CN109706165A - The lactic acid bacteria excretion vector and its construction method, application of recombinant screen can be realized in Escherichia coli - Google Patents
The lactic acid bacteria excretion vector and its construction method, application of recombinant screen can be realized in Escherichia coli Download PDFInfo
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- CN109706165A CN109706165A CN201811582790.3A CN201811582790A CN109706165A CN 109706165 A CN109706165 A CN 109706165A CN 201811582790 A CN201811582790 A CN 201811582790A CN 109706165 A CN109706165 A CN 109706165A
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Abstract
The invention belongs to bioengineering fields.The invention discloses the lactic acid bacteria excretion vectors that one kind can realize recombinant screen in Escherichia coli comprising ampicillin resistance sequence Escherichia coli replicon ori, Escherichia coli constitutive promoter P32, lactic acid bacteria secreting signal peptide USP45, multiple cloning sites and Escherichia coli heat-labile LTB gene order；The invention also discloses the construction methods that one kind can realize the lactic acid bacteria excretion vector of recombinant screen in Escherichia coli；The invention also discloses the applications that one kind can realize the lactic acid bacteria excretion vector of recombinant screen in Escherichia coli.The construction method of the lactic acid bacteria excretion vector of recombinant screen can obtain a kind of lactic acid bacteria secretory type vaccine carrier that recombinant screen can be realized in Escherichia coli to can realize in Escherichia coli in through the invention.
Description
Technical field
The present invention relates to bioengineering fields, can realize the cream of recombinant screen in Escherichia coli more particularly, to one kind
Sour bacterium excretion vector and its construction method, application.
Background technique
Lactic acid bacteria (lactic acid bacteria, LAB) is a group energy bulk fermentation carbohydrate and generates cream
The gram-positive bacteria of acid, is the common bacteria in people and most animals enteron aisle, safe and non-toxic element is acknowledged as safety level
(generaly recoas safe, GRAS) microorganism.Lactic acid bacteria vector oral vaccine acts on the specific machine of Mucosal system
System, which is to be adhered on body mucous membrane, can stimulate generation in the continual synthesis of mucosal sites and released antigen albumen
Specific S IgA, and the antibody titer for stimulating the antigen protein of the IgA antibody potency ratio direct injection purifying generated to generate
It is high.Also some researches show that lactic acid bacteria vector vaccine is detected by the immune cellular immunity for inducing antibody specificity of nasal feeding
The variation of the expression quantity of Th1 cell factor (Th1 cytokines), proleulzin (IL-2) and interferon-γ (IFN-γ).Make
With lactic acid bacteria vector vaccine inhibiting tumor cell growth, the main TC cell effect approach relied on by CD4+ and CD8+.
High efficient expression of the foreign protein in lactic acid bacteria vector adjust can by the promoter of composing type and induction type come
Experiment, the lactic acid bacteria expression system of constitutive expression, foreign protein can use the lasting table of protein expression system of lactic acid bacteria
It reaches, and the induction for then needing inducer, mortifier environmental factor living of induction type is just able to achieve great expression.Utilize lactic acid bacteria egg
White expression system, the signal peptide being inserted into protein secretion system in lactic acid bacteria expression vectors, may be implemented foreign protein in cream
Sour bacterium surface or extracellular expression secretion, meet different requirement of experiment.
Summary of the invention
To solve the above problems, the present invention provides the lactic acid bacterias point that one kind can realize recombinant screen in Escherichia coli
Secrete type carrier；
The present invention also provides the building sides that one kind can realize the lactic acid bacteria excretion vector of recombinant screen in Escherichia coli
Method；
The present invention also provides the applications that one kind can realize the lactic acid bacteria excretion vector of recombinant screen in Escherichia coli.
To achieve the above object, The technical solution adopted by the invention is as follows:
A kind of lactic acid bacteria excretion vector that recombinant screen can be realized in Escherichia coli comprising ampicillin resistance sequence
Escherichia coli replicon ori, Escherichia coli constitutive promoter P32, lactic acid bacteria secreting signal peptide USP45, multiple cloning sites and
Escherichia coli heat-labile LTB gene order.
Preferably, can realize that the lactic acid bacteria excretion vector of recombinant screen further includes ammonia benzyl resistance in Escherichia coli
Gene order.
Preferably, can realize that the lactic acid bacteria excretion vector of recombinant screen further includes PBR322 multiple in Escherichia coli
System.
Preferably, after Escherichia coli constitutive promoter P32 and lactic acid bacteria secreting signal peptide USP45, there are polyclonal positions
Point.
Preferably, after Escherichia coli heat-labile LTB gene order is located at multiple cloning sites.
Preferably, can realize that the lactic acid bacteria excretion vector of recombinant screen is located at Escherichia coli in Escherichia coli
In Dh5 α.
Preferably, it is electroporated to realize that the lactic acid bacteria excretion vector of recombinant screen passes through in Escherichia coli
Mode is present in Lactobacillus casei.
It is a kind of to realize that the construction method of the lactic acid bacteria excretion vector of recombinant screen includes following in Escherichia coli
Step:
It is sequentially prepared and obtains ampicillin resistance sequence Escherichia coli constitutive promoter P32, lactic acid bacteria secreting signal peptide
USP45, multiple cloning sites, the acquisition of Escherichia coli heat-labile LTB gene order can realize recombination in Escherichia coli
The lactic acid bacteria excretion vector of son screening.
Preferably, can realize that the lactic acid bacteria excretion vector of recombinant screen can be applied in large intestine in Escherichia coli
Screening lactobacillus expression vector foreign gene recon in bacillus.
8149 be the galactococcus carrier an of food-grade, but it needs to be transferred in lactic acid bacteria after vitro recombination to screen, difficulty
It is very big, it is therefore desirable to be transformed, the purpose of transformation is to add a replicon that can be replicated in Escherichia coli and a resistance
Gene, and add restriction enzyme site at the segment both ends that this is added, in this way, colony screening can be carried out in Escherichia coli,
Then the sequence being added is gone with digestion again, forms terminal viscosity, is transferred in galactococcus and interconnects, form transformant.
Therefore, the invention has the following advantages: can realize recon sieve in Escherichia coli in through the invention
The construction method of the lactic acid bacteria excretion vector of choosing can obtain a kind of lactic acid that recombinant screen can be realized in Escherichia coli
Bacterium secretory type vaccine carrier.
Detailed description of the invention
Fig. 1 is that pNZES8149-degQ converts E. Coli results figure；
Fig. 2 is that pNZES8149-degQ converts bacterium colony PCR verification result figure after Escherichia coli.
Specific embodiment
Further description of the technical solution of the present invention With reference to embodiment.
Obviously, the described embodiments are merely a part of the embodiments of the present invention, instead of all the embodiments.Based on this
Embodiment in invention, all other reality obtained by those of ordinary skill in the art without making creative efforts
Example is applied, shall fall within the protection scope of the present invention.
In the present invention, if not refering in particular to, all equipment and raw material is commercially available or the industry is common,
Method in following embodiments is unless otherwise instructed conventional method in that art.
Plasmid extraction, genomic DNA are carried out using the kit of Omega Bio-Tek (GA) in the embodiment of the present invention
Extraction, the purifying that DNA fragmentation and PCR product are recycled from Ago-Gel, operating method are carried out according to specification.TA clone carries
Body pEASY-T1 simple is bought from Quan Shijin Bioisystech Co., Ltd.High-fidelity DNA polymerase, T4 DNA connection
Enzyme, DL2000 Marker, DL5000 Marker, restriction enzyme, RT-PCR Kit are purchased from Fermentas company.Draw
Object is synthesized by the raw work biology Co., Ltd in Shanghai, remaining experiment agents useful for same is that domestic analysis is pure.
Embodiment 1 can realize the building of the lactic acid bacteria excretion vector pNZES8149 of recombinant screen in Escherichia coli
The clone of AMP and PBR322 sequence in 1.1 PPIC9K
Using plasmid PPIC9K as template, using primer AP F1 (5 '-GTCGACATGAGTATTCAACATTTCCGTGT -3 ',
SEQ ID NO.1, scribing line base are I restriction enzyme site of Sal) and AP R1 (5 '-GTCGACCGCGTTGCTGGCGTTTTT-
3 ', SEQ ID NO.2, scribing line base is I restriction enzyme site of Sal) it is that primer carries out PCR amplification, obtain AMP and PBR322 sequence
(SEQ ID NO.5).PCR condition are as follows: 94 DEG C of 5min initial denaturation template DNAs, then 94 DEG C of 30s, 58 DEG C of 1min, 72 DEG C
1min is adjusted to 94 DEG C of 30s, 68 DEG C of 1min, 72 DEG C of 1min after 5 circulations, 72 DEG C of extension 10min after 25 circulations；It will
PCR product is connect 10 minutes with carrier pEASY-Simple-T1 room temperature after purification, and mixed liquor is converted to bacillus coli DH 5 alpha
In, containing 100 μ g/ml ampicillins, 40 μ g/ml X-Gal and 24 μ g/ml isopropyl-beta D-thio galactopyranoses
The means screening positive clone of PCR amplification is used after cultivating 16 hours on the LB solid medium of glycosides, sequence verification is recombinated
Plasmid pEASY AP.
The building of 1.2 lactic acid bacteria vectors that can be replicated in Escherichia coli
After distinguishing digested plasmid PNZ8149 and pEASY AP under the conditions of 37 DEG C of I restriction enzyme of Sal, glue recovery purifying
Target fragment；It is connected using T4 ligase, AMP and PBR322 sequence is inserted into PNZ8149 plasmid, it is heat-shock transformed to large intestine bar
In bacterium DH5 α, the hand of PCR amplification is used after cultivating 16 hours on the LB solid medium containing 100 μ g/ml ampicillins
Section screening positive clone, sequence verification obtain recombinant plasmid PNZAMP8149.
The acquisition of 1.3 P32 promoters, Usp45 and LTB sequence
P32 sequence is obtained according to Pmg36e plasmid map, usp45 the and LTB sequence design synthesis announced according to gene bank contains
There is the sequence of these three genes, which contains the I digestion with restriction enzyme position Nco
Point includes V restriction enzyme site of BamH I and EcoR between usp45 and LTB sequence.
1.4 can realize the building of the lactic acid bacteria excretion vector pNZAES8149 of recombinant screen in Escherichia coli
Under the conditions of foregoing fusion gene order and plasmid PNZAMP8149 are used 37 DEG C of I restriction enzyme of Nco respectively, digestion is simultaneously
Glue recycles target fragment.Using T4 ligase, foregoing fusion gene order and plasmid PNZAMP8149 are attached, heat shock turns
Change into bacillus coli DH 5 alpha, is used after being cultivated 16 hours on the LB solid medium containing 100 μ g/ml ampicillins
The means screening positive clone of PCR amplification, sequence verification obtain recombinant plasmid pNZES8149.
Screening of lactic acid bacteria excretion vector of the embodiment 2 containing Aeromonas hydrophila degQ gene in Escherichia coli
The clone of 2.1 Aeromonas hydrophila degQ genes
According to the Aeromonas hydrophila degQ gene order (SEQ ID NO.7) that gene bank is announced, using primer degQF1
(5 '-GGATCCATGCGTAAACCTCTTTCTGT -3 ', SEQ ID NO.3, scribing line base be I restriction enzyme site of BamH) and
DegQR1(5 '-CCCGGGACGGATCACCAGATAGAGG -3 ', SEQ ID NO.4, scribing line base are the V digestion position EcoR
Point), it is cloned using Aeromonas hydrophila genome as template and obtains degQ gene, and be connected to pEASY-Simple-T1, heat shock
Conversion is cultivated 16 hours on the LB solid medium containing 100 μ g/ml ampicillins to obtaining in bacillus coli DH 5 alpha
The means screening positive clone of PCR amplification is used afterwards, and sequence verification obtains recombinant plasmid pEASY/degQ.
The building of 2.2 pNZES8149-degQ
Digested plasmid pNZES8149 and pEASY/degQ are distinguished using restriction enzyme, and recycling obtains target fragment, T4 connection
Enzyme connection, it is heat-shock transformed to obtain bacillus coli DH 5 alpha in, containing 100 μ g/ml ampicillins, 40 μ g/ml X-Gal and
The hand of PCR amplification is used after cultivating 16 hours on the LB solid medium of 24 μ g/ml isopropyl-beta D-thio galactopyranosides
Section screening positive clone, sequence verification obtain recombinant plasmid pNZES8149-degQ, and testing result is as shown in figures 1 and 2.
It should be understood that those skilled in the art, can be improved or be become according to the above description
It changes, and all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Sequence table
<110>Hangzhou Pedagogic University
<120>the lactic acid bacteria excretion vector and its construction method, application of recombinant screen can be realized in Escherichia coli
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gtcgacatga gtattcaaca tttccgtgt 29
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtcgaccgcg ttgctggcgt tttt 24
<210> 3
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggatccatgc gtaaacctct ttctgt 26
<210> 4
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cccgggacgg atcaccagat agagg 25
<210> 5
<211> 1635
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240
cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540
cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840
tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 900
ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 960
accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 1020
aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 1080
ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 1140
gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 1200
ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 1260
ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 1320
ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 1380
gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg 1440
cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 1500
cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 1560
cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 1620
aacgccagca acgcg 1635
<210> 6
<211> 708
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
caatctgcct cctcatcctc ttcatcctct tcgtcttggt agctttttaa atatgggtcg 60
atcgaattcg gtcctcggga tatgataaga ttaatagttt tagctattaa tcttttttta 120
tttttattta agaatggctt aataaagcgg ttactttgga tttttgtgag cttggactag 180
aaaaaaactt cacaaaatgc tatactaggt aggtaaaaaa atattcggag gaattttgaa 240
atgaaaaaga agattatctc agctattctt atgtcaacag ttatcttatc agctgctgct 300
ccactttcag gtgtttatgc tgacacaaac tcagatcttg aaatctcatc aacttgtgac 360
gctgtcgacc tgcagggtgg atcaggaggt gctccccaga ctattacaga actatgttcg 420
gaatatcgca acacacaaat atatacgata aatgacaaga tactatcata tacggaatcg 480
atggcaggca aaagagaaat ggttatcatt acatttaaga gcggcgaaac atttcaggtc 540
gaagtcccgg gcagtcaaca tatagactcc cagaaaaaag ccattgaaag gatgaaggac 600
acattaagaa tcacatatct gaccgagacc aaaattgata aattatgtgt atggaataat 660
aaaaccccca attcaattgc ggcaatcagt atggaaaact agaagctt 708
<210> 7
<211> 1362
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atgcgtaaac ctctttctgt gctcagtgtg ttagccctca gtgtcggtat cgccatgtct 60
gcggcccctg tacaggctgc actgccctct ctgctggcga acaatcagga gatgccgagc 120
ctggcgccgg tgctcgagca ggtcacccct gccgtggtca acatttcagt ctccggcaag 180
aagatcaccc gccagcgtct gccggaacag ttccgtttct tcttcggccc caacatgccc 240
gacgagcagg tgagcgagca gcccttccag gcgctgggct ccggcgtcat catcgatgcc 300
aagaagggct acgtgatcac caacgcccac gtggtgcacg aggcggacga gatcaaggtc 360
aacctgaagg atggccgtga atacgccgcc aagaagatcg gcgaggacaa gcagtccgac 420
atcgcgctgc tgcaaatcaa ggccgaggat ctggtgcaga tcaagtttgc cgactccgac 480
gagctgcggg tcggtgacta tgcactggcc atcggcaacc cgtttggcct cggccagacc 540
gtcacctccg gcatcgtcag tgcgcttggc cgcagcggcc tcaacatcga aaatctggaa 600
aacttcatcc agaccgatgc cgccatcaac tccggcaact caggtggcgc cctgctcaac 660
ctgcgcggcg agctgatcgg catcaacacc gccatcctgg gcccgaacgg cggcaacatc 720
ggcatcggct tcgccattcc gtccaacatg gtgcgcgacc tgtccgagca gatcgtgaaa 780
tacggtgaag tgcgccgcgg ccaactgggc atcaccggca ccgagcttac ctccgaaatc 840
gccaagacct tcggctacaa caagaaagat ggcgccttcg tcaaccaggt catgcctgat 900
tccgccgccg ccaaggcggg catcaaggcc ggcgatatca tcatcagcat cgatggcaag 960
gccatccgct cgtttggtga gctgcgcgcc aagatcgcca ccatgggggc tgacaagcag 1020
gtcgctctgg gcctcatccg cgatggcaag gagcagaccg tcaaggtcac gctgaagaag 1080
gcggatgaca gcgagatcct ggccagcgcc ctgcacccgg cactggaagg ggccaagctg 1140
agcaccaccg ccgagccggt gaccggtgtg gccgtggccg acatagatcc acgctcgcct 1200
gccgccgctt ccggcctgca gaagggtgac atcatcatcg gggtcaaccg cctgcgcatc 1260
aactcgctgg aagagctgag caaggcgttg aagaacaagc cggatgtgct ggcgctgaac 1320
atccagcgcg gggaatcctc cctctatctg gtgatccgtt aa 1362
Claims (9)
1. the lactic acid bacteria excretion vector that one kind can realize recombinant screen in Escherichia coli, it is characterised in that:
It includes ampicillin resistance sequence Escherichia coli replicon ori, Escherichia coli constitutive promoter P32, lactic acid bacteria point
Pil signal peptide USP45, multiple cloning sites and Escherichia coli heat-labile LTB gene order.
2. one kind according to claim 1 can realize the lactic acid bacteria excretion vector of recombinant screen in Escherichia coli,
It is characterized by also including ampicillin resistance sequences.
3. one kind according to claim 1 can realize the lactic acid bacteria excretion vector of recombinant screen in Escherichia coli,
It is characterized by also including PBR322 replicons.
4. one kind according to claim 1 can realize the lactic acid bacteria excretion vector of recombinant screen in Escherichia coli,
It is characterized by: after the Escherichia coli constitutive promoter P32 and lactic acid bacteria secreting signal peptide USP45, there are polyclonal positions
Point.
5. one kind according to claim 1 or 4 can realize that the lactic acid bacteria secreting type of recombinant screen carries in Escherichia coli
Body, it is characterised in that: after the Escherichia coli heat-labile LTB gene order is located at multiple cloning sites.
6. one kind according to claims 1 to 4 can realize that the lactic acid bacteria secreting type of recombinant screen carries in Escherichia coli
Body, it is characterised in that: it is located in Escherichia coli Dh5 α.
7. one kind according to claims 1 to 4 can realize that the lactic acid bacteria secreting type of recombinant screen carries in Escherichia coli
Body, it is characterised in that: it is present in Lactobacillus casei by electroporated mode.
8. a kind of lactic acid bacteria secreting type that can realize recombinant screen in Escherichia coli according to claims 1 to 4 carries
The construction method of body, it is characterised in that the following steps are included:
It is sequentially prepared and obtains ampicillin resistance sequence Escherichia coli constitutive promoter P32, lactic acid bacteria secreting signal peptide
USP45, multiple cloning sites, the acquisition of Escherichia coli heat-labile LTB gene order can realize recombination in Escherichia coli
The lactic acid bacteria excretion vector of son screening.
9. one kind according to claims 1 to 4 can realize that the lactic acid bacteria secreting type of recombinant screen carries in Escherichia coli
Body, it is characterised in that: it can be applied to the screening lactobacillus expression vector foreign gene recon in Escherichia coli.
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Cited By (1)
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| CN115521891A (en) * | 2021-06-24 | 2022-12-27 | 中国科学院青岛生物能源与过程研究所 | Recombinant bacterium for producing acetaminophen and application thereof |
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