CN103146739A - Establishing method of bifidobacterium functional gene no-trace knockout method - Google Patents
Establishing method of bifidobacterium functional gene no-trace knockout method Download PDFInfo
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Abstract
The invention discloses an establishing method of a bifidobacterium functional gene no-trace knockout method. The establishing method comprises the following steps of: constructing a serpin gene suicide type targeting vector UPD; loading a promoter of the bifidobacterium by using pDG7 as a framework to construct a bifidobacterium-escherichia coli shuttle expression vector pDG-Hup; constructing a bifidobacterium-escherichia coli shuttle expression vector Pdg-hup-cre of a Cre enzyme gene based on pDG-Hup; transferring the targeting vector into the bifidobacterium and obtaining a bifidobacterium serpin gene defective strain through homologous recombination; and removing the spectinomycin spc gene introduced in the bifidobacterium genome by utilizing a site-specific recombination Cre-loxp system to realize no-trace knockout of the bifidobacterium resistance gene. The establishing method of the bifidobacterium functional gene no-trace knockout method disclosed by the invention is used for obtaining the defective strain of the bifidobacterium functional gene by utilizing a homologous recombination principle, can be applied to the knockout of the bifidobacterium functional gene and can be used for improving the strains.
Description
Technical field
The invention belongs to the application in the bifidus bacillus genetically engineered, specifically relate to the seamless knockout technique of a kind of bifidus bacillus functional gene.
Background technology
Gene knockout is a kind of mode of modifying native gene of utilizing DNA homology restructuring principle to carry out, make between the target gene homologous sequence on foreign DNA and recipient cell genomic dna and recombinate, foreign vector DNA site-directed integration is entered on the predetermined site of target gene, or replace with a certain DNA fragmentation of target gene, complete accurate modification and transformation to the genomic dna target gene, cause the structure of target gene or the sudden change of composition, cause gene function to be lost, and the gene of modified and transformation can be with copying of genomic dna and stable copying.It has, and polarization is strong, target gene can overcome blindness and the randomness of the random integration of mutagenesis with the characteristics of genomic dna genetic stability after knocking out, and is a kind of desirable modification, the method for transformation microorganism hereditary material.In the Research for Industrial Microbial Germ improvement, flow by pathways metabolism and metabolism that knocking out of target gene changes microorganism, can not only improve the output of leavened prod and produce new meta-bolites, can also improve the artificial regulatory of fermentation industry fermentation approach and process, be the most promising microbial strains improved means of 21 century.
The application of Cre-loxp system in gene targeting can be said to gene target position technology and bring a revolution.The Cre recombinase is a kind of activated protein of molecular weight 38kd that derives from the recombinase family of pl phage.It not only has catalytic activity, and similar with restriction enzyme, identifies special DNA sequence dna, as: the Loxp site causes restructuring.The Loxp site is the specificity recombination site of Cre recombinase effect, formed by 34 bp, that is: and ATAACTTCGTATAGCATACATTATACGAAGTTAT, the interval region of the foldback sequence of two 13 bp and 8 bp consists of.The Cre recombinase has 70% recombination efficiency, not by any cofactor, acts on the DNA substrate of various structures, as linear, ring-type, and superhelix (being become linear structure by the uncoiling of Cre recombinase) even.The Cre recombinase by with the combination in the upper Loxp of DNA site, bring into play its recombination.The Cre/Loxp system is short and pithy, the effect of Cre enzyme is single-minded, and do not affect normal expression and the regulation and control of gene, these characteristics make it in the gene targeting operation, very important effect be arranged, and this system successfully is used for the gene knockout of multiple eukaryotic cell and bacterium at present.
Bifidus bacillus (
Bifidobacterium)Can keep the microecological balance of host's enteron aisle, improve human body to the lactose tolerance, have the blood fat of reduction, antineoplastic action, and can synthesize multivitamin, promote body's immunity.In recent years, preparation and food development that state includes bifidobacteria viable bacteria are rapid, and the bifidus bacillus product puts goods on the market.Abroad, the food that contains bifidus bacillus is also all the fashion, particularly in Japan, Europe and North America.Yet bifidus bacillus is as a kind of gram-positive microorganism, and cell walls is thicker, and it is carried out genetic manipulation is a difficult point always, has limited the progress in bifidus bacillus genetically engineered field.
The present invention is by the conversion of bifidus bacillus, targeting vector and shuttle expression carrier are imported bifidus bacillus step by step, and first with the Cre-loxp system applies in the genetically engineered of gram-positive microorganism bifidus bacillus, realized that successfully the seamless of bifidus bacillus functional gene knocks out, provide means for studying its gene function.
Summary of the invention
The purpose of this invention is to provide the seamless knockout technique of a kind of bifidus bacillus functional gene; The method first with the Cre-loxp system applies in the research of gram-positive microorganism bifidus bacillus, obtained the characteristics such as the seamless gene-deleted strain of bifidus bacillus functional gene.
The concrete technical scheme of the present invention is as follows:
The establishment method of the seamless gene-deleted strain of a kind of bifidus bacillus functional gene comprises the following steps:
(1) SEQ ID.NO.1 is passed through
EcoR V site and
PstThe I enzyme is cut and is connected on the pUC57 carrier, obtains the pUC57-LoxP carrier;
(2) take the pER8 plasmid as template, by primer SEQ. ID.NO.2 and SEQ. ID.NO.3 amplification, amplified production is passed through
XhoI and
ApaTwo the cutting afterwards of I is connected with the pUC57-LoxP carrier, builds and obtains carrier pUC-Spc;
(3) with the genome of bifidus bacillus as template, by primer SEQ. ID.NO.4 and SEQ. ID.NO.5 amplification, amplified production is passed through
EcoR I and
SpeTwo the cutting afterwards of I is connected with the pUC-Spc carrier, builds and obtains carrier pUC-spc-serpin
Up800
(4) with the genome of bifidus bacillus as template, by primer SEQ. ID.NO.6 and SEQ. ID.NO.7 amplification, amplified production is passed through
BglII and
HindIII is two cuts afterwards and pUC-spc-serpin
Up800Carrier connects, and builds to obtain targeting vector plasmid pUC-spc-serpin
Up+down, i.e. UPD;
(5) SEQ. ID.NO.8 sequence warp
EcoRI and
SalThe I double digestion is connected to pDG7, builds shuttle expression carrier pDG-Hup;
(6) take the pCrepA plasmid that contains Cre enzyme gene is template, by primer SEQ. ID.NO.9 and SEQ. ID.NO.10 amplification Cre enzyme gene; The amplified production process
XhoI and
SalI is two cut after, with pDG-Hup, is connected, structure obtains pDG-Hup-Cre;
(7) conversion of targeting vector: add targeting vector plasmid pUC-spc-serpin in the bifidus bacillus protoplastis
Up+down, merge conversion; Washing, recovery after recovery are coated on bacterium liquid and cultivate 2-3 d on the regeneration culture medium with spectinomycin, obtain
⊿ serpinStrain;
(8) will
⊿ serpinThe protoplastis competent cell is made in strain, and shuttle expression carrier pDG-Hup-Cre is imported
⊿ serpinConversion is merged in strain, and bacterium colony after transforming is coated on the regeneration culture medium of amicillin resistance, screens the bifidus bacillus of no longer carrying spectinomycin resistance gene
SerpinThe seamless gene-deleted strain of gene
⊿ serpin ⊿ spcStrain.
The condition that fusion described in step (7) and step (8) transforms is: join containing in the solution that magnesium ion concentration is 20 ± 1mM/L, 40 ± 1% (v/v) PEG6000 of preheating, merge conversion.
In step (7), described bifidus bacillus protoplast preparation is: the bifidus bacillus of preservation is transferred on the MRS flat board, and 37 ± 1 ℃ of anaerobism are cultivated 20-26 h; Choose a single bacterium colony from flat board, be inoculated in the MRS liquid nutrient medium, 37 ± 1 ℃ of anaerobism are cultivated 20-26h; Transfer in fresh MRS liquid nutrient medium with 3 ± 1%(v/v) inoculum size, 37 ± 1 ℃ of anaerobism are cultivated 20-26 h; Centrifugal collection thalline, resuspended; Adding mutanolysin to final concentration is 4.5-5.5 mg/L, 36-38 ℃ enzymolysis 25-35 minute; Be resuspended in SMM solution, obtain the bifidus bacillus protoplastis.
Preferably, described bifidus bacillus is bifidus longum bb.
Another object of the present invention is to provide a kind of seamless gene-deleted strain of bifidus bacillus functional gene that does not carry spectinomycin resistance gene.
Preferably, the seamless gene-deleted strain of described bifidus bacillus functional gene, it is to be prepared by above-mentioned method.
Advantage of the present invention is: present method substep imports bifidus bacillus with targeting vector and shuttle expression carrier, utilize the principle of homologous recombination, screening has obtained gene-deleted strain, express the Cre recombinase after importing shuttle expression carrier, removed resistant gene, thereby realized that the seamless of bifidus bacillus functional gene knocks out, utilize this method to carry out gene knockout, the recombinant bacterium that obtains does not at last carry any resistance screening mark, can avoid Antibiotic use in its use procedure, for the strain improvement of bifidus bacillus provides means.
Description of drawings
Fig. 1 is the structure schematic diagram of targeting vector UPD.
Fig. 2 is recombinant vectors pUC-Spc double digestion the result, M:1 K marker; The 1:pUC-spc carrier; 2,3:pUC-spc carrier is used respectively
XhoI，
ApaI double digestion result.
Fig. 3 is recombinant vectors pUC-spc-serpin
Up800The double digestion the result, M:1 K marker; 1,2:pUC-spc-serpin
Up800Carrier; 3,4: pUC-spc-serpin
Up800Carrier is used respectively
EcoR I,
SpeI double digestion result.
Fig. 4 is targeting vector pUC-spc-serpin
Up+downDouble digestion checking electrophoresis result, 1 K marker; 1:pUC-spc-serpin
Up+downCarrier is used respectively
BglII,
HindIII double digestion result; 2:pUC-spc-serpin
Up+downCarrier is used respectively
XhoI,
HindIII double digestion result.
Fig. 5 is the Cre-loxP system schematic.
Fig. 6 is recombinant plasmid pDG-Hup-Cre structural representation.
Fig. 7 is that the pDG-Hup enzyme is cut the result, pDG-HUP transformant double digestion checking 1: γ Hind III 2:pDG7 double digestion (
BamThe H I with
SalI) the 3:pDG-HUP double digestion (
BamThe H I with
SalI) 4:pDG7 not enzyme cut 5:pDG-HUP not enzyme cut the 6:pDG-HUP double digestion (
BglII with
EcoThe R I) the 7:pDG7 double digestion (
BglII with
EcoThe R I) 8:DL2000.
Fig. 8 is that the pDG-Hup-Cre enzyme is cut the result, from left to right 1:DL2000; 2:pDG-Hup-Cre; 3:pDG-Hup-Cre; 4: λ Hind III.
Fig. 9 is that pDG-Hup-Cre transforms
⊿ serpinThe dull and stereotyped result of bifidus bacillus.
Figure 10 is the positive transformant collection of illustrative plates of PCR checking, 1:500-15000 bp Wide range maker; 2:
spcPositive control; 3/4:
spcPrimer PCR detects
⊿ serpinStrain; 5:
spcPrimer PCR detects
⊿ serpin ⊿ spcStrain; 6:
Serpin Positive control 7/8:
SerpinPrimer PCR detects
⊿ serpinStrain; 9:
SerpinPrimer PCR detects
⊿ serpin ⊿ spcStrain.
Figure 11 is that PCR checking and enzyme are cut checking pDG-Hup-Cre conversion
⊿ serpinThe bifidus bacillus electrophoretogram.PCR verifies electrophoretogram (left side), the recombinant plasmid electrophoretogram (right side) after enzyme is cut and transformed in the bifidus bacillus body.
Figure 12 bifidus bacillus genome dna electrophoresis figure, 1/2:NCC2705 prime strain genome; 3/4:
⊿ serpinThe pnca gene group; 5/6:
⊿ serpin ⊿ spcThe pnca gene group; M:Wide range marker 500-15000 bp.
The positive transformant dotting of Figure 13 proof diagram A:T-spc is probe, A1: blank, and A2:
⊿ serpin ⊿ spc, A3
⊿ serpinStrain, A4
⊿ serpinStrain, A5 Bifidobacterium longum NCC2705 genome (negative control), A6
spc+ (positive control), B:T-
SerpinBe probe; B1: blank B2:
⊿ serpin ⊿ spcStrain, B3:
⊿ serpinStrain, B 4:
⊿ serpinStrain, B5: Bifidobacterium longum NCC2705 genome (positive control), B6:
Serpin+ (positive control).
Figure 14 is Southern hybridization analysis figure, A: the Southern hybridization figure take T-serpin as probe, B: the Southern hybridization figure take T-spc as probe; A1:
⊿ serpinStrain; A2:
⊿ serpin ⊿ spcStrain; A3:NCC2705 bifidus longum bb genome; A0: negative control; B1:
⊿ serpinStrain; B2:
⊿ serpin ⊿ spcStrain; B3: Bifidobacterium longum NCC2705 genome; B0: negative control.
Figure 15 promotor.
Embodiment
Embodiment 1: the structure of bifidus bacillus targeting vector and shuttle expression carrier:
1. in view of the characteristics of LoxP site sequence, directly synthesize on the pUC57 carrier in LoxP that both direction is identical site, sequence (totally 108 bp) from
EcoR V site and
PstInsert in the I site, will insert respectively spectinomycin resistance gene from centre and the two ends in two LoxP sites afterwards
spcHomology arm sequence with upstream and downstream.
This step carrier construction pUC57-LoxP.Two synthetic LoxP site sequences are:
5 '-CGG
ACTAGTATAACTTCGTATAATGTATGCTATACGAAGTTAT
CTCGAGAACGATCGGTT
GGGCCCATAACTTCGTATAATGTATGCTATACGAAGTTAT
AGATCTCC-3 ' (SEQ ID.NO.1), restriction endonuclease is followed successively by
SpeI,
XhoI,
ApaI,
BglII.
2. take the pER8 plasmid as template, design primer P-Spc-F:5 '-CCG
CTCGAGTGGTCCAGAACCTTGACCG-3 ' (
XhoI), (SEQ. ID.NO.2)
P-Spc-R:5 '-CCC
GGGCCCTTATTTGCCGACTACCTTGGTG-3 ' (
ApaI),, (SEQ. ID.NO.3) spectinomycin resistance gene that increases, amplification system (40 μ L: comprise pER8 plasmid dilution template 4 μ L, 2 * Taq mix, 20 μ L, 10 μ M primer pair 4 μ L, ddH
2O 12 μ L), the pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 50 seconds, 60 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, PCR product process
XhoI and
ApaTwo the cutting afterwards of I is connected with the pUC57-LoxP carrier, carrier construction pUC-Spc.Fig. 1 is targeting vector building process schematic diagram, and Fig. 2 is pUC-Spc double digestion the result.
3. design primer Serpin-up800-F:5 '-CCG
GAATTCAACCAGCACGTCAGGCC-3 ' (
EcoR I), (SEQ. ID.NO.4), Serpin-up800-R:5 '-CGG
ACTAGTGGTTGGCCCCTTTGCTTG-3 ' (
SpeI), (SEQ. ID.NO.5) with the genome of Bifidobacterium longum NCC2705 as masterplate, amplification
SerpinUpstream 800 bp fragments, amplification system (40 μ L: comprise the genome dilution template 4 μ L of NCC2705,2 * Taq mix, 20 μ L, 10 μ M primer pair 4 μ L, ddH
2O 12 μ L), the pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 30 seconds, 57 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, PCR product process
EcoR I and
SpeTwo the cutting afterwards of I is connected with pUC-Spc, successful carrier construction pUC-spc-serpin
Up800Fig. 3 is pUC-spc-serpin
Up800The double digestion the result.
4. design primer Serpin-down800-F:5 '-GGA
AGATCTTACTTTCCTGGCAGTCCGTC-3 ' (
BglII) (SEQ. ID.NO.6)
Serpin-down800-R:5 '-CCC
AAGCTTCGTACACATACCAGGCGCG-3 ' (
HindIII), (SEQ. ID.NO.7) with the genome of Bifidobacterium longum NCC2705 as masterplate, amplification
SerpinDownstream 800 bp gene fragments, amplification system 40 μ L: comprise the genome dilution template 4 μ L of Bifidobacterium longum NCC2705,2 * Taq mix, 20 μ L, 10 μ M primer pair 4 μ L, ddH
2O 12 μ L, the pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 30 seconds, 56 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, PCR product process
BglII and
HindIII is two cuts afterwards and pUC-spc-serpin
Up800Connect successful carrier construction pUC-spc-serpin
Up+downBe UPD.Fig. 4 is targeting vector pUC-spc-serpin
Up+downIt is UPD double digestion checking electrophoresis result.
5. take the recombinant plasmid pDG7 that contains bifidus bacillus endogenous replicon as skeleton, 200 bp(of synthetic Hup gene start codon and upstream thereof comprise the Hu promoter sequence), and add multiple clone site (MCS) after its sequence, add respectively restriction enzyme site in its two ends
EcoRI and
SalI, double digestion is connected to pDG7, successfully builds shuttle expression carrier pDG-Hup.Fig. 7 is that the pDG-Hup enzyme is cut the result.
Synthetic promoter sequence (totally 283 bp) (as Figure 15)
6. design primer Cre-F:5 '-CCGCTCGAGATGTCCAATTTACTGACCGTACACC-3 '
XhoI, (SEQ. ID.NO.9)
Cre-R:5 '-CGCGTCGACCTAATCGCCATCTTCCAGCAG-3 '
SalI, (SEQ. ID.NO.10)
Take pCrepA plasmid (containing Cre enzyme gene) as template, amplification Cre enzyme gene.Amplification system 40 μ L: comprise pCrepA plasmid dilution template 4 μ L, 2 * Pfu mix, 20 μ L, 10 μ M primer pair 4 μ L, ddH
2O 12 μ L, the pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 30 seconds, 62 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, PCR product process
XhoI and
SalI is two cut after, with pDG-Hup, be connected, successfully build pDG-Hup-Cre.Fig. 5 is the Cre-loxP system schematic, and Fig. 6 is recombinant plasmid pDG-Hup-Cre collection of illustrative plates.
Embodiment 2: bifidus longum bb protoplast preparation and conversion:
1. the conversion of targeting vector: Bifidobacterium longum NCC2705 is transferred to (MRS nutrient agar: peptone 10 g, extractum carnis 10 g, yeast extract paste 5 g, Tween-80 1 g, K on the MRS flat board from the glycerine pipe of-70 ℃ of preservations
2HPO
42 g, CH
3COONa3H
2O 5 g, Citric Acid three ammonium 2 g, MgSO
47H
2O 0.2 g, MnSO
4H
2O 0.05 g, Agar 15 g, glucose 20 g, adding distil water to 1000 mL transfers 6.4,121 ℃ of autoclaving 15 min of pH 6.2-.The MRS broth culture: the same agar that do not add of composition), 37 ℃ of anaerobism are cultivated 24 h.Choose a single bacterium colony from flat board, be inoculated in 5 mL MRS liquid nutrient mediums, 37 ℃ of anaerobism are cultivated 24 h; Then transfer in 25 fresh mL MRS liquid nutrient mediums with the inoculum size of 3% (V/V), 37 ℃ of anaerobism are cultivated 24 h; Centrifugal collection thalline, resuspended; Adding mutanolysin to final concentration is 4.5-5.5 mg/L, 36-38 ℃ enzymolysis 25-35 minute; Be resuspended in SMM(Hepes (4-hydroxyethyl piperazine ethanesulfonic acid) 0.02 M/L, magnesium chloride 1 mM/L, raffinose 0.5 M/L) in solution, obtain the bifidus bacillus protoplastis.The targeting vector plasmid pUC-spc-serpin that adds 50 μ L precoolings in the protoplastis of 500 μ L bifidus longum bbs
Up+down(plasmid DNA amount＜5 μ g) add 40% (v/v) PEG6000 solution, 1.5 mL(of preheating to contain magnesium ion concentration 20 mM/L) the fusion conversion.Add SMMP(liquid regeneration culture medium after conversion: SMM=3:1) solution washing is 2 times.Add 1 mLSMMP recovery 1 h.After recovery, bacterium liquid is coated on spectinomycin (final concentration 100 μ g/mL) regeneration solid medium (tryptone 5.0 g, yeast extract 5.0 g, aminothiopropionic acid hydrochloride 0.4 g, glucose 5.0 g, KH
2PO
42.5 g, CH
3COONa 10.0 g, CaCl
22H
2O 5.0 g, MgCl
26H
2O 5.0 g, agar 15.0 g, raffinose 0.2-0.3M/L transfers 6.5,115 ℃ of autoclaving 20 min of pH) the upper 2-3 of cultivation d; Obtain
⊿ serpinStrain;
2. picking
⊿ serpinBacterium colony is activated in the MRS liquid nutrient medium of 100 μ g/mL spectinomycin resistances.Extract
⊿ serpinStrain bifidus bacillus genomic dna, as template, design primer serpin-F:5 '-ACC AAT CGC TGC TAA GTT CG-3 ' (SEQ. ID.NO.11), R:5 '-GAA CCG GTG TCG ATC ATC TT-3 ' (SEQ. ID.NO.12) amplification serpin gene, amplification system (10 μ L: the genome dilution template 1 μ L that comprises transformant, 2 * Taq mix, 5 μ L, 10 μ M primer pair 1 μ L, ddH
2O 3 μ L), the pcr amplification condition be 95 ℃ 10 minutes, then through 95 ℃ 30 seconds, 60 ℃ 40 seconds, 72 ℃ 1 minute 30 times circulations, 72 ℃ 10 minutes, detect before and after restructuring
SerpinThe variation of gene signal; With primer spc-F/R(SEQ. ID.NO.2 and SEQ. ID.NO.3) amplification
spcGene, amplification system is with amplification system and the response procedures in 2 steps in embodiment 1, after detecting restructuring
spcGene signal.Be verified as positive
⊿ serpinStrain is activated in the MRS liquid nutrient medium of 100 μ g/mL spectinomycin resistances.
3. will
⊿ serpinThe protoplastis competent cell is made in strain, and shuttle expression carrier pDG-Hup-Cre is imported
⊿ serpinStrain adds 40% (v/v) PEG6000 solution, 1.5 mL(of preheating to contain magnesium ion concentration 20 mM/L) the fusion conversion.Bacterium colony after transforming is coated on regeneration solid medium (tryptone 5.0 g, yeast extract 5.0 g, aminothiopropionic acid hydrochloride 0.4 g, glucose 5.0 g, the KH of 25 μ g/mL amicillin resistances
2PO
42.5 g, CH
3COONa 10.0 g, CaCl
22H
2O 5.0 g, MgCl
26H
2O 5.0 g, agar 15.0 g, raffinose 0.2-0.3M/L transfers 6.5,115 ℃ of autoclaving 20 min of pH) on, obtain possible
⊿ serpin ⊿ spcStrain.
4. picking
⊿ serpin ⊿ spcBacterium colony is activated in 25 μ g/mL amicillin resistance MRS liquid nutrient mediums.Extract
⊿ serpinStrain bifidus bacillus genomic dna is as template, with primer spc-F/R(SEQ. ID.NO.2 and SEQ. ID.NO.3) amplification
spcGene, amplification system is with amplification system and the response procedures in 2 steps in embodiment 1, after detecting restructuring
spcGene signal.The bacterial strain that no longer carries spectinomycin resistance gene is positive
⊿ serpin ⊿ spcStrain.Screening obtains
⊿ serpin ⊿ spcStrain is activated to the MRS substratum of not being with resistance and uploads culture, further checking.
Embodiment 3: bifidus bacillus
SerpinThe checking of gene-deleted strain:
With
⊿ serpin ⊿ spcPnca gene group DNA is template, with primer serpin-F/R(SEQ. ID.NO.11 and SEQ. ID.NO.12) amplification
SerpinGene (amplification system and response procedures are with step 2 in embodiment 2) detects in regrouping process
SerpinThe variation of gene signal; With primer SEQ. ID.NO.2 and SEQ. ID.NO.3 amplification
spcGene detects shuttle expression carrier and transforms front and back
spcGene signal.Figure 10 is the PCR the result after substep transforms, warp
SerpinThe amplification of genetically deficient fragment PCR and 1% agarose gel electrophoresis show
⊿ serpinStrain can amplify 1000 bp, shows in the transformant genome to contain
spcGene;
⊿ serpinStrain fail the to increase fragment of 1400 bp shows that homologous recombination has occured the homologous fragment on target practice fragment and karyomit(e),
SerpinThe gene quilt
spcGene substitution has built
SepinGene-deleted strain; Yet
⊿ serpin ⊿ spcStrain is failed to increase and is arrived
SerpinGene has also lost
spcThe signal of gene, this illustrates under the help of shuttle expression carrier, the resistant gene between Cre recombinase LoxP site that both direction is identical
spcFragment has been deleted.
With
⊿ serpin ⊿ spcThe genomic dna of strain is template, with primer Cre-F/R amplification cre gene, and PCR checking pDG-Hup-Cre positive transformant (amplification system is with 6 step implementation steps in embodiment 1), and extract the recombinant plasmid enzyme and cut checking.Fig. 9 is for transforming dull and stereotyped photo, and the positive transformant PCR of Figure 10 proof diagram, Figure 11 are that pDG-Hup-Cre transforms the PCR checking and enzyme is cut proof diagram.Result shows, shuttle expression carrier pDG-Hup-Cre has successfully imported bifidus bacillus.
3. Figure 12 is that glass bead method is extracted the bifidus longum bb genomic dna, and electrophoresis result shows.Electrophoretic band concentrates on 15000 bp, and the genome effect of extraction is better, can be used for further dotting or Southern hybridization check.Take spectinomycin spc digoxigenin labeled fragment T-spc as probe,
SerpinLabeled fragment T-serpin is that probe carries out respectively the dotting checking.Figure 13 shows
⊿ serpinStrain
He ⊿ serpin ⊿ spcStrain pair
SerpinGene all is revealed as feminine gender,
⊿ serpinStrain pair
spcThe spectinomycin gene is aobvious positive, and
⊿ serpin ⊿ spcStrain pair
spcThe spectinomycin gene is revealed as feminine gender, the result demonstration, and the Cre recombinase successfully will
spcGene has removed from the genome of bifidus bacillus recombinant bacterial strain.
4. will screen
⊿ serpinStrain and
⊿ serpin ⊿ spcSouthern hybridization checking is carried out in strain.Figure 14 shows that positive control A3 presents single band when hybridizing with the T-serpin probe, and A1/A2 is all without band, namely
⊿ serpinStrain and
⊿ serpin ⊿ spcIn strain without
SerpinGene signal; When hybridizing with the T-spc probe, B1
⊿ serpinStrain presents single band, and B3 is that the bifidus bacillus prime strain does not present band, namely
⊿ serpinIntroduced the spectinomycin resistance gene of external source in strain transformant genome, band does not appear in B2, explanation
⊿ serpin ⊿ spcOn the pnca gene group
spcResistant gene has been removed by the restructuring of Cre recombinase, has realized that successfully the seamless of bifidus bacillus knocks out.
These are only specific embodiments of the invention, do not limit protection scope of the present invention with this; Any replacement and the improvement done on the basis of not violating the present invention's design all belong to protection scope of the present invention.
Sequence table
＜110〉University Of Nanchang
＜120〉foundation of the seamless knockout technique of bifidus bacillus functional gene
<160> 12
<170> PatentIn version 3.1
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agatctcc 108
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<210> 7
<211> 28
<212> DNA
＜213〉artificial sequence
<400> 7
cccaagcttc gtacacatac caggcgcg 28
<210> 8
<211> 283
<212> DNA
＜213〉artificial sequence
<400> 8
cgcggatccc attccgctgg gcgcggcggc catgaagtgg cttgacaagc 50
ataatcttgt ctgattcgtc tattttcata cccccttcgg ggaaatagat 100
gtgaaaaccc ttataaaacg cgggttttcg cagaaacatg cgctagtatc 150
attgatgaca acatggacta agcaaaagtg cttgtcccct gacccaagaa 200
ggatgcttta tgctcgagga gctcgggccc ggtacctacg taagatctgc 250
ggccgccctg caggactagt gtcgacgtcg gcc 283
<210> 9
<211> 34
<212> DNA
＜213〉artificial sequence
<400> 9
ccgctcgaga tgtccaattt actgaccgta cacc 34
<210> 10
<211> 30
<212> DNA
＜213〉artificial sequence
<400> 10
cgcgtcgacc taatcgccat cttccagcag 30
<210> 11
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 11
accaatcgct gctaagttcg 20
<210> 12
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 12
gaaccggtgt cgatcatctt 20
Claims (6)
1. the establishment method of the seamless gene-deleted strain of bifidus bacillus functional gene, is characterized in that, comprises the following steps:
(1) SEQ ID.NO.1 is cut by EcoR V site and Pst I enzyme be connected on the pUC57 carrier, obtain the pUC57-LoxP carrier;
(2) take the pER8 plasmid as template, by primer SEQ. ID.NO.2 and SEQ. ID.NO.3 amplification spectinomycin resistance gene spc, amplified production be connected with Apa I through Xho I is connected with the pUC57-LoxP carrier after cutting, structure obtains carrier pUC-Spc;
(3) with the genome of bifidus bacillus as template, by primer SEQ. ID.NO.4 and SEQ. ID.NO.5 amplification, amplified production be connected with Spe I through EcoR I be connected with the pUC-Spc carrier after cutting, structure obtains carrier pUC-spc-serpin
Up800
(4) with the genome of bifidus bacillus as template, by primer SEQ. ID.NO.6 and SEQ. ID.NO.7 amplification, with amplified production through Bgl II and the Hind III is two cut after with pUC-spc-serpin
Up800Carrier connects, and builds to obtain targeting vector plasmid pUC-spc-serpin
Up+down, i.e. UPD;
(5) SEQ. ID.NO.8 sequence is connected to pDG7 through EcoR I and Sal I double digestion, builds shuttle expression carrier pDG-Hup;
(6) take the pCrepA plasmid that contains Cre enzyme gene as template, by primer SEQ. ID.NO.9 and SEQ. ID.NO.10 amplification Cre enzyme gene; Amplified production through Xho I and Sal I are two cut after, is connected with pDG-Hup, structure obtains pDG-Hup-Cre;
(7) conversion of targeting vector: with targeting vector plasmid pUC-spc-serpin
Up+downJoin in the bifidus bacillus protoplastis, merge conversion; Washing, recovery after recovery are coated on bacterium liquid and cultivate 2-3 d on the regeneration culture medium with spectinomycin, and De is Dao ⊿ serpin strain;
(8) make the protoplastis competent cell Jiang ⊿ serpin strain, with shuttle expression carrier pDG-Hup-Cre Dao Ru ⊿ serpin strain, merge and transform, bacterium colony after transforming is coated on the regeneration culture medium of amicillin resistance, screens the seamless disappearance of the bifidus bacillus serpin gene Zhu ⊿ serpin ⊿ spc strain of no longer carrying spectinomycin resistance gene.
2. establishment method according to claim 1, it is characterized in that, the condition that fusion described in step (7) and step (8) transforms is: join containing in the solution that magnesium ion concentration is 20 ± 1mM/L, 40 ± 1% (v/v) PEG6000 of preheating, merge conversion.
3. establishment method according to claim 1, is characterized in that, in step (7), described bifidus bacillus protoplast preparation is: the bifidus bacillus of preservation is transferred on the MRS flat board, and 37 ± 1 ℃ of anaerobism are cultivated 20-26 h; Choose a single bacterium colony from flat board, be inoculated in the MRS liquid nutrient medium, 37 ± 1 ℃ of anaerobism are cultivated 20-26h; Transfer in fresh MRS liquid nutrient medium with 3 ± 1%(v/v) inoculum size, 37 ± 1 ℃ of anaerobism are cultivated 20-26 h; Centrifugal collection thalline, resuspended; Adding mutanolysin to final concentration is 4.5-5.5 mg/L, 36-38 ℃ enzymolysis 25-35 minute; Be resuspended in SMM solution, obtain the bifidus bacillus protoplastis.
4. according to claim 1-3 described establishment method of any one, is characterized in that, described bifidus bacillus is bifidus longum bb.
5. seamless gene-deleted strain of bifidus bacillus functional gene that does not carry spectinomycin resistance gene.
6. the seamless gene-deleted strain of described bifidus bacillus functional gene according to claim 5, it is to be prepared by the described method of claim 1-4 any one.
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