VP4, VP2, VP3, VP1 base
Cause), P2 nonstructural protein genes (including 2A, 2B, 2C gene) and P3 nonstructural protein genes (3A, 3B, 3C, 3D gene) group
Into.Wherein, critical epitopes of the VP1 albumen with induction FMD immune responses, 141-160,200-213 and 21-40 including VP1
Amino acids.VP1 full length gene 636bp are the immunogenic genes for encoding FMDV-VP albumen.Around VP1 molecules, grind at present
Study carefully hotter FMD recombinant vaccines and include subunit vaccine, edible vaccine, protein carrier vaccine, gene-deleted vaccine, work
Carrier bacterin and nucleic acid vaccine etc..The anti-system of aftosa is mainly taken at present and is immunized with slaughtering the policy of being combined, and safety has
The vaccine of effect is successfully anti-system or even the final prerequisite for eliminating aftosa.Aftosa attenuated vaccine has preferable immunity,
The immune duration is long, and vaccine inoculation amount is few, but attenuated vaccine exists and dissipates poison and virulence atavism, have in the world more by
Cause the report of aftosa in using attenuated vaccine, therefore, many countries are forbidden to use attenuated vaccine in plain text.Inactivated vaccine
Though overcoming some shortcomings of attenuated vaccine, vaccination doses are larger, and immune duration is short；Non- complete inactivation in vaccine
Virus may cause the new outburst of disease；In addition the broad spectrum activity of the antiviral immunity mechanism of inactivated vaccine induction is limited, limitation
Property is outstanding day by day, and therefore, the correlative study room of countries in the world all is being dedicated to developing safe and effective Vaccine for hoof-and-mouth disease.
Mucous membrane is distributed in the surface layer of the organs internal organs such as respiratory tract, alimentary canal, urogenital tract, is that foreign substance enters body
Portal and body immune system the first line of defence.Mucous membrane is not only removed and degraded with powerful physics, chemical mechanism
The foreign substance of invasion, it may have the congenital invasion and sense for body being protected to resist pathogenic microorganism with acquired immune system
Dye.Mucosal immune system is the first line of defence that the important component of body immune system and body resist cause of disease invasion.
For the infectious disease by alimentary canal and respiratory tract infection, mucosa-immune has great importance：Part can be induced glutinous
The generation of film immune response and the secretion of sIgA resist pathogen, while also can induce humoral immunity in invasion and infection site
And cellular immunity.Research shows that the antibody that mucosal immune response generates is more early than the time that the antibody in serum occurs, the duration
It is long, play the role of in terms of preventing and resisting pathogenic microorganism very important.
Lactic acid bacteria plays its promotion nutriment molecule as the normal flora in body, can be long-term be colonized in body
Degradation and absorption maintain bacterium colony balance in enteron aisle, adjust the prebiotic effects such as immune system effect.People are to the benefit of lactic acid bacteria
Raw effect gives sizable concern, particularly in dairy processing industry.At present, it can buy containing probiotics in the market
Dairy produce, Yoghourt are even more to become general goods.In today that the prebiotic effect of lactic acid bacteria has been confirmed, more about lactic acid
The research of the bacterium mechanism of action and binding mode still needs development.
The research and development of mucosa-immune makes viable bacteria carrier vaccine become the hot spot in vaccine research.Bacterial vector vaccines living
The generation of mucosal immune response can be induced on mucous membrane surface layer, stimulate the generation of sIgA, by pathogen blocking in vitro.Lactic acid bacteria makees
It is the normal flora in animal and human body for the microorganism of aliment security level, there is prebiotic effect and immunoadjuvant function, this makes
Obtaining it becomes strong candidate bacterial strain of carrier in bacterial live vector vaccine research.Lactic acid bacteria is ground as carrier for live vector vaccine
The advantages of studying carefully is as follows：
(1) it is easy to support, and genetic manipulation (such as electroporation transgenosis) method is ripe, easy and efficient, reproducible；
(2) this does carrier non-immunogenicity, can effectively improve the safety of vaccine as food grade bacteria, safety, and
The aftertreatment technology of expression product can be simplified to a certain extent；
(3) it can express in the cell and also express and be secreted into extracellular in cell surface；
(4) lactic acid bacteria has body mucous membrane extremely strong adhesive attraction, therefore the lactic acid bacillus genic engineering bacterium built can
Constantly to breed at mucous membrane, continue to discharge purpose antigen albumen to body；
(5) lactic acid bacteria can directly take orally, inoculation safety, simple and effective, while external purification for eliminating destination protein etc.
Post-processing treatment process；
(6) there is immunoadjuvant function, the generation of mucosa-immune, humoral immunity and cellular immunity can be induced.
In recent years, the intensification with the development of molecular biology and to lactic acid bacteria understanding, lactic acid bacteria are used for live vector vaccine
Research obtain remarkable progress.Lactic acid bacteria expression system has been used successfully to pig epidemic diarrhea, newcastle disease, lockjaw, colyliform
The research of the live vector vaccines such as virus.Xu etc. constructs coexpression classics swine fever CD8+CTL epitopes 290 and pig parvoviral VP2
The genetic engineering Bacillus acidi lactici of antigen simultaneously analyzes its immune effect as pig oral vaccine.Data shows do not appointing
In the case of what adjuvant, recombinant Lactobacillus can effectively induce mucous membrane and systemic immunity response to prevent classical swine fever.Meanwhile
Recombinant Lactobacillus also can induce the generation of the serum antibody IgG and mucosal antibodies IgA of anti-ppv-vp2.These results imply that,
In the development of following classical swine fever and pig parvoviral disease vaccine, recombinant Lactobacillus probiotics are a kind of valuable
Strategy.
Belkis Marelli etc. are successfully constructed rotavirus spike protein subunit VP8 being expressed in cytoplasm,
Recombinant Lactobacillus that is extracellular, being shown to phage surface is secreted into, is immunized after mouse and evaluates its immune response.Study table
It is bright, the mouse of oral immunity LL1 produce the intestinal mucosa antibody of the level of signifiance and be inoculated with LL3 mouse then produce intestinal mucosa and
The anti-VP8 antibody of system level.The protective rate of the intestinal mucosa antibody of LL1 immune groups reaches the serum antibody of 50%, LL3 immune groups
Protective rate reaches 100%.These encouraging results depict the new direction of Rotavirus Vaccine development.
Lei etc. successfully constructs the Recombinant Lactococcus lactis for expressing and be illustrated in bird flu HA1 antigens phage surface.
Recombinant Lactobacillus individually or with cholera toxin unit B together oral immunity experimental animal.Analyze immune response as a result, last
Mouse attacks the H5N1 virus of malicious lethal dose.The HA specific serums of apparent titre are detected in the experimental group of addition CTB
IgG and excrement IgA.Cell proliferation experiment and the result of INF- γ Enzyme linked immunospots disclose cellullar immunologic response
Occur.Importantly, the mouse of CTB and recombinant Lactobacillus combined immunization group can resist the H5N1 virus sense of lethal dose
Dye.
Invention content
Relative to escherichia coli prokaryotic expression system, lactic acid bacteria expression system is not mature enough, and is mainly manifested in external source egg
White expression quantity is relatively low, certain genetic backgrounds of lactic acid bacteria are not clear enough, and operation is comparatively laborious, and expression condition is opposite to be not sufficiently stable
Deng the technical problems to be solved by the invention are the defects of overcoming the prior art, provide a kind of breast of efficiently expressing exogenous gene
Acidfast bacilli carrier system improves exogenous gene expression amount, so as to fulfill oral immunity.
The technical problems to be solved by the invention are achieved through the following technical solutions：
A kind of recombinant Lactobacillus of high efficient expression foot-and-mouth disease virus antigen gene, which is characterized in that the recombination lactic acid
Bacillus, which contains, to be introduced the Bacillus acidi lactici signal peptide gene of transformation in N-terminal and carries out the VP 1 Gene of Foot-and-Mouth Disease virus of codon optimization
Sequence, nucleotide sequence such as SEQ ID NO：Shown in 1.
Expression vector pSIP/opti-FMDV-A/VP1 containing the nucleotide sequence also would naturally fall within the protection of the present invention
Within scope.
In the present invention, it is preferred to, improved Bacillus acidi lactici signal peptide gene borrows a connection small peptide GSGGSGG and password
VP 1 Gene of Foot-and-Mouth Disease virus after son optimization is merged.
In the present invention, it is preferred to, the foot and mouth disease virus is foot and mouth disease A-type virus.
The method for preparing the recombinant Lactobacillus, which is characterized in that include the following steps：
(1) using the hydrophobic method of N-terminal positive charge and H areas is improved, the structure of Bacillus acidi lactici signal peptide is transformed,
Borrow improved Bacillus acidi lactici signal peptide gene to a connection small peptide GSGGSGG and the FMDV VP1 after codon optimization
Gene is merged, nucleotide sequence such as SEQ ID NO：Shown in 1；
(2) gene order after fusion is inserted into Escherichia coli-lactic acid bacteria shuttle expression vector, construction recombination plasmid
pSIP/opti-FMDV-A/VP1；
(3) by pSIP/opti-FMDV-A/VP1 recombinant plasmid transformeds to Bacillus acidi lactici.
In the present invention, it is preferred to, the conversion of recombinant plasmid pSIP/opti-FMDV-A/VP1 electricity is imported into breast by step (3)
In acidfast bacilli NC8, the Bacillus acidi lactici NC8 after electricity conversion is coated on MRS culture mediums, and -72h for 24 hours is cultivated in 37 DEG C of incubators,
Bacterium colony on tablet is chosen and is incubated overnight respectively in MRS fluid nutrient mediums after spot, by above-mentioned bacterium solution with 1:100 rate of vaccination connects
For kind in 50mL MRS fluid nutrient mediums, 37 DEG C of cultures to OD values are 0.6-0.8, and with a concentration of 50-100ng/mL lactobacillus
Peptide (SppIP) is inducer, and induced expression 4-7h obtains recombinant protein.
In the present invention, it is preferred to, the recombinant protein is secreted into extracellular under the guiding of signal peptide, has preferable
Immunogenicity, molecular weight 25kD.
In the present invention, it is preferred to, the voltage parameter of the electricity conversion is 2500V, 5ms.
The present invention optimizes VP 1 Gene of Foot-and-Mouth Disease virus according to Bacillus acidi lactici codon preference, and in VP1 genes
N-terminal increases the signal peptide gene and connection small peptide of one section of transformation, and an artificial synthesized overall length is that the FMDV-A/VP1 of 741bp is excellent
Change gene, be named as opti-FMDV-A/VP1, utilize the gene constructed breast of secretion expression's VP 1 Gene of Foot-and-Mouth Disease virus
Acidfast bacilli recombinant expression carrier pSIP/opti-FMDV-A/VP1.It is imported into Bacillus acidi lactici NC8 by electricity conversion, to transformed
Electric Transformation Parameters optimize in journey, are induced through lactobacillus peptide SppIP, that target gene is stablized in Bacillus acidi lactici,
Efficient expression, for SDS-PAGE analysis shows fusion protein molecule amount is about 25kD, densitometric scan analysis understands the weight expressed
Histone accounts for the 18% of bacterial protein.Supernatant of bacteria solution has a small amount of purpose egg through SDS-PAGE electrophoresis and Western-blot detections
White expression, preliminary proof destination protein can be expressed effectively and extracellular, final acquisition height are secreted under the guiding of signal peptide
The recombinant Lactobacillus NC8 of effect expression VP 1 Gene of Foot-and-Mouth Disease virus, the present invention by inducer concentration during expression, lure
The exploration that agent adds in multiple key factors for influencing expression such as time and induction time is led, provides a kind of high efficient expression external source base
The Bacillus acidi lactici carrier system of cause.
Lactic acid bacteria is probiotics, and using safer compared with traditional vaccine, and it is immunized conveniently as vaccine carrier, many
Research shows that lactic acid bacteria can significantly increase the immune effect of other vaccines such as bird flu, newcastle disease vaccine as probiotics
Power, therefore, lactic acid bacteria live vector vaccine are upper with certain competitive advantage in application.This research is directed to the weight that foot and mouth disease virus is developed
Group bacterium lactic acid bacteria vaccine, has not been reported both at home and abroad, lays a good foundation for the novel mucosa-immune vaccine development of aftosa.
In one particular embodiment of the present invention, immune guinea pig is taken orally after recombinant lactic acid bacteria NC8 is resuspended with fresh milk, simultaneously
If empty carrier and fresh milk are negative control group, indirect ELISA detection specific antibody.The result shows that recombinant lactic acid bacteria is to cavy
Two exempt from rear 9d, and the anti-FMDV specific antibody levels of cavy are more first to be exempted from rear 9d and be significantly improved；Three exempt from rear 9d, and antibody level significantly rises
Height illustrates that the antigen protein expressed in recombinant lactic acid bacteria NC8 has good immunogenicity.Recombinant lactic acid bacteria immune group it is disease-resistant
Malicious ability is far longer than blank control group, and blank control group cavy all falls ill, and empty carrier immune group half cavy falls ill,
Demonstrating lactic acid bacteria has the function of Mucosal Adjuvants.
Therefore it is further, the present invention provides the recombinant Lactobacillus in A type aftosa oral vaccines are prepared
Application.And
Application of the recombinant Lactobacillus in A type aftosa Mucosal Adjuvants are prepared.
Compared with prior art, beneficial effects of the present invention are embodied in：
(1) destination protein expressed of the present invention can be expressed effectively, and be secreted under the guiding of signal peptide extracellular.Cause
We can obtain soluble expression product in culture supernatant for this.Both it can be immunized with the expression product of purifying,
Can oral immunity directly be carried out with recombinant bacterium, thus eliminate the complicated purifying after conventional prokaryotic expression
Journey and renaturation process, oral immunity is then more suitable for large-scale cultivation.
(2) antigen of recombinant Lactobacillus NC8 expression prepared by the animal immune experiment confirmation present invention, which has, preferably exempts from
Epidemic focus；Bacillus acidi lactici belongs to beneficial bacteria of intestinal tract, is the normal flora in animal and human body as food security level microbe,
With prebiotic effect and immunoadjuvant function, this causes it to become the strong candidate bacterium of carrier in bacterial live vector vaccine research
Strain, to safety of human and livestock, no pathogen contamination；With lactic acid bacteria express albumen after need not move through extraction purification process, have it is simple for process,
Good immune effect and it is strong to animal protection power the advantages that, suitable for the immune use of domestic animal.
Description of the drawings
Fig. 1 is the structure route map of recombinant vector pSIP/opti-FMDV-A/VP1；
Fig. 2 is the digestion qualification figure of recombinant plasmid pSIP/opti-FMDV-A/VP1；Wherein, M is DNA molecular quality mark
It is accurate；1 for plasmid pSIP/opti-FMDV-A/VP1 after I/Xhol of Nco, I double digestions；
Fig. 3 is the PCR analysis charts of recombinant Lactobacillus NC8 target gene；Wherein, M is DNA molecular amount standard；2 is are weights
Group Bacillus acidi lactici NC8PCR products；
Fig. 4 is the SDS-PAGE electrophoresis of recombinant Lactobacillus NC8 expression albumen；In wherein, M is pre-dyed marker；1 is
Empty carrier pSIP411 induces the expression product after 7h in NC8；2 for recombinant plasmid pSIP411-VP1 in NC8 without induction
Expression product after 7h；3 be expression products of the recombinant plasmid pSIP411-VP1 in NC8 after inducing 7h；4-5 is recombination matter
Expression product supernatant bacterial sediments by supersound process after of the grain pSIP411-VP1 in NC8 after inducing 7h；
Fig. 5 is the Western-blot detection figures of recombinant Lactobacillus NC8 expression albumen；Wherein, 1 is induces through SppIP
Recombinant Lactobacillus NC8 express express target proteins；2 express albumen for the Bacillus acidi lactici NC8 containing empty carrier；3 be without induction
Recombinant Lactobacillus NC8 expression albumen；M is Protein Marker；
Fig. 6 is the dynamic change figure of sIgA antibody levels in cavy saliva；
Fig. 7 is the dynamic change figure of sIgA antibody levels in cavy excrement leachate；
Fig. 8 is the dynamic change figure of IgA antibody level in cavy blood；
Fig. 9 is the dynamic change figure of IgG antibody level in cavy blood；
Figure 10 is cavy splenic T lymphproliferation response figure.
Specific embodiment
The invention will now be further described with reference to specific embodiments, 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.Those skilled in the art should
It should be appreciated that the details and form of technical solution of the present invention can be repaiied without departing from the spirit and scope of the invention
Change or replace, but these modifications and replacement are each fallen in protection scope of the present invention.
The acquisition of 1 opti-FMDV-A/VP1 genes of embodiment
1 materials and methods
1.1 materials and source
Foot and mouth disease A-type virus is preserved for this laboratory.Escherichia coli-lactic acid bacteria shuttle plasmid expression vector pSIP411 and
Bacillus acidi lactici NC8 wins Bioisystech Co., Ltd purchased from Beijing bit, and cloning vector pUC57 is had by Nanjing Jin Sirui biotechnologies
Limit company provides.
1.2 test method
1.2.1 the transformation and synthesis of gene
Gene order of the Bacillus acidi lactici signal peptide Usp45 original series for the retrieval of GenBank databases online tool, sequence
Number for ADJ57691, using the hydrophobic method of Usp45 signal peptide N-terminal positive charges and H areas is increased, final design goes out overall length 81bp
Improved USP45 signal peptide structures；The original series overall length 639bp of A type FMDV VP1 genes, by VP1 genes and Usp45
Gene is connected together using a connection small peptide (GSGGSGG), the fusion is carried out according to lactic acid bacteria codon preference excellent
Change, sequence is synthesized by Nanjing Jin Sirui Bioisystech Co., Ltd full genome after transformation, is cloned on carrier pUC57, recipient bacterium
For bacillus coli DH 5 alpha, pUC/opti-FMDV-A/VP1 is named as, optimization serves extra large Sani company and carries out sequencing.
2 result of the tests
The base sequence of the opti-FMDV-A/VP1 genes of acquisition such as SEQ ID NO:Shown in 1：
Sequencing result shows that artificial synthesized opti-FMDV-A/VP1 is identical with intrinsic DNA sequence dna, altogether
741bp.Usp45 signal peptide overall length 81bp, encode 27 amino acid (MEKKIISAILMSTVILSAAAPLSGVYA), and connection is short
Peptide 21bp encodes 7 amino acid sequences (GSGGSGG).VP1 full length gene 639bp encode 213 amino acid.Generally as people
The connection peptide of work fusion protein is all amino acid simple in structure or with specific function, such as Gly, Ser, Pro, Ala and Thr,
Especially Gly and Ser are the most commonly used, they can provide enough spaces and flexibility.In wild type VP1 genes 639bp, change
Become 420bp, account for the 66.04% of complete sequence, so as to which ten C contents of G be made to be reduced to 47.49% by 59.53%, 213 codons
(Codon) in, change 140 codons, optimization codon accounts for 66.04%.
The acquisition of 2 recombinant expression carrier pSIP/opti-FMDV-A/VP1 of embodiment
1 materials and methods
1.1 materials and source
Restriction enzyme NcoI, Xhol and T4 ligase is purchased from NEB companies, Taq enzyme, dNTP, DNAMarker
DL2,000, DL15,000, Agarose Gel DNA Purification Kit, MiniBEST Plasmid
Purification Kit are purchased from Dalian treasured biotech firm.Cloning vector pUC57 is by Nanjing Genscript Biotechnology Co., Ltd.
It provides.
1.2 test method
The plasmid pUC/opti-FMDV-A/VP1 of embodiment 1 is similary with warp with the small fragment after Nco I/Xhol double digestions
The Escherichia coli of double digestion-lactic acid bacteria shuttle expression vector pSIP411 large fragments connection (Fig. 1), conversion, alkaline lysis upgrading
Grain identifies transformant with electrophoresis, enzymatic cleavage methods, obtains positive recombinant plasmid and be named as pSIP/opti-FMDV-A/VP1.
2 result of the tests
Segments of the recombinant plasmid pSIP/opti-FMDV-A/VP1 through digestion is with it is expected that size is consistent, illustrating codon optimization
VP1 genes Escherichia coli-lactic acid bacteria shuttle expression vector pSIP/opti-FMDV-A/VP1 structure it is correct (Fig. 2).
In pSIP/opti-FMDV-A/VP1 carriers, Usp45 signal peptide genes connect small peptide, VP1 genes totally 741 nucleotide, coding
247 amino acid.
Embodiment 3 expresses the preparation and detection of A type VP 1 Gene of Foot-and-Mouth Disease virus
1 materials and methods
1.1 materials and source
(1) inducer：Bacitracin (SppIP) (20mg/mL) is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd；
(2) antibiotic：Ammonia benzyl (Amr), erythromycin (Emr) are purchased from Baeyer enlightening Bioisystech Co., Ltd.
1.2 test method
1.2.1 electricity conversion and the screening of resistant strain of the target gene in Bacillus acidi lactici NC8：Lactic acid bar after electricity conversion
Bacterium NC8 is coated on MRS culture mediums, and -72h for 24 hours is cultivated in 37 DEG C of incubators, by the bacterium colony on tablet choose after spot respectively at
It is incubated overnight in MRS fluid nutrient mediums；
1.2.2 the PCR detections of recombinant Lactobacillus NC8：5mL is taken to be incubated overnight the extraction that bacterium solution carries out plasmid, PCR amplification
Testing goal gene, while negative control is done with the carrier pSIP411 of non-transgenosis；
1.2.3 the influence of various concentration inducer concentration, induction time to target gene induced expression：By above-mentioned bacterium solution with
1:100 rate of vaccination is inoculated in 50mL MRS fluid nutrient mediums, and 37 DEG C of cultures to OD values are 0.6-0.8 (about 3h), are remembered at this time
It is induced for 0h, adds in inducer SppIP in the medium, the final concentration of inducer is respectively 10ng/mL, 20ng/mL, 30ng/
ML, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 100ng/mL determine to take bacterium solution every 1h after the concentration of inducer, until
7h.Similary recombinant Lactobacillus of the induction containing empty carrier pSIP411, as a control group, handles thalline respectively after induction
Precipitation and culture solution supernatant do electrophoretic analysis, carry out quantitative analysis to expression product by densitometric scan, obtain best induction
Object concentration.
1.2.4 the SDS-PAGE analyses of expression product：Since Bacillus acidi lactici pSIP/opti-FMDV-A/VP1 is extracellular
Expression, can be by protein secretion to culture medium, therefore after taking the 37 DEG C of bacterium solutions being incubated overnight centrifugations, retains supernatant and carry out 4 respectively
It DEG C dialysis and is freeze-dried with 50 times of protein concentrates, 1 X sds gel sample loading buffer (containing DTT) is added in after concentration, it is fully mixed
It is even, 10min is boiled, 1000g centrifugation 10min take supernatant 10ul loadings.Simultaneously by bacterium solution centrifugation after gained bacterial sediment also into
Row SDS-PAGE electrophoretic analysis.
1.2.5 recombinant protein expression efficiency is analyzed：Gel after decoloration is made wafer with dry glue film and preserves, to wafer
With dual wavelength TCL scanners (shimadzu CS-930) analyze, can quantitative analysis expression albumen content
1.2.6 the Western-blot detections of recombinant Lactobacillus NC8：Take the recombination lactic acid bar that 10mL is induced through SppIP
Bacterium supernatant adds in the sample-loading buffer of certain volume, boils 10min, routinely method electrophoresis, transfer, closing, rinsing, and FMD is positive
37 DEG C of incubation 1h of Swine serum, 1h, drift are incubated in 37 DEG C after rinsing with the anti-pig IgG of goat of horseradish peroxidase (HRP) label
ECL colour developings are carried out after washing.
2 result of the tests
The acquisition of 2.1 recombinant Lactobacillus NC8：Culture passes through the bacterium colony of the chief in liquid medium after electricity conversion coated plate
Double digestion is identified after extracting plasmid, and stripe size is suitable；Experiment proves 2500V, and the voltage parameter of 5ms is most suitable, and electricity turns
Change efficiency highest.
The PCR detections of 2.2 recombinant Lactobacillus NC8：It extracts recombinant Lactobacillus plasmid and carries out PCR detections, recombinant plasmid
It is amplifiable go out target fragment, empty plasmid do not amplify any band (Fig. 3).
2.3 various concentration inducer concentration, influence of the induction time to target gene induced expression：The kind daughter bacteria of activation with
1% is inoculated in the MRS fluid nutrient mediums of improvement, and between 6.0~7.0,37 DEG C of culture to OD values are initial medium pH value
0.6-0.8, and with lactobacillin (SppIP) for inducer, induction expression protein amount is most in 50-100ng/mL for inducer concentration
It is high；Lactic acid bacteria starts higher in 4h or so expression protein contents after addition inducer, reaches highest level in 7h and tends towards stability；It is logical
Cross 18% (Fig. 4) that the recombinant protein that densitometric scan analysis understands to express accounts for bacterial protein.
2.4 secretion expression：The supernatant of bacteria solution of induction respectively through 4 DEG C dialysis and freeze-drying concentration 50 times after, SDS-
PAGE electrophoresis and Westblot detections have a small amount of destination protein to express, and preliminary proof destination protein can be expressed effectively and believed
It is secreted into extracellular (Fig. 4) under the guiding of number peptide.Therefore we can obtain soluble expression product in culture supernatant.Both may be used
To be immunized with the expression product of purifying, can also oral immunity directly be carried out with recombinant bacterium, thus eliminate conventional original
Complicated purification process and renaturation process after nuclear expression system expression, oral immunity is then more suitable for large-scale cultivation.Experiment
By the exploration to Bacillus acidi lactici pSIP411 plasmid expression system ideal expression conditions more preferably to utilize the system expression activity egg
It lays a good foundation in vain.
The Western-blot detections of 2.5 recombinant Lactobacillus NC8：Recombinant Lactobacillus protein extract is through electrophoresis and turns
After film hybridization, as a result show：Expression albumen can identify that the recombinant bacterial strain through induction exists by foot and mouth disease virus positive Swine serum antibody
Visible fainter specific immune response band at about 25KD, and the bacterial strain that does not induce and and the bacterial strain containing empty carrier it is miscellaneous without this
Hand over band (Fig. 5).Illustrate that VP1 genes have been expressed in transgenosis recombinant Lactobacillus, and expressed albumen has immune response
Activity.
4 animal immune experiment of embodiment
1 materials and methods
1.1 materials and source
Healthy guinea pig (300-500g) is provided by experimental animal farm of this institute.Envelope antigen, horseradish peroxidase
(HRP) reagents such as the rabbit Anti-cavy IgG of label, OPD, hydrogen peroxide are provisioned in ELISA diagnostic kits.
1.2 test method
1.2.1 animal packet and immune with attacking poison
Cavy is randomly divided into 5 groups, every group 6, the NC8 (A of the respectively pSIP/opti-FMDV-A/VP1 containing recombinant plasmid
Group), the NC8 (B groups) of the pSIP411 containing plasmid, the WCFS1 (C groups) of the pSIP/opti-FMDV-A/VP1 containing recombinant plasmid, containing plasmid
The WCFS1 (D groups) and negative control (only gavaging milk, E groups) of pSIP411 is immunized three times, and immunization time is one week per minor tick,
Each continuous immunity three days, once a day, 4th week attack poison, kill cavy within the 5th week, detect indices.
Table 1
Immune programme
The 7h recombinant Lactobacillus supernatants induced are resuspended in fresh milk；Divide every cavy injection 0.2ml；Respectively at 0d,
9d, 19d, 29d take a blood sample, and indirect elisa method measures serum antibody；28d attacks poison after third time is immune, the intradermal puncture in foot plantar after cavy
Poison is attacked, 7d is observed continuously in the A type FMDV of every cavy inoculation 100ID50/0.2mL, using the severity that lesion occurs as guarantor
Protect the judgement of effect：Is there is not any lesion in protection completely；Part protection occurs only at single foot for attacking poison inoculation for lesion；
It does not protect and only reaches more than foot for two and lesion occur.
1.2.2 in mucous membrane sample (in saliva and excrement) sIgA detection
5d, 7d, 10d, 15d, 17d, 20d, 25d, 27d, 30d are acquired exempt from respectively after (0d) and initial immunity before immune
Fecal specimens and saliva sample the detection sIgA of epidemic disease cavy are horizontal.Method is as follows：
A type aftosa rabbit anti-serums PBST 1:1000 dilutions, 96 orifice plates are per 50 μ L of hole, room temperature sealing plate 8h.It is washed with PBST
After 96 orifice plates are cleaned 5 times and had the final say, A type foot and mouth disease virus inactivation antigens (PBST 1 is added in:6 dilutions) per 100 μ L of hole, 37 DEG C of trainings
Support 1h.It is washed after 96 orifice plates clean 5 times and have the final say with PBST, the measuring samples (serum 1 of 100 μ L is added in per hole:10 dilutions, saliva
1:2 dilutions, stool supernatant do not dilute), set up negative hole, positive hole and blank control wells, 37 DEG C of culture 1h.96 are washed with PBST
After orifice plate is cleaned 5 times and had the final say, the rabbit-anti cavy IgA secondary antibodies (1 of HRP labels are added in:1000PBST dilutes) per 100 μ L of hole, 37
DEG C culture 50min.It is washed after 96 orifice plates clean 5 times and have the final say with PBST, the OPD color developing agents of 50 μ L are added in per hole, and (H2O2 is per mL100
μ L), 37 DEG C are cultivated addition terminate liquid (dilute sulfuric acid) after 15min, and OD values are read under 492nm wavelength in 15min.
1.2.3 in blood IgA, IgG detection
10d, 20d, 30d acquisition immune guinea pig heart blood detection IgA, IgM after (0d) and initial immunity before immune,
Method is same as above.IgG detection methods are as follows：
96 orifice plates are per 100 μ L foot-and-mouth disease a types inactivation antigen of hole (with PBST with 1:6 dilutions), 4 DEG C of incubation 8h.It is washed with PBST
Plate 5 times and after having the final say, the PBST solution that 200 μ L contain 1% skimmed milk power, 37 DEG C of culture 2h are added in per hole.PBST board-washings 5 times are simultaneously clapped
After plate, the serum to be checked (1 of 100 μ L is added in per hole:8 dilute, successively doubling dilution).Negative, positive and blank pair is set up simultaneously
According to hole, 37 DEG C of culture 1h.PBST board-washings 5 times and after having the final say, the rabbit Anti-cavy IgG secondary antibody of the HRP labels of 100 μ L is added in per hole
(1:1000 dilutions), 37 DEG C of culture 50min.PBST board-washings 5 times and after having the final say, add in the OPD color developing agents of 50 μ L per hole, 37 DEG C
15min.50 μ L terminate liquids (dilute sulfuric acid) are added in per hole, OD values are read under 492nm.
1.2.4 spleen lymphocyte proliferation is tested
Immune guinea pig spleen is acquired in 30d, and dissection cavy takes its spleen under aseptic condition, spleen is ground with asepsis injector
It grinds, cell suspending liquid (30mL or so) is made, be put in Tissue Culture Dish and detect lymphopoiesis with CFSE methods decoration method
Experiment.
The RPMI-1640 culture mediums without serum is used to be resuspended the lymphocyte of separation, be added in 6 orifice plates, 37 DEG C of temperature
Case is incubated 15min, draws supernatant (discarding attached cell), and cell number is diluted to as 1.0~2.0 × 107/mL (one with 1640
8 pipe of a cavy spleen point, each 5mL)；Then CFSE liquid (2.5 μ L 2mM) is added in cell suspension；37 DEG C of items after mixing
After cultivating 15min under part, the FBS with cell suspension isometric (5mL) is added in；Then PBSs of the 5mL containing 3%FBS is added in, fully
After mixing, 2000r/min centrifugation 10min are discarded supernatant；It repeats the above steps 3 times；Finally with dilution (containing 10%FBS,
The RPMI-1640 culture solutions of 15mM HEPES liquid and 0.05mM β-ME) diluting cells are to a concentration of 2 × 106/mL.To 24 holes
Add in the good cell suspension of the above-mentioned dilutions of 1mL in tissue culture plate per hole, each immune group sets up three experimental groups, every group 3
Parallel repetition, experimental group are respectively：
Antigenic stimulus group：The FMDV inactivation antigens that 200 μ L are added in per hole are (final concentration of with the dilution of RPMI-1640 culture mediums
10μg/mL)；
Positive controls：ConA solution (the final concentration of 10 μ g/ of RPMI-1640 culture mediums dilution of 200 μ L are added in per hole
mL)；
Negative control group：The RPMI-1640 culture mediums of 200 μ L are added in per hole；
It marks and collects cell flow cytomery after culture 60h in 37 DEG C of CO2 incubators (5%).
2 result of the tests
In 2.1 mucous membrane samples (in saliva and excrement) sIgA testing result
It can clearly be seen that oral immunity recombinant Lactobacillus NC8 can induce cavy to generate sIgA from figure, and
Antibody level reaches highest after the third immunization.Blank control group antibody level dynamic changing curve is substantially unchanged, and contains
The lactic acid bacteria control group for having empty carrier has slowly raising during immune but peak value is relatively low, which proves with lactic acid fungus oral
Immune animal can generate slight immune response (Fig. 6, Fig. 7) at animal mucous membrane.
The testing result of IgA, IgG in 2.2 blood
IgA is drawn with the average value of the blood serum sample OD values of each experimental group, the dynamic changing curve of IgG antibody level (is schemed
8, Fig. 9).The variation of antibody is clear that from figure, three kinds of antibody are all induced to generate and when three exempt from contain after exempting from one
Amount reaches highest, but IgG rises level compared with IgA high.
2.3 spleen lymphocyte proliferation test result analysis
Shown with the proliferation results of CFSE methods detection cavy spleen lymphocyte：All locate substantially when no exogenous antigen stimulates
In quiescent condition, each immune group Guinea pig lymphocyte can't detect proliferation；Receiving non-specific mitogen concanavalin A
Then lymphocyte shows rapid and apparent splitting status when (Concanavalin A, ConA) is stimulated.
From fig. 10 it can be seen that there is breeder reaction all in the splenic T lymphocyte of recombination NC8 and WCFS1 immune group cavys
It is obvious that recombination WCFS1 immune groups are slightly weaker than recombination NC8 immune groups, two control groups containing empty carrier also have slight increasing
Grow reaction, but unobvious.The T lymphocytes of blank control group do not occur breeder reaction, remain static.
2.4 protest test results
Table 2
Challenge viral dosage was carried out to every group of 4 cavy in the 30th day after just exempting from.Two groups of recombination lactic acids can be analyzed from upper table
Bacterium has all played good protective effect after animal attacks poison, and recombinates WCFS1 groups and play the role of full guard.Negative control
Group has one not fall ill the possible reason is the cavy slight of stature constitution is weaker, when clinical symptoms show unobvious or delayed onset
Between.Control group containing empty carrier is also demonstrated by certain protection and demonstrates lactic acid bacteria that the prebiotic work of immunity of organism can be adjusted
With.
Finally it should be noted that：The foregoing is merely the preferred embodiments of the present invention, are not intended to restrict the invention, to the greatest extent
Pipe is with reference to the foregoing embodiments described in detail the present invention, for those skilled in the art, still can be with
It modifies to the technical solution recorded in foregoing embodiments or equivalent replacement is carried out to which part technical characteristic.It is all
Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in the guarantor of the present invention
Within the scope of shield.
Claims (8)
- A kind of 1. recombinant Lactobacillus of high efficient expression foot-and-mouth disease virus antigen gene, which is characterized in that the recombination lactic acid bar Bacterium contains the Bacillus acidi lactici signal peptide gene in N-terminal introducing transformation and carries out the VP 1 Gene of Foot-and-Mouth Disease virus of codon optimization Nucleotide sequence, the Bacillus acidi lactici signal peptide gene of transformation is by connecting small peptide GSGGSGG and the aftosa after codon optimization Virus VP 1 gene is merged, the nucleotide sequence such as SEQ ID NO：Shown in 1.
- 2. recombinant Lactobacillus according to claim 1, which is characterized in that the foot and mouth disease virus is foot-and-mouth disease a type Virus.
- 3. a kind of recombinant vector pSIP/opti-FMDV-A/VP1, which is characterized in that include nucleotides sequence described in claim 1 Row.
- 4. prepare the method for the recombinant Lactobacillus described in claims 1 or 2, which is characterized in that include the following steps：(1) using the hydrophobic method of N-terminal positive charge and H areas is improved, the structure of Bacillus acidi lactici signal peptide is transformed, will be changed The Bacillus acidi lactici signal peptide gene made by the VP 1 Gene of Foot-and-Mouth Disease virus after connecting small peptide GSGGSGG and codon optimization into Row fusion, the nucleotide sequence such as SEQ ID NO of the gene after fusion：Shown in 1；(2) by the nucleotide sequence SEQ ID NO after fusion：1 is inserted into Escherichia coli-lactic acid bacteria shuttle expression vector, Construction recombination plasmid pSIP/opti-FMDV-A/VP1；(3) by pSIP/opti-FMDV-A/VP1 recombinant plasmid transformeds to Bacillus acidi lactici.
- 5. according to the method described in claim 4, it is characterized in that, step (3) is by recombinant plasmid pSIP/opti-FMDV-A/ The conversion of VP1 electricity is imported into Bacillus acidi lactici NC8, and the Bacillus acidi lactici NC8 after electricity conversion is coated on MRS culture mediums, in 37 DEG C of trainings Support in case and cultivate -72h for 24 hours, the bacterium colony on tablet is chosen and is incubated overnight respectively in MRS fluid nutrient mediums after spot, by bacterium solution with 1:100 rate of vaccination is inoculated in 50mL MRS fluid nutrient mediums, and 37 DEG C of cultures to OD values are 0.6-0.8, and with a concentration of 50- 100ng/mL lactobacillus peptides SppIP is inducer, and induced expression 4-7h obtains recombinant protein.
- 6. according to the method described in claim 5, it is characterized in that, the voltage parameter of the electricity conversion is 2500V, 5ms.
- 7. application of the recombinant Lactobacillus in A type aftosa oral vaccines are prepared described in claims 1 or 2.
- 8. application of the recombinant Lactobacillus in A type aftosa Mucosal Adjuvants are prepared described in claims 1 or 2.
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