CN105754919B - A kind of EHEC O157:H7 recombination lactobacillus acidophilus carrier bacterin and its preparation method and application - Google Patents
A kind of EHEC O157:H7 recombination lactobacillus acidophilus carrier bacterin and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of EHEC O157:H7 lactobacillus acidophilus carrier bacterins and its preparation method and application; the recombination lactobacillus acidophilus is the live vector bacterium for being transformed into the bis- epitope protective antigens of the M of O157:H7EspA-Tir containing EHEC; the recombination lactobacillus acidophilus strain that the present invention constructs can steadily subculture in vitro separately under whether there is or not selection pressure; mouse is immunized by oral vaccine; and carry out challenge viral dosage; prove that the lactobacillus acidophilus live vector vaccine has good immunogenicity and immune protective effect, the live vector vaccine is safe and non-toxic, immunoprophylaxis works well.
Description
Technical field
The invention belongs to gene engineering technology fields, are related to a kind of exploitation of live vector vaccine, and in particular to a kind of to prepare
The methods and applications of EHEC O157:H7 bivalent recombination lactobacillus acidophilus carrier bacterin.
Background technique
Enterohemorrhagic escherichia coli (enterohaemorrhagic E.coli, EHEC) O157:H7 mainly passes through digestion
Road is propagated, causing bleeding property of clinic enteritis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura etc., or even can
Lead to death.Since nineteen eighty-three Riley etc. confirms severe haemorrhage colitis caused by EHEC O157:H7 for the first time, EHEC
Sporadic cases caused by O157:H7 and outbreak of epidemic worldwide constantly occur, and whole world morbidity is in rising trend.It is right at present
EHEC O157:H7 infection still lacks effectively preventing measure, although most of antibiotic can be killed, releases
Shiga toxin will increase the danger of concurrent hemolytic uremic syndrome.Seriousness in view of EHEC O157:H7 outbreak of epidemic and anti-
The risk of raw extract for treating, safely and effectively vaccine is the first choice for preventing its infection, but there is no the vaccine that can be used for people at present, because
The research and development of this vaccine are particularly important.
The pathogenic major embodiment of EHEC O157:H7 in bacterium host it is intracorporal stick field planting and generate toxin two
Aspect.Stick be with field planting bacterium infection the first step, Tir and EspA are in bacterial adhesion and are formed and characteristic stick and smooth out
It damages (attaching and effacing lesion, A/E lesion), is i.e. is played in the effector molecule transmitting of A/E damage
Important function served as bridge, if block O157:H7 bacterium infection stick initial phase if can be to avoid the hair damaged with wiping
It is raw, to terminate infection.Wherein adhesin Intimin, Intimin receptor Tir and III type excretory system T3SS GAP-associated protein GAP
The important virulence factor of EHEC O157:H7, some researches show that human bodies after infecting EHEC O157:H7 to Tir, Intimin,
EspA, EspB have strong immune response, these albumen is prompted to can be used as potential vaccine candidate antigen.Therefore, we select
Vaccine candidate antigen of the key field planting functional protein of EHEC O157:H7Tir and EspA two as its infection of development prevention,
Construct the vaccine with protective novel O157:H7Tir and EspA bivalent antigen.
In recent years, lactic acid bacteria expression system is widely used in expression external source target gene, is that mucosa-immune antigen and drug pass
The ideal chose of delivery carrier has a good application prospect in food, medicine and veterinarily.Lactic acid bacteria is expressed as carrier
It can stick with transmitting antigen and be colonized in mucosal epithelial cells, point of certain Immunity active factors such as IL-2, TNF-α, INF can be improved
Secrete, induce strong mucosal immune response, and it is safe and non-toxic, do not destroyed by bile, constitute in vivo type high efficient expression and
Foreign protein is secreted, the active bacteria formulation developed can be applied directly, be eliminated loaded down with trivial details, complicated needed for general genetic engineering bacterium
Extraction process.But the research report of lactobacillus acidophilus expression EHEC O157:H7 antigen so far, is had no both at home and abroad.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of EHEC O157:H7 to recombinate lactobacillus acidophilus live vector vaccine
Preparation method
The present invention is to be achieved through the following technical solutions:
(1) EspA-Tir M di-chain fusion gene is prepared by Overlap extension PCR method；
(2) in the pMG36e carrier after EspA-Tir M di-chain fusion gene to be inserted into double digestion, recombinant plasmid is obtained
(pMG36e-EspA-Tir M)；
(3) recombinant plasmid (pMG36e-EspA-Tir M) electrotransformation is entered into lactobacillus acidophilus, building recombination lactobacillus acidophilus
(ATCC 4356/pMG36e-EspA-Tir M), and identify；
(4) EspA-Tir M protein expression and immunogenicity in the recombination lactobacillus acidophilus that detecting step (3) obtains obtain
The recombination lactobacillus acidophilus candidate vaccine strain of EHEC O157:H7；
(5) subculture in vitro separately culture is carried out with and without selection pressure, identification recombination lactobacillus acidophilus vaccine strain
Stability.
Above-mentioned preparation method, it is characterised in that: to recombination double-strand EspA-Tir M sequence and pMG36e carrier in step (2)
Respectively through Pst I and HindIII double digestion, it is attached with T4 ligase；Lactobacillus acidophilus described in step (3) is
ATCC4356。
Recombination lactobacillus acidophilus as described in the above technical scheme is the live vector for being used to prevent EHEC O157:H7 infection
Vaccine.
The EspA-Tir M di-chain fusion gene order is as shown in SEQ ID No.1:
atggatacatcaaatgcaacatccgttgttaatgtgagtgcgagttcttcgacatcgacgatctatga
cttaggtaatatgtcgaaggatgaggtggttaagctatttgaggaactcggtgtttttcaggctgcgattctcatg
ttttcttatatgtatcaggcacaaagtaatctgtcgattgcaaagtttgctgatatgaatgaggcatctaaagcgt
caaccacggcacaaaagatggctaatcttgtggatgccaaaattgctgatgttcagagtagcactgataagaatgc
gaaagccaaacttcctcaagacgtgattgactatataaacgatccacgtaatgacataagtgtaactggtattcgt
gatcttagtggtgatttaagcgctggtgatctgcaaacagtgaaggcggctatttcagctaaagcgaataacctga
caacggtagtgaataatagccagctcgaaattcagcaaatgtcgaatacattaaatctcttaa cgagtgcacgtt
ctgatgtgcaatctctacaatatagaactatttcagcaatatcccttggtaaaggaggcggaagtggaggaggtag
cagcccaaccacgaccgaccctgatgcagctgcaagtgcaactgaaactgcgacaagagatcagttaacgaaagaa
gcgttccagaacccagataatcaaaaagttaatatcgatgagctcggaaatgcgattccgtcaggggtattgaaag
atgatgttgttgcgaatatagaagagcaggctaaagcagcaggcgaagaggccaaacagcaagc SEQ ID No.1。
One aspect of the present invention provides a kind of weight as enterohemorrhagic escherichia coli EHEC O157:H7 vaccine
Group lactobacillus acidophilus, it is characterised in that: the recombination lactobacillus acidophilus is the lactobacillus acidophilus for being transformed into foreign vector, described outer
Source carrier is the carrier of the albumen coded sequence of M containing EspA-Tir.
Further, the carrier of the albumen coded sequence of M containing EspA-Tir is pMG36e-EspA-Tir M carrier.
Further, the pMG36e-EspA-Tir M carrier is connected by pMG36e carrier and EspA-Tir M fusion
Connect composition.
Further, the EspA-Tir M fusion by EHEC O157:H7 protective antigens EspA and Tir M
It merges, sequence is SEQ ID No.1.
Another aspect of the present invention provides a kind of vaccine for preventing EHEC O157:H7, the vaccine include activity at
Point and pharmaceutically acceptable carrier, it is characterised in that: the active constituent be aforementioned recombination lactobacillus acidophilus.
Another aspect of the invention provides a kind of preparation method for preparing above-mentioned recombination lactobacillus acidophilus, including following
Step:
(1) EspA-Tir M di-chain fusion gene is prepared by Overlap extension PCR method；
(2) in the pMG36e carrier after EspA-Tir M di-chain fusion gene to be inserted into double digestion, connection product is obtained；
(3) by connection product electrotransformation lactobacillus acidophilus, identification contains pMG36e-EspA-Tir M recon acidophilus cream bar
Bacterium；
(4) EspA-Tir M protein expression and immunogenicity in the recombination lactobacillus acidophilus that detecting step (3) obtains obtain
The recombination lactobacillus acidophilus of EHEC O157:H7 vaccine；
(5) subculture in vitro separately culture, identification recombination lactobacillus acidophilus are carried out respectively in the case where having and/or without selection pressure
Vaccine stability.
Further, in step (2) to recombination double-strand EspA-Tir M sequence and pMG36e carrier respectively through Pst I and
Hind III carries out double digestion, is attached with T4 ligase.
Further, lactobacillus acidophilus described in step (3) is ATCC 4356；
The aforementioned recombination lactobacillus acidophilus ATCC 4356/pMG36e-espA-Tir M of another aspect of the invention is making
Application in standby prevention EHEC O157:H7 vaccine.
The device have the advantages that are as follows: the lactobacillus acidophilus of building expression EHEC O157:H7EspA-Tir M albumen
Oral live vector vaccine strain, the bacterial strain under whether there is or not selection pressure can steadily subculture in vitro separately, by being taken orally to mouse
Inoculation and challenge viral dosage, it was demonstrated that the vaccine has good immunogenicity and immune protective effect.The prebiotic bacteria vaccine can stick
Be colonized in mucosal epithelial cells, and it is safe and non-toxic, do not destroyed by bile, high efficient expression while growth and breeding in enteron aisle
EspA-Tir M albumen, the localized accumulated stimulation and absorption of EspA-Tir M albumen, which are expected to induce in host, in enteron aisle produces
Raw immune response plays the effect of prevention O157:H7 infection to generate specific antibody protection host.This method development
Active bacteria formulation can directly apply, eliminate extraction process loaded down with trivial details, complicated needed for general genetic engineering bacterium, oral vaccine
Both drug administration by injection is not needed, it is also relatively inexpensive, it is a kind of mode of new prevention EHEC O157:H7 infection.
For above and other objects of the present invention, feature and advantage can be clearer and more comprehensible, example is cited below particularly, and be equipped with
Attached drawing is described in detail as follows.
Detailed description of the invention
Fig. 1: recombinant plasmid pMG36e-EspA-Tir M building route map；
Fig. 2: for Overlap extension PCR as a result, wherein swimming lane M is nucleic acid (DNA) molecular weight standard (Marker), swimming lane 1 is Tir
M (210bp), swimming lane 2 are espA (576bp), and swimming lane 3 is EspA-Tir M (826bp)；
Fig. 3: recombinant plasmid pMG36e-EspA-Tir M identification with multi-plex PCR result 1, swimming lane M are nucleic acid (DNA) molecular weight
Standard (Marker), swimming lane 2 are PCR result (816bp)；
Fig. 4: recombinant plasmid pMG36e-EspA-Tir M identification with multi-plex PCR result 2, swimming lane M are nucleic acid (DNA) molecular weight
Standard (Marker), swimming lane 2 are PCR result (670bp)；
Fig. 5: the product sequencing result of recombinant plasmid pMG36e-EspA-Tir M identification with multi-plex PCR result 1；
Fig. 6: recombination lactobacillus acidophilus Western Blot as a result, swimming lane 1 is lactobacillus acidophilus (ATCC 4356), swimming lane 2
To recombinate lactobacillus acidophilus (ATCC 4356/pMG36e-EspA-Tir M)；
Fig. 7: mice serum IgG testing result, T0, T1, T2, T3 are respectively referred to before being immunized, immune for the first time 7 days latter, and second
Secondary immune 7 days latter, 10 days after third time is immune, PBS group is PBS negative control group, and LA is lactobacillus acidophilus control group, LA-et
It (i.g.) is recombination lactobacillus acidophilus intragastric group, LA-et (i.p.) is that immune group is injected intraperitoneally in recombination lactobacillus acidophilus；
Fig. 8: stool in mice IgA testing result；
Fig. 9: mouse intestinal juice IgA testing result；
Figure 10: the testing result of mouse cytokine IL-4, IL-10 and INF- γ；
Figure 11: mouse survival curve after poison is attacked；
Figure 12: O157 field planting result in Mice Body is attacked after poison.
Specific embodiment
In following embodiment and test example unless otherwise indicated, agents useful for same and experimental method are technology commonly used in the art
Method and material.
The preparation of embodiment 1:EHEC O157:H7 recombination lactobacillus acidophilus live vector vaccine strain
As shown in Figure 1, the specific method of the present invention are as follows:
1. the design and synthesis of primer
EspA gene order (KJ549678.1) and Tir M gene order (NC_002655.2) are searched from GenBank,
The end of EspA upstream primer 5 ' introduces Pst I restriction enzyme site, and downstream primer removes terminator codon TAA, introduces linker
(ggaggcggaagtggaggaggtagc), design of primers is as follows: EspA-P1:5 '-
AAAACTGCAGGATGGATACATCAAATGCA(SEQ ID No.2),EspA-P2:5′-GCTACCTCCTCCACTT
CCGCCTCCTTTACCAAGGGATATTGCTG(SEQ ID No.3)；Tir M upstream primer introduces linker, and downstream primer is drawn
Enter restriction enzyme site Hind III, Tir M-P1:5 '-GGAGGCGGAAGTGGAGGAGGTAGC AGCCCAACCACGACCGAC
(SEQ ID No.4), Tir M-P2:5 '-CCCAAGCTTTTAGGCTTGCTGTTT GGCCTCTT (SEQ ID No.5), primer
By Shanghai, Sheng Gong gene technology Co., Ltd is synthesized.
2. Overlap extension PCR method prepares EspA-Tir M di-chain fusion gene
2.1 extract EHEC O157:H7 type strain EDL933 dyeing using genome DNA extracting reagent kit (Tiangeng biology)
Body DNA is template, expands espA gene order and Tir M gene order respectively, is carried out in PCR instrument by following loop parameter anti-
Answer: 94 DEG C initial denaturation 5 minutes, then 94 DEG C of 30sec → 58 DEG C 30sec → 72 DEG C 45sec carry out 30 circulation, last 72 DEG C are prolonged
It stretches 10 minutes.Reaction terminates 1% agarose gel electrophoresis, (it is public to be purchased from the precious biology in Dalian using Ago-Gel QIAquick Gel Extraction Kit
Department) recycling EspA and Tir M after carry out Overlap extension PCR.
2.2 Overlap extension PCRs: first by EspA and Tir M in high fidelity enzyme reaction system 94 DEG C of 30sec → 58 DEG C
30sec → 72 DEG C 45sec reacts 10 circulations, and upstream and downstream primer espA-P1 and Tir M-P2 are added later in reaction condition and is
94 DEG C of 30sec → 58 DEG C 30sec → 72 DEG C 1min reaction, 30 circulation, last 72 DEG C extend 10 minutes.1% agarose of reaction result
Gel electrophoresis, Ago-Gel QIAquick Gel Extraction Kit recycle EspA-Tir M di-chain fusion gene (Fig. 2).
3. constructing pMG36e-EspA-Tir M recombinant plasmid
3.1 will import the bacillus coli DH 5 alpha of pMG36e plasmid after the recovery of the LB solid medium of erythromycin containing 200ug/ml
Picking single colonie after expanding culture in the LB liquid medium containing erythromycin, proposes plasmid kit (Dalian treasured life using small
Object) extract pMG36e plasmid.
3.2 use Pst I, Hind III restriction endonuclease (the silent winged generation that of match is scientific and technological), and double digestion EspA-Tir M double-strand is melted respectively
Close gene and pMG36e plasmid, 37 DEG C of water bath digestion 2h, 1% agarose gel electrophoresis, using Ago-Gel reclaim reagent
Box recycles the product after double digestion.
3.3 use T4 ligase (match is silent to fly) after 16 DEG C of connection 4h, will be containing the recombination double-strand EspA-Tir after double digestion
M fusion connects the pMG36e carrier (10:1) after double digestion, constructs pMG36e-EspA-Tir M recombinant plasmid.
4. building recombination lactobacillus acidophilus (ATCC 4356/pMG36e-EspA-Tir M)
The preparation of 4.1 lactobacillus acidophilus competent cells:
4.1.1 about 50 μ l of the single colonie Lactobacillus acidophilus species of preservation 5ml MRS liquid culture medium is inoculated into (to contain
In 0.05%CysteineHCl), 37 DEG C Anaerobic culturel 48 hours；
4.1.2 become muddy to bacterium solution, when OD600 is 0.6, turn to be inoculated into MRS culture medium (containing 0.5M by 1:25 dilution
Sucrose and 0.05%CysteineHCl) in, 37 DEG C of anaerobism are about cultivated 24 hours, until OD600 is 0.8；
4.1.3 bacterium solution is set is pre-chilled 10 minutes on ice, and 4 DEG C 5000 revs/min are centrifuged 20 minutes, abandon supernatant；It is dense with 0.5M
The sterile sucrose solution of degree suspends, and 4 DEG C 5000 revs/min are centrifuged 20 minutes, abandon supernatant, washes repeatedly 1 time；
4.1.4 bacterium is resuspended with the pre-cooling electroporation buffer (acid amide containing lemon, citric acid, sucrose, DMSO) of appropriate volume,
4 DEG C 5000 revs/min are centrifuged 20 minutes, abandon supernatant；
4.1.5 bacterium is resuspended with the pre-cooling 1x electroporation buffer (lemon acid amide, citric acid, sucrose) of 1/250 initial volume,
It is prepared into lactobacillus acidophilus competence, 4 DEG C of refrigerators is set and saves backup.
4.2 recombinant plasmids (pMG36e-EspA-Tir M) electrotransformation lactobacillus acidophilus
4.2.1 recombinant plasmid (pMG36e-EspA-Tir M) is converted into lactobacillus acidophilus competence, in the thermophilic of 60-80 μ l
About 10-20 μ l recombinant plasmid is added in Lactobacillus lactis competent cell, adds the 2x electroporation buffer isometric with added plasmid
(lemon acid amide, citric acid, sucrose)；
4.2.2 it is added after mixing in the electric shock cup of 2mm wide, carries out electrotransformation (voltage 2.5kV, capacitor after setting pre-cooling on ice
25 μ F, 200 Ω of resistance)；
4.2.3 electric shock terminates, and adds 600 μ l antibiotic-free MRS meat soups to suspend, rinses bacterium, after 37 DEG C of Anaerobic culturel 1h,
It respectively takes 200ul to be applied to the MRS plate of Erythromycinresistant containing 0.1ug/ml, sets 37 DEG C of Anaerobic culturels 24-48 hours.
5. recombinating the screening and identification of lactobacillus acidophilus (ATCC 4356/pMG36e-EspA-Tir M)
The smooth complete bacterium colony of 5.1 picking milkys, circular edge carries out Gram's staining, microscopically observation.
5.2 screening single colonies are inoculated with 37 DEG C of Anaerobic culturels of the MRS of erythromycin containing 0.1ug/ml culture medium, after bacterium solution is muddy,
Plasmid is extracted using the big extraction reagent kit of Gram-positive bacteria plasmid (Beijing Suo Laibao company) and carries out multiplex PCR (Fig. 3,4) and survey
Sequence (Fig. 5) screens positive recombinant.
5.3PCR identifies 1 (Fig. 3): identifying that gene order is EspA-Tir M di-chain fusion gene, upstream primer: EspA-
P1:5′-AAAACTGCAGGAtggatacatcaaatgca (SEQ ID No.2), downstream primer: 5 '-CCCAAGCTTTTA
GGCTTGCTGTTTGGCCTCTT(SEQ ID No.5)；94 DEG C of reaction condition initial denaturation 5 minutes, then 94 DEG C of 30sec → 58 DEG C
30sec → 72 DEG C 1min carries out 30 circulations, and last 72 DEG C extend 10 minutes.Reaction terminates 1% agarose gel electrophoresis, agar
Sugared gel reclaims kit recycles PCR product, serves extra large Invitrogen sequencing identification (Fig. 5).
5.4PCR identifies 2 (Fig. 4): identifying that gene order is to be inserted into the company of the upstream foreign gene EspA-Tir M and pMG36e
The genetic fragment of place 670bp is connect, upstream primer: GCTCTAGAGATGGATACATCAAATGCA (SEQ ID No.6), downstream is drawn
Object: CCCAAGCTTTTAGGCTTGCTGTTTGGCCTC TT (SEQ ID No.5), 94 DEG C of reaction condition initial denaturation 5 minutes, so
94 DEG C of 30sec → 52 DEG C 30sec → 72 DEG C 45sec carries out 30 circulations afterwards, and last 72 DEG C extend 10 minutes, and reaction terminates 1%
Agarose gel electrophoresis.
6. recombinant protein EspA-Tir M expression and Immunity identification
6.1 will screen the recombination lactobacillus acidophilus containing positive recombinant in the MRS culture medium of erythromycin containing 0.1ug/ml
Anaerobic culturel 36h, is collected by centrifugation culture supernatant.
6.2TCA (trichloroacetic acid) precipitation method are concentrated: the TCA solution of 10% volume, vortex concussion being added in supernatant
Ice bath 60min after mixing, 12000 turns of centrifugation 10min abandon supernatant, and the pre-cooling acetone washing that 1/4 volume is added precipitates, and 12000 turns
It is centrifuged 5min, supernatant is abandoned, is repeated once.
6.2.1 appropriate 1XSDS sample-loading buffer soluble protein is added after drying at room temperature precipitating.
6.2.2 after boiling 15min for 100 DEG C of sample, 12000 turns of centrifugation 10min take supernatant sample-adding to carry out SDS-PAGE electricity
Swimming.
6.3 using the recombinant protein EspA-Tir M in routine Western blot detection lactobacillus acidophilus cells' lysate
Purpose band can be detected in expression and immunogenicity (Fig. 6) at 35KD as the result is shown, it was demonstrated that successfully building recombination acidophilus cream
Coli vaccine strain.
7. recombinating lactobacillus acidophilus (ATCC 4356/pMG36e-EspA-Tir M) Detection of Stability
Lactobacillus acidophilus will be recombinated and pass on random picking 100 after 50 generations under conditions of having selection pressure and without selection pressure
A colony inoculation in Anaerobic culturel on the MRS plate without erythromycin and MRS plate containing erythromycin for 24 hours.As the result is shown: no matter
Whether there is or not 100 bacterium colonies 98% of selection pressure institute picking to grow.Prove that the recombination lactobacillus acidophilus strain is having no pressure selection
Down can steadily subculture in vitro separately, illustrate it is oral after can express EspA-Tir M albumen in enteron aisle, stimulation mouse generates antibody, from
And mouse is protected to be infected by O157:H7, it is a kind of potential O157:H7 oral vaccine.
Illustrate present invention recombination lactobacillus acidophilus expression EspA-Tir M albumen preparation below by way of specific test example
O157:H7 vaccine generates good application effect in Mice Body.
S embodiment 2: the recombination lactobacillus acidophilus of expression EHEC O157:H7EspA-Tir M albumen infects O157:H7
Mouse have immanoprotection action
1. the MRS meat soup of lactobacillus acidophilus inoculation erythromycin containing 0.1ug/ml will be recombinated, 37 DEG C of Anaerobic culturels are raw to logarithm
For a long time, bacterial sediment is collected, adjustment cell density is 1010Mouse is immunized in CFU/ml.
2. recombinating the research of lactobacillus acidophilus immunogenicity: SPF grades of BALB/c mouses of 4-5 week old are chosen, by it respectively with every
15 progress stomach-fillings of group, intraperitoneal injection 0.1ml 1010CFU/ml recombinates lactobacillus acidophilus, while PBS negative control is arranged, and is immunized
Program are as follows: 0~3d, 7~10d, 21~24d.
2.1 are immunized the blood sampling of docking in 10 days after preceding and final immunization every time, and centrifuging and taking serum (1:50) is used after being stored at room temperature 2h
Indirect elisa method measures specific IgG class antibody titer, and acquires stool in mice detection mouse specificity SIgA class antibody.
10 days after 2.2 third times are immune, Cytokine of Serum IFN-γ, IL-4 and IL-10 are surveyed in blood sampling
In addition 5 mouse are put to death for every group, take intestinal tissue sample, intestinal juice, utilized by expressions such as (Elabascience kits)
Indirect ELISA detects mouse intestinal juice specificity SIgA class antibody, investigates the immune effect that recombination lactobacillus acidophilus induces mouse, really
Fixed best immunization route, evaluates its safety and immunoprophylaxis effect.
3. attacking toxic bacterial strain preparation: infecting insensitive, first to be selected with antibiotic side to solve the problems, such as mouse to O157 bacterium
Method makes O157 obtain the drug resistance to streptomysin, and the effect of streptomysin is the other normal floras for inhibiting enteron aisle, makes O157 in intestines
Become dominant microflora in road, is conducive to its infection.The EHEC O157:H7 bacterium of resistance to streptomycin is inoculated in LB culture medium, 37 DEG C of vibrations
Culture 8h is swung, supernatant is abandoned after centrifugation, then with sterile LB washing 2 times, thallus is finally resuspended with sterile LB liquid, adjustment concentration is
1.0×1010CFU/ml。
4. recombinating the research of lactobacillus acidophilus immune protective effect: mouse is given containing 5g/L chain for 10 days after Full-access immunization
The aseptic aqueous solution of mycin drinks 3d, then with 5 × 109The full bacterium solution of the O157 of CFU/ dosage in two times, be spaced 6 hours, orally
Mouse GI tract is poured into, the activity of close observation mouse after bacterium is attacked, ingests and the change of the state of mind, at the end of 15 day observation period
Calculate the death rate and discharge of bacteria amount of mouse.
5. attacking the culture identification of stool in mice O157 after bacterium: 3 after taking mouse to attack poison, 5,7,9,11,13,15d excrement number,
It is suspended with LB culture medium, after 4 DEG C of refrigerator 1h of broken postposition of shaking, is coated on and buys generous agar (SMAC) selective medium plate, with
100mg/CFU≤100 are considered as discharge of bacteria and terminate.
6. statistical method: carrying out One-way ANOVA LSD statistical method using SPSS21.0, be statistics with P < 0.05
It is variant, it is mapped using GraphPad Prism5.
As a result: results of animal shows that recombinating lactobacillus acidophilus is immunized mouse, after third time booster immunization, two groups
Immune group have compared with control group IgG improve (p < 0.000 * *), and intragastric group effect better than intraperitoneal injection group (* * p <
0.000).Specificity SIgA class antibody can be detected in excrement, intestinal juice in two immune groups, and antibody level is higher than control group (* * p
<0.000) IgA, but in excrement detected, antibody level indifference (p>0.05) between two immune groups, but in intestinal juice
Testing result but shows variant (p < 0.05 *) between the two that intragastric group effect is better than intraperitoneal injection group.Two immune groups
IL-4 cytokine content improves (p < 0.05 *) compared with control group, but indifference between two immune groups；Intragastric group IL-10
Cytokine content is improved (p < 0.000 * *) compared with control group, intraperitoneal injection group, and intraperitoneal injection group is mentioned compared with PBS control group
High (p < 0.05 *), but nothing significantly improves compared with LA control group, and PBS control group and LA control group indifference.
Mouse is attacked after poison and observe within 15 days, within the observation period, PBS control group survival rate is that 10%, LA control group is deposited
Motility rate is 40%, and intraperitoneal injection immune group survival rate is 50%, intragastric group survival rate 80%.And it is carried out in survival mice
The monitoring of O157 discharge of bacteria amount, as the result is shown intragastric group discharge of bacteria amount substantially less than LA control group and intraperitoneal injection group (* p <
0.000), and in the 11st day O157 discharge of bacteria terminate, and the discharge of bacteria amount of intraperitoneal injection group most of the time less than LA control group (* p <
0.000), but within 15 day observation period not yet terminate discharge of bacteria.
Comprehensive, mouse is immunized in recombination lactobacillus acidophilus live vector vaccine, and oral vaccination can induce mouse and generate compared with high titre
Specific antibody and cell factor, attack poison with the O157 bacterium with streptomycin resistance, reinforced immune mouse can after attacking poison
Field planting amount and resident time of the O157 in enteron aisle are reduced, protective rate is up to 80%, and it is poor to be injected intraperitoneally immune effect, protection
Rate is 50%；Show that oral immunity recombination lactobacillus acidophilus live vector vaccine can generate good immune protective effect to mouse.
The above, only presently preferred embodiments of the present invention, not to the present invention make in any form with substantial limit
System, all those skilled in the art, without departing from the scope of the present invention, when using disclosed above
Technology contents, and the equivalent variations for few modifications, modification and the differentiation made, are equivalent embodiment of the invention；Meanwhile it is all
Change, modification and the differentiation of any equivalent variations to the above embodiments of substantial technological according to the present invention, still fall within
In the range of technical solution of the present invention.
Claims (6)
1. a kind of recombination lactobacillus acidophilus as enterohemorrhagic escherichia coli EHEC O157:H7 vaccine, it is characterised in that:
The recombination lactobacillus acidophilus is the lactobacillus acidophilus for being transformed into foreign vector, and the foreign vector is the albumen of M containing EspA-Tir
The carrier of coded sequence；
The carrier of the albumen coded sequence of M containing EspA-Tir is pMG36e-EspA-Tir M carrier；
The pMG36e-EspA-Tir M carrier is connected with EspA-Tir M fusion by pMG36e carrier and is formed；
The EspA-Tir M fusion is merged by protective antigens EspA and the Tir M of EHEC O157:H7, sequence
It is classified as SEQ ID No.1.
2. a kind of vaccine for preventing EHEC O157:H7, the vaccine includes active constituent and pharmaceutically acceptable carrier,
Be characterized in that: the active constituent is recombination lactobacillus acidophilus described in claim 1.
3. a kind of preparation method for preparing recombination lactobacillus acidophilus as described in claim 1, includes the following steps:
(1) EspA-Tir M di-chain fusion gene is prepared by Overlap extension PCR method；
(2) in the pMG36e carrier after EspA-Tir M di-chain fusion gene to be inserted into double digestion, connection product is obtained；
(3) by connection product electrotransformation lactobacillus acidophilus, identification contains pMG36e-EspA-Tir M recon lactobacillus acidophilus；
(4) EspA-Tir M protein expression and immunogenicity in the recombination lactobacillus acidophilus that detecting step (3) obtains obtain EHEC
The recombination lactobacillus acidophilus of O157:H7 vaccine.
4. preparation method according to claim 3, it is characterised in that: to recombination double-strand EspA-Tir M sequence in step (2)
Column and pMG36e carrier carry out double digestion through Pst I and Hind III respectively, are attached with T4 ligase.
5. the preparation method according to claim 4, it is characterised in that: lactobacillus acidophilus described in step (3) is
ATCC4356。
6. application of the recombination lactobacillus acidophilus described in claim 1 in preparation prevention EHEC O157:H7 vaccine.
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| WO1999045136A1 (en) * | 1998-03-05 | 1999-09-10 | University Of British Columbia | Methods for assaying type iii secretion inhibitors |
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| CN101234197A (en) * | 2008-01-29 | 2008-08-06 | 中国人民解放军第三军医大学 | Recombination attenuation salmonella typhimurium vector vaccine and preparation |
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|---|---|---|---|---|
| WO1999045136A1 (en) * | 1998-03-05 | 1999-09-10 | University Of British Columbia | Methods for assaying type iii secretion inhibitors |
| WO2002074812A2 (en) * | 2001-03-15 | 2002-09-26 | Valorisation-Recherche, Societe En Commandite | Antibodies for preventing and treating attaching and effacing escherichia coli (aeec) associated diseases |
| CN101234197A (en) * | 2008-01-29 | 2008-08-06 | 中国人民解放军第三军医大学 | Recombination attenuation salmonella typhimurium vector vaccine and preparation |
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| Oral immunization with Lactococcus lactis-expressing EspB induces protective immune responses against Escherichia coli O157:H7 in a murine model of colonization;B.Ahmed 等;《Inflammation ﹠Vaccination》;20121231;第1页 |
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