CN107557372A - For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme SacI - Google Patents

For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme SacI Download PDF

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Publication number
CN107557372A
CN107557372A CN201710764317.6A CN201710764317A CN107557372A CN 107557372 A CN107557372 A CN 107557372A CN 201710764317 A CN201710764317 A CN 201710764317A CN 107557372 A CN107557372 A CN 107557372A
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China
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saci
restriction enzyme
alui
bacterial strain
enzyme saci
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张坤晓
龚雪梅
韩挺翰
许恒皓
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Jiangsu Yugong Life Science & Technology Co Ltd
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Jiangsu Yugong Life Science & Technology Co Ltd
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Abstract

The present invention is a kind of screening technique for the protection bacterial strain that methylates for being used to express restriction enzyme SacI; it passes through the foundation of transmethylase M.AluI recombinant strains, the restriction enzyme SacI step such as recombination expression; correct recombinant expression plasmid pBAD R.SacI transformed competence colibacillus cell will be sequenced and carry out screening and culturing, obtain restriction enzyme SacI recombinant strains；The bacterial strain is induced after expanding and cultivating with arabinose, obtains restriction enzyme SacI recombinant protein.The inventive method realizes the high efficiency recombinant expressed of restriction enzyme SacI；The harsh control expressed using arabinose operon expression system background carries out restriction enzyme SacI recombination expression；Digestions of the restriction enzyme SacI to host genome DNA is resisted, method is stable and efficient；The M.AluI filtered out, which methylates, protects bacterial strain to be similarly applied to other recombination expressions with the restriction enzymes similar to recognition site.

Description

For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme SacI
Technical field
The present invention relates to a kind of screening technique of bacterial strain, particularly a kind of methyl for being used to express restriction enzyme SacI Change the screening technique of protection bacterial strain.
Background technology
Restriction enzyme be can identify double chain DNA sequence distinguished sequence and be cut nuclease (Ch.8, Eds.De Bruijin, et al., Chapman＆Hall, New York, 78-92.1998), its discovery promotes DNA recombinant techniques Birth so as to greatly promote the development of modern molecular biology and genetic engineering, be that contemporary genetic engineering research is indispensable Master tool.In bacterium, restriction enzyme and its corresponding transmethylase are construed as limiting-modification system, micro- to protect Biology is not infected or plasmid by external bacteriophage.The restriction enzyme and transmethylase of same system have phase on DNA Same recognition sequence, but different, the non-recognition sequence to methylate of restriction enzyme cleavage is acted on, transmethylase is in identification sequence Special sex modification on row can prevent the cutting of restriction enzyme.The normal close linkage of limitation-modifier, bacterium pass through regulation and control Both expression, makes itself DNA be first subjected to the protection that methylates, and expresses restriction enzyme after the completion of protection again, and the allogeneic dna sequence DNA of invasion It is not protected thus is degraded by restriction enzyme.I.e. the related transmethylase pair of intracellular is matched in restricted with it The identical particular sequence of enzyme cutting identification carries out the modification that methylates, and has the DNA of modification and resist cutting for its restriction enzyme The characteristic cut, protect host DNA and exogenous DNA (Geoffery G.W., the Nucleic Acids not being methylated that degrades Res,2539-2564.1991)。
Show on the research that restriction modification system is expressed in prokaryotic, the foundation and maintenance of system are with methyl Premised on the protective effect of transferase.Structure composition and characteristic based on DNA molecular, three class DNA first are primarily present in nature Based transferase, it is C respectively5Cytimidine methylase, N4Cytimidine methylase and N6Adenine methylase.Wherein, N4Cytimidine and N6 Adenine methylase belongs to amino-methyl transferase (Malone et al.J.Mol.Biol.253:618-632.1995).When After the DNA of one restricted enzyme recognition site is modified by transmethylase, this DNA molecular, which will have, resists corresponding restriction enzyme Digestion characteristic.I.e. under normal circumstances, if DNA recognition sequences identical (or recognition sequence is included) and methylase Decorating site is also consistent with mode, then this DNA by the modification of transmethylase specific methylation just has and can supported Resist the effect of the restriction enzyme cleavage of similar recognition sequence, so as to protect the DNA damages of host cell.Such as EagI Recognition sequence be 5 ' CGGCCG3 ', (5 ' G among NotI recognition sequenceCGGCCGC3 '), and its position that methylates Point (C consistent with modification mode5Cytimidine methylase), thus restriction enzyme can be resisted by expressing M.EagI in Host Strains Digestions of the NotI to DNA.
From late 1960s after first restriction enzyme is found, increasing restriction enzyme quilt It was found that, purify and obtain application.On the research of restriction enzyme, their practical valencys as toolenzyme are focused on for many years Value, including discovery and the screening of new enzyme, the structure of high-yielding engineering bacterial strain, the Optimal improvements etc. of enzyme purification technology and program.Wherein SacI is a kind of widely used restriction enzyme, can recognize that the nucleotide sequence (5 ' GAGC of six basesTC3 '), no Molecular cloning only can be applied to as common restriction enzyme, also there is important application in other directions of biological field Value and research potential.It is but presently commercially available due to the problem such as expression quantity is low, it is cumbersome to isolate and purify program, albumen yield is low Not only price is high for SacI restriction enzymes, and also limit its extensive use and further investigation.It is meanwhile above-mentioned a series of Problem also limit the further investigation of other many widely used restriction enzymes.
Restriction enzyme has the characteristic for being capable of cutting DNA, once expression will cut host cell DNA, causes host The damage of cell is even dead.Therefore, it is necessary to the DNA to host cell could realize it after carrying out the specific modification that methylates Recombination expression.In the scheme of prior art one, natural specificity transmethylase M.SacI corresponding to SacI make use of to carry out place Main DNA protection.Specific steps include：Extract natural origin bacterial strain Streptomyces achromogenes chromosomal DNAs simultaneously Prepare DNA library；Characteristics of the DNA not by SacI cuttings is protected using M.SacI, M.SacI genes are filtered out from library；By band The plasmid conversion for having M.SacI genes enters in expressive host engineered strain.Obtaining expression places of the DNA by the protection that methylates After main works bacterial strain, SacI restriction enzyme alleles are implemented in expression vector, conversion enters above-mentioned bacterial strains, induces SacI restructuring table Reach.Purification step after expression is related to the affinity chromatography of Ni-based matter, ion-exchange chromatography, hydrophobic chromatography, reversed phase chromatography, heparin fine jade The chromatographic purifying process such as layer and gel molecular exclusion that lipolysaccharide is affine.Its shortcoming is：The protection that methylates to host cell DNA exists After cumbersome DNA library construction and screening, the protection bacterial strain that methylates obtained specific can only protect DNA from SacI The cutting of restriction enzyme, the rare expression that can be used for other restriction enzymes, application are extremely narrow.
The content of the invention
The technical problems to be solved by the invention are in view of the shortcomings of the prior art, there is provided one kind can be used for expression limitation Property restriction endonuclease SacI the new protection host strain that methylates.It is right first that DNA cuttings are needed according to restriction enzyme simultaneously The principle that recognition sequence is identified, the restriction enzyme that obtaining has similar recognition sequence to same class carry out the guarantor that methylates The bacterial strain of shield, i.e. one kind, which methylate, protects bacterial strain to carry out a variety of restriction enzyme expression of enzymes.
The technical problems to be solved by the invention are realized by following technical scheme.The present invention is
A kind of screening technique for the protection bacterial strain that methylates for being used to express restriction enzyme SacI, is characterized in,
(1) foundation of transmethylase M.AluI recombinant strains
A, the acquisition of transmethylase M.AluI genes
The acquisition of transmethylase M.AluI genes is using PCR amplifications, gene chemical synthesis or extracts chromosomal DNA and establishes library Method, the specific primer of transmethylase M.AluI genes is used from strains A rthrobacter luteus genome Obtain transmethylase M.AluI gene；
B, transmethylase M.AluI recombinant strains are built
M.AluI gene orders are inserted in low-copy expression vector pACYC184 plasmids in a manner of digestion-connection, connection production Thing conversion, which enters clone strain DH5 α and is coated on chlorampenicol resistant flat board, is cultivated；
C, methylate and protect the screening of bacterial strain
To bacterial strain after conversion methylate the screening of degree of protection；
D, the acquisition of restriction enzyme SacI genes
The acquisition of restriction enzyme SacI genes is using PCR amplifications, gene chemical synthesis or extracts chromosomal DNA and establishes the side in library Method, obtained from bacterial strain Streptomyces achromogenes genome using the specific primer of restriction enzyme SacI genes Obtain restriction enzyme SacI gene；
E, restriction enzyme SacI recombinant strains are built
Using arabinose operon expression system, SacI gene orders are inserted to pBAD expression matter in a manner of digestion-connection In grain, connection product conversion, which enters DH5 alpha expressions bacterial strain and is coated on ammonia benzyl chloramphenicol resistance flat board, is cultivated；By being sequenced, really Conversion enters R.SacI specificity protection bacterial strains [pACYC184-M.AluI, ER2566], structure after recognizing the successfully constructing of recombinant plasmid Build restriction enzyme SacI recombinant strains；
(2) restriction enzyme SacI recombination expression
To be sequenced correct recombinant expression plasmid pBAD-R.SacI transformed competence colibacillus cell [pACYC184-M.AluI, ER2566], screening and culturing on the flat board of the mycin of benzyl containing ammonia and chloramphenicol antibiotics is coated on, obtains restriction enzyme SacI restructuring table Up to bacterial strain；The bacterial strain is induced after expanding and cultivating with arabinose, obtains restriction enzyme SacI recombinant protein, and confirms bacterium Protein crude extract has restriction enzyme SacI activity.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above It is a kind of be used for express restriction enzyme SacI methylate protection bacterial strain screening technique, be characterized in, in step (1), Expanded from strains A rthrobacter luteus genome using the specific primer PCR of transmethylase M.AluI genes Increase the gene for obtaining transmethylase M.AluI；The method for obtaining transmethylase M.AluI genes is as follows：
After strains A rthrobacter luteus flat board culture bacterium colonies are resuspended with deionized water, 95 DEG C of incubation 10min, as PCR Template；Sense primer 5 ' of the primer sequence based on the transmethylase M.AluI genes-terminal sequence and anti-sense primer of PCR amplifications 3 '-end reverse complementary sequence is designed synthesis；
The primer sequence of synthesis is as follows:
Forward primer：5’-CATGCATATGGAGCTCATGGGTGATCAACTG-3’
NdeI SacI
Reverse primer：5’-TATGCTCGAGTTAGAGCTCTTCTGCTGCGCCCAGTGC-3’
XhoI SacI
This pair of primers is used for specific amplification M.AluI genes, while introduces NdeI and XhoI restriction enzyme site, convenient follow-up Digestion-connection and screening experiment；Pcr amplification reaction uses KOD-Pure+ DNA Polymerase, is reacted according to standard PCR Flow is carried out, and is obtained and theoretical value M.AluI gene fragment orders 1 of the same size；Above-mentioned pcr amplification product by digestion- The mode of connection forms recombinant expression plasmid with pACYC184 carriers.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above It is a kind of be used for express restriction enzyme SacI methylate protection bacterial strain screening technique, be characterized in, in step (1), In step (1), the pACYC184 expression vectors for preparing M.AluI are carried out according to below scheme：
PACYC184 plasmids are with LightningTMNdeI and LightningTMAfter XhoI restriction enzyme double digestions, then with Alkaline Phosphatase (Fast) carry out dephosphorylation process, and digestion products enter row agarose gel electrophoresis, and tap rubber The pACYC184 carrier segments of linearisation are reclaimed, are dissolved in TE Buffer；
Enter performing PCR amplification to M.AluI genes using KOD-Pure+DNA Polymerase, fine jade is carried out to the PCR primer of acquisition Sepharose electrophoresis, confirm after obtaining specific amplification fragment, use LightningTMNdeI and LightningTMXhoI is limited It is connected after enzyme double digestion with the pACYC184 carrier segments of linearisation, according to molal weight than 1:3 concentration, through T4 DNA Ligase (Fast) is entered in DH5 α competent cells after 1h is reacted at 22 DEG C using chemical transformation conversion；Sieved in chloramphenicol Cell culture is carried out under the conditions of choosing, obtains M.AluI recombinant expression plasmid pACYC184-M.AluI；Plasmid pACYC184- The correctness of M.AluI sequences confirms by sequencing.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above It is a kind of be used for express restriction enzyme SacI methylate protection bacterial strain screening technique, be characterized in, in step (1), The screening of M.AluI protection bacterial strains is carried out according to below scheme：Correct plasmid pACYC184-M.AluI will be sequenced and pass through chemistry Conversion method conversion enters coli strain ER2566, and cell culture is carried out under chloramphenicol screening conditions；Monoclonal is selected, Rule on agar plate containing 35 μ g/ml chloramphenicol antibiotics, and carry out corresponding numbering after 37 DEG C, 200rpm culture 16h, Bacterium solution is collected to propose plasmid again；In 10 μ l reaction systems, 0.2 μ l restriction enzymes are taken Under buffer condition, 1h is incubated at 37 DEG C with 100ng recombinant plasmids pACYC184-M.AluI, then using 0.8% Agarose gel electrophoresis detection substrate quiltThe situation of digestion；Confirm the recombinant plasmid in host bacterial by The complete protection of M.AluI transmethylases, acquiescence Host genomic DNA equally can be by expressed by pACYC184-M.AluI M.AluI transmethylases protection；The bacterial strain accordingly numbered on flat board is used into CaCl after verifying protectiveness again2Processing, It is prepared into competent cell [pACYC184-M.AluI, ER2566].
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above It is a kind of be used for express restriction enzyme SacI methylate protection bacterial strain screening technique, be characterized in, in step (1), The PCR amplification method of restriction enzyme SacI genes is as follows：
After bacterial strain Streptomyces achromogenes flat board culture bacterium colonies are resuspended with deionized water, 95 DEG C of incubation 10min, As pcr template；Sense primer 5 ' of the primer sequence based on the restriction enzyme SacI genes-terminal sequence and anti-sense primer of PCR amplifications 3 '-end reverse complementary sequence；The primer sequence of synthesis is as follows:
Forward primer：5’-GAACCATGGGCATTACC-3’
NcoI
Reverse primer：5’-GCCAAGCTTAGGTTTCC-3’
HindIII
This pair of primers is used for specific amplification R.SacI genes, and introduces NcoI, HindIII restriction enzyme site, facilitates follow-up enzyme Cut-connect；Pcr amplification reaction uses KOD-Pure+ DNA Polymerase, is carried out according to standard PCR reaction process, obtains With theoretical value R.SacI gene fragment orders 2 of the same size；Above-mentioned pcr amplification product by way of digestion-connection with PBAD carriers form recombinant expression plasmid.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above It is a kind of be used for express restriction enzyme SacI methylate protection bacterial strain screening technique, be characterized in, in step (1), The pBAD expression vectors for preparing SacI are carried out according to below scheme：
PBAD plasmids are with LightningTMNcoI and LightningTMAfter HindIII restriction enzyme double digestions, then with Alkaline Phosphatase (Fast) carries out dephosphorylation process；Digestion products enter row agarose gel electrophoresis, and recovery linearisation of tapping rubber PBAD carrier segments, be dissolved in TE Buffer；
Enter performing PCR amplification to R.SacI genes using KOD-Pure+DNA Polymerase, fine jade is carried out to the PCR primer of acquisition Sepharose electrophoresis, confirm after obtaining specific amplification fragment, use LightningTMNcoI and LightningTM HindIII It is connected after restriction enzyme double digestion with the pBAD carrier segments of linearisation, according to molal weight than 1:3 concentration, through T4 DNA Ligase (Fast) is entered in DH5 α competent cells after 1h is reacted at 22 DEG C using chemical transformation conversion；In ammonia benzyl mycin Cell culture is carried out under the conditions of resistance screening, obtains R.SacI recombinant expression plasmid pBAD-R.SacI；Plasmid pBAD- The correctness of R.SacI sequences confirms by sequencing.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above It is a kind of be used for express restriction enzyme SacI methylate protection bacterial strain screening technique, be characterized in, in step (2), Restriction enzyme SacI recombinant expression method is as follows：Correct recombinant expression plasmid pBAD-R.SacI conversions will be sequenced [pACYC184-M.AluI, ER2566] competent cell, it is coated on the flat board of the mycin of benzyl containing ammonia and chloramphenicol antibiotics and screens Culture, obtain restriction enzyme SacI recombinant strains；In LB inoculation of medium restriction enzymes SacI recombinant strains Afterwards, cultivated in 37 DEG C of shaking tables with 200rpm to OD 0.8, then add derivant arabinose, final concentration 0.2%, and at 16 DEG C Shaking table cultivates 16h with 200rpm.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above It is a kind of be used for express restriction enzyme SacI methylate protection bacterial strain screening technique, be characterized in, in step (2), Restriction enzyme SacI vigor testing methods are as follows：After gradient dilution being carried out to the restriction enzyme SacI obtained after purified, In 1X CutOneTMUnder Buffer buffer conditions, in 20 μ l reaction systems, with substrate λ DNA 37 DEG C incubate 15min, then with The digestion situation of 1% agarose gel electrophoresis detection substrate；Restriction enzyme SacI enzyme activity is defined as：At 37 DEG C, in 20 μ In l reaction systems, 1 μ l can digest 1 μ g λ DNA completely in 15min.
The inventive method recombinates restriction enzyme for clonal expression and provides a kind of efficiently feasible new method and thinking, Success Prepare restructuring restriction enzyme SacI also provides valuable reference for the lifting of restriction enzyme production technology. Compared with prior art, the beneficial effect that technical solution of the present invention is brought is：The inventive method realizes restriction enzyme SacI height Effect recombination expression；The harsh control expressed using arabinose operon expression system background carries out restriction enzyme SacI restructuring Expression；Resist digestions of the restriction enzyme SacI to host genome DNA, the relative expression system without the protection that methylates, can stablize and Efficiently carry out restriction enzyme SacI recombination expression；The M.AluI filtered out the protection bacterial strains that methylate are similarly applied to other and had The recombination expression of the restriction enzyme of similar recognition site, such as AluI, HindIII, PvuII.
Brief description of the drawings
Fig. 1 is pACYC184-M.Alu I Vector map；
Fig. 2 is pET28b-R.SacI Vector map；
Fig. 3 methylates for restriction enzyme SacI protects bacterial strain screening, in Fig. 3：
Swimming lane 1：SacI digested plasmids pUC19；Swimming lane 2：Control plasmid pUC19；Swimming lane 3：SacI digested plasmids pACYC184- M.AluI；Swimming lane 4：Control plasmid pACYC184-M.AluI
Fig. 4 is SacI viability examination electrophoretogram, in Fig. 4：
Swimming lane 1 is DNAMarker；Swimming lane 2 is λ DNA (HindIII digest).
It is the specific cleavage map that SacI stostes are carried out to different gradient dilutions respectively that swimming lane 3-11, which is, and final vigor is set to 10,240U/ul。
3：80 times of dilution；4：160 times of dilution；5：320 times of dilution；6：640 times of dilution；7：1280 times of dilution；8；2560 times of dilution； 9：5120 times of dilution；10：10240 times of dilution；11：20480 times of dilution.
Embodiment
Referring to the drawings, the concrete technical scheme of the present invention is further described, in order to which those skilled in the art enters One step the present invention is understood, without forming the limitation to its right.
1st, material
1.1 bacterial strains and plasmid Arthrobacter luteus, Streptomyces achromogenes
Bacterial strain is all from American Type Culture Collecti (ATCC)；
DH5 α are purchased from Beijing Quanshijin Biotechnology Co., Ltd, commercially available prod；
ER2566 is purchased from Thermo Fisher Scientific Inc. (Thermo Fisher Scientific), commercially available prod；pBAD、 PACYC184, pUC19 are purchased from the vast biological plasmid platform of spirit；
Enzyme activity assay substrate λ DNA (HindIII digest) are purchased from Thermo Fisher Scientific Inc., commercially available prod (Thermo Fisher Scientific)；
1.2 instruments crush instrument with reagent JN-02C low-temperature ultrahigh-pressures continuous flow cell：Purchased from Guangzhou cumulative nano biological high-tech stock Part Co., Ltd；Protein Marker：PAGE-MASTER Protein Standard Plus are purchased from Jin Sirui biotechnologies Co., Ltd
Restriction enzyme LightningTM NdeI,LightningTM NcoI,LightningTM HindIII, LightningTMXhoI (Jiangsu the Foolish Old Man's Life Science Co., Ltd, commercially available prod)；Purchased from New England Biolabs(NEB)
KOD-Pure+DNA Polymerase (Jiangsu the Foolish Old Man's Life Science Co., Ltd, commercially available prod)；
T4 DNA Ligase (Fast) (Jiangsu the Foolish Old Man's Life Science Co., Ltd, commercially available prod)；
Alkaline Phosphatase (Fast) (Jiangsu the Foolish Old Man's Life Science Co., Ltd, commercially available prod)；
10×CutOneTMBuffer Jiangsu the Foolish Old Man's Life Science Co., Ltd, commercially available prod.
The foundation of 2.2 transmethylase M.AluI recombinant strains
The present invention establishes transmethylase M.AluI recombinant strains according to method described below.
2.2.1 the acquisition of transmethylase M.AluI genes
From the strains A rthrobacter luteus transmethylase M.AluI disclosed (GeneBank of gene order： HM_569710.1).The acquisition of transmethylase M.AluI genes can use PCR amplifications, gene chemical synthesis, extraction chromosomal DNA And establish the row such as library technology known in the industry.The method that invention uses PCR amplifications, from strains A rthrobacter Enter performing PCR amplification using the specific primer of transmethylase M.AluI genes in luteus genome and obtain methyl transfer Enzyme M.AluI gene.Gene chemical synthesis, extract chromosomal DNA and establish other technologies such as library and can also reach same purpose.
Experiment 1：Obtain transmethylase M.AluI genes
After strains A rthrobacter luteus flat board culture bacterium colonies are resuspended with deionized water, 95 DEG C of incubation 10min, as PCR Template.5 '-terminal sequence (sense primer) and 3 ' of the primer sequence based on transmethylase M.AluI genes of PCR amplifications-end are anti- Synthesis is designed to complementary series (anti-sense primer).
The primer sequence of synthesis is as follows:
Forward primer：5’-CATGCATATGGAGCTCATGGGTGATCAACTG-3’
NdeI SacI
Reverse primer：5’-TATGCTCGAGTTAGAGCTCTTCTGCTGCGCCCAGTGC-3’
XhoI SacI
This pair of primers can specific amplification M.AluI genes, while introduce NdeI and XhoI restriction enzyme site, facilitate follow-up enzyme Cut-connect and screening experiment.Pcr amplification reaction uses KOD-Pure+ DNA Polymerase, according to standard PCR reaction streams Cheng Jinhang, obtain and theoretical value M.AluI genetic fragments of the same size.Above-mentioned pcr amplification product passes through digestion-connection Mode forms recombinant expression plasmid with pACYC184 carriers.
2.2.2 transmethylase M.AluI recombinant strains are built
M.AluI recognition sequence is 5 ' AGCT 3 ', the modification that methylates is carried out to the 3rd cytimidine.SacI knows to DNA specificity Other sequence is 5 ' GAGCTC3 ', cleavage site is between TC bases.M.AluI recognition sequence is included in AluI, HindIII, In PvuII, SacI recognition sequence, when M.AluI is to the AG of sequence 5 'CT 3 ' methylate after modification, it is possible to resists The cutting of AluI, HindIII, PvuII, SacI to the modification sequence that methylates.M.AluI is make use of in the present invention to 5 ' AGCT Base C specific methylation modification, equally can reach the purpose for the protection that to host genome DNA methylate, keeps away in 3 ' Exempt from express SacI after lethal host.But the expression and modification of too high methylase equally have lethal effect for host, therefore And the present invention inserts M.AluI gene orders in low-copy expression vector pACYC184 plasmids in a manner of digestion-connection, even Thing of practicing midwifery conversion, which enters clone strain DH5 α and is coated on chlorampenicol resistant flat board, is cultivated.
Experiment 2：Build transmethylase M.AluI recombinant strains
The pACYC184 expression vectors for preparing M.AluI are carried out according to below scheme.PACYC184 plasmids are with LightningTM NdeI and LightningTMAfter XhoI restriction enzyme double digestions, then with alkaline phosphatase Alkaline Phosphatase (Fast) Carry out dephosphorylation process.Digestion products enter row agarose gel electrophoresis, and the pACYC184 carrier-pellets for recovery linearisation of tapping rubber Section, is dissolved in TE Buffer.
Enter performing PCR amplification to M.AluI genes using KOD-Pure+DNA Polymerase, the PCR primer of acquisition is entered Row agarose gel electrophoresis, confirm after obtaining specific amplification fragment, use LightningTMNdeI and LightningTM XhoI It is connected after restriction enzyme double digestion with the pACYC184 carrier segments of linearisation, according to molal weight than 1:3 concentration, through T4DNA Ligase (Fast) is entered in DH5 α competent cells after 1h is reacted at 22 DEG C using chemical transformation conversion.Sieved in chloramphenicol Cell culture is carried out under the conditions of choosing, obtains M.AluI recombinant expression plasmid pACYC184-M.AluI (reference picture 1).Plasmid The correctness of pACYC184-M.AluI sequences confirms (Jin Wei intelligence bio tech ltd) by sequencing.
2.2.3 methylate and protect the screening of bacterial strain
The carrier pACYC184 used in the present invention is low-copy plasmid, thus the M.AluI genes built thereon are in low-level Expression status.In order to which the not lethal host of the high level expression of restriction enzyme in subsequent process to bacterial strain after conversion, it is necessary to enter The screening of the capable degree of protection that methylates.
Experiment 3：
The screening of M.AluI protection bacterial strains is carried out according to below scheme：Correct plasmid pACYC184-M.AluI will be sequenced to pass through Chemical transformation conversion enters coli strain ER2566, and cell culture is carried out under chloramphenicol screening conditions.Select Dan Ke It is grand, rule on the agar plate containing 35 μ g/ml chloramphenicol antibiotics, and corresponding numbering is carried out after 37 DEG C, 200rpm is trained 16h is supported, bacterium solution is collected and proposes plasmid again.In 10 μ l reaction systems, 0.2 μ l restriction enzymes SacI- is taken Under buffer condition, 1h is incubated at 37 DEG C with 100ng recombinant plasmids pACYC184-M.AluI, is then used 0.8% agarose gel electrophoresis detection substrate is by SacI-The situation of digestion.As a result show, with the restructuring matter of SacI digestions Grain pACYC184-M.AluI with without SacI-The control plasmid band of digestion is completely the same (reference picture 3), shows that this is heavy For group plasmid by the complete protection of M.AluI transmethylases in host bacterial, acquiescence Host genomic DNA equally can be by To the M.AluI transmethylases protection expressed by pACYC184-M.AluI.Will be corresponding on flat board after verifying protectiveness again The bacterial strain of numbering is prepared into competent cell [pACYC184-M.AluI, ER2566] using CaCl2 processing.
2.2.4 the acquisition of restriction enzyme SacI genes
Gene order from bacterial strain Streptomyces achromogenes restriction enzyme SacI is disclosed (GeneBank：AF_027867.1).The acquisition of restriction enzyme SacI genes can use PCR amplifications, gene chemical synthesis, extraction dyeing Body DNA simultaneously establishes the row such as library technology known in the industry.The method that the present invention is expanded using PCR, from bacterial strain Streptomyces Enter performing PCR amplification using the specific primer of restriction enzyme SacI genes in achromogenes genome and obtain restriction enzyme SacI gene.Gene chemical synthesis, extract chromosomal DNA and establish other technologies such as library and can also reach same purpose.
Experiment 4：The PCR amplifications of restriction enzyme SacI genes
After bacterial strain Streptomyces achromogenes flat board culture bacterium colonies are resuspended with deionized water, 95 DEG C of incubation 10min, As pcr template.5 '-terminal sequence (sense primer) and 3 ' of the primer sequence based on restriction enzyme SacI genes of PCR amplifications-end are anti- To complementary series (anti-sense primer).This pair of primers can specific amplification restriction enzyme SacI gene.The primer sequence of synthesis is such as Shown in lower:
Forward primer：5’-GAACCATGGGCATTACC-3’
NcoI
Reverse primer：5’-GCCAAGCTTAGGTTTCC-3’
HindIII
This pair of primers can specific amplification R.SacI genes, and introduce NcoI, HindIII restriction enzyme site, facilitate follow-up digestion- Connection.Pcr amplification reaction has used KOD-Pure+DNA Polymerase, and is carried out according to standard PCR reaction process, obtains With theoretical value R.SacI genetic fragments of the same size.Above-mentioned pcr amplification product is carried by way of digestion-connection with pBAD Body forms recombinant expression plasmid.
2.2.5 restriction enzyme SacI recombinant strains are built
Lactose operon expression system is used in the present invention, SacI gene orders are inserted into pBAD tables in a manner of digestion-connection Up in plasmid, connection product conversion, which enters DH5 alpha expressions bacterial strain and is coated on ammonia benzyl chloramphenicol resistance flat board, is cultivated.Pass through survey Sequence, confirm after the successfully constructing of recombinant plasmid conversion enter R.SacI specificity protections bacterial strain [pACYC184-M.AluI, ER2566], structure restriction enzyme SacI recombinant strains.
Experiment 5：Build restriction enzyme R.SacI recombinant strains
The pBAD expression vectors for preparing SacI are carried out according to below scheme：PBAD plasmids are with LightningTMNcoI and LightningTMAfter HindIII restriction enzyme double digestions, then with Alkaline Phosphatase (Fast) carry out dephosphorylation at Reason.Digestion products enter row agarose gel electrophoresis, and the pBAD carrier segments for recovery linearisation of tapping rubber, and are dissolved in TE Buffer.
R.SacI genes are entered using KOD exo+ polymerases (Jiangsu the Foolish Old Man's Life Science Co., Ltd, commercially available prod) Performing PCR is expanded, and row agarose gel electrophoresis are entered to the PCR primer of acquisition, is confirmed after obtaining specific amplification fragment, with NcoI and PBAD carrier-pellets after HindIII restriction enzymes (Jiangsu the Foolish Old Man's Life Science Co., Ltd, commercially available prod) double digestion with linearisation Section connection, according to molal weight than 1:3 concentration, chemistry is used after T4 DNA Ligase (Fast) react 1h at 22 DEG C Conversion method conversion enters in DH5 α competent cells.Cell culture is carried out under ammonia benzyl chloramphenicol resistance screening conditions, is obtained R.SacI recombinant expression plasmid pBAD-R.SacI (reference picture 2).The correctness of plasmid pBAD-R.SacI sequences is by sequencing Confirm (Jin Wei intelligence bio tech ltd).
Restriction enzyme SacI recombination expression
To be sequenced correct recombinant expression plasmid pBAD-R.SacI transformed competence colibacillus cell [pACYC184-M.AluI, ER2566], screening and culturing on the flat board of the mycin of benzyl containing ammonia and chloramphenicol antibiotics is coated on, obtains restriction enzyme SacI restructuring table Up to bacterial strain.The bacterial strain is induced after expanding and cultivating with arabinose, obtains restriction enzyme SacI recombinant protein, and confirms bacterium Protein crude extract has restriction enzyme SacI activity.
Experiment 6：Restriction enzyme SacI recombination expression
It is thin that correct recombinant expression plasmid pBAD-R.SacI conversions [pACYC184-M.SacI, ER2566] competence will be sequenced Born of the same parents, screening and culturing on the flat board of the mycin of benzyl containing ammonia and chloramphenicol antibiotics is coated on, obtains restriction enzyme SacI recombination expression bacterium Strain.After LB inoculation of medium restriction enzymes SacI recombinant strains, cultivated in 37 DEG C of shaking tables with 200rpm to OD 0.8, derivant arabinose, final concentration 0.2% are then added, and 16h is cultivated with 200rpm in 16 DEG C of shaking tables.
Restriction enzyme SacI viability examination
Restriction enzyme SacI enzyme activity is defined as：At 37 DEG C, in 20 μ l reaction systems, 1 μ l can be in 15min completely Digest 1 μ g λ DNA (HindIII digest).After the present invention carries out gradient dilution to the restriction enzyme SacI obtained after purified, 1X CutOneTMUnder Buffer (the Foolish Old Man's Life Science Co., Ltd) buffer condition, in 20 μ l reaction systems, with substrate λ DNA (HindIII digest) incubates 15min at 37 DEG C, then with the digestion situation (ginseng of 1% agarose gel electrophoresis detection substrate According to Fig. 4).After tested, purifying the restriction enzyme SacI of acquisition has good digestion vigor, and Rate activity is about 2,900,000U/mg Induction bacterium solution.

Claims (8)

  1. A kind of 1. screening technique for the protection bacterial strain that methylates for being used to express restriction enzyme SacI, it is characterised in that
    （1）The foundation of transmethylase M.AluI recombinant strains
    A, the acquisition of transmethylase M.AluI genes
    The acquisition of transmethylase M.AluI genes is using PCR amplifications, gene chemical synthesis or extracts chromosomal DNA and establishes library Method, from bacterial strainArthrobacter luteusGenome in use transmethylase M.AluI genes specific primer Obtain transmethylase M.AluI gene；
    B, transmethylase M.AluI recombinant strains are built
    M.AluI gene orders are inserted in low-copy expression vector pACYC184 plasmids in a manner of digestion-connection, connection production Thing conversion, which enters clone strain DH5 α and is coated on chlorampenicol resistant flat board, is cultivated；
    C, methylate and protect the screening of bacterial strain
    To bacterial strain after conversion methylate the screening of degree of protection；
    D, the acquisition of restriction enzyme SacI genes
    The acquisition of restriction enzyme SacI genes is using PCR amplifications, gene chemical synthesis or extracts chromosomal DNA and establishes the side in library Method, from bacterial strainStreptomyces achromogenesGenome in obtained using the specific primer of restriction enzyme SacI genes Obtain restriction enzyme SacI gene；
    E, restriction enzyme SacI recombinant strains are built
    Using lactose operon expression system, SacI gene orders are inserted in pBAD expression plasmids in a manner of digestion-connection, Connection product conversion, which enters DH5 alpha expressions bacterial strain and is coated on ammonia benzyl chloramphenicol resistance flat board, is cultivated；By sequencing, weight is confirmed Conversion enters R.SacI specificity protection bacterial strains [pACYC184-M.AluI, ER2566], structure limit after group the successfully constructing of plasmid Enzyme SacI processed recombinant strains；
    （2）Restriction enzyme SacI recombination expression
    To be sequenced correct recombinant expression plasmid pBAD-R.SacI transformed competence colibacillus cell [pACYC184-M.AluI, ER2566], screening and culturing on the flat board of the mycin of benzyl containing ammonia and chloramphenicol antibiotics is coated on, obtains restriction enzyme SacI restructuring table Up to bacterial strain；The bacterial strain is induced after expanding and cultivating with arabinose, obtains restriction enzyme SacI recombinant protein, and confirms bacterium Protein crude extract has restriction enzyme SacI activity.
  2. A kind of 2. screening side of protection bacterial strain that methylates for being used to express restriction enzyme SacI according to claim 1 Method, it is characterised in that step（1）In, from bacterial strainArthrobacter luteusGenome in use transmethylase The specific primer PCR amplifications of M.AluI genes obtain transmethylase M.AluI gene；Obtain transmethylase The method of M.AluI genes is as follows：
    Bacterial strainArthrobacter luteusAfter flat board culture bacterium colony is resuspended with deionized water, 95 C incubate 10 min, as Pcr template；Sense primer 5 ' of the primer sequence based on transmethylase M.AluI genes-terminal sequence of PCR amplifications and downstream are drawn Thing 3 '-end reverse complementary sequence is designed synthesis；
    The primer sequence of synthesis is as follows:
    Forward primer：5’-CATGCATATGGAGCTCATGGGTGATCAACTG-3’
    NdeI SacI
    Reverse primer：5’-TATGCTCGAGTTAGAGCTCTTCTGCTGCGCCCAGTGC-3’
    XhoI SacI
    This pair of primers is used for specific amplification M.AluI genes, while introduces NdeI and XhoI restriction enzyme site, convenient follow-up Digestion-connection and screening experiment；Pcr amplification reaction uses KOD-Pure+ DNA Polymerase, is reacted according to standard PCR Flow is carried out, and is obtained and theoretical value M.AluI gene fragment orders 1 of the same size；Above-mentioned pcr amplification product by digestion- The mode of connection forms recombinant expression plasmid with pACYC184 carriers.
  3. A kind of 3. screening side of protection bacterial strain that methylates for being used to express restriction enzyme SacI according to claim 1 Method, it is characterised in that step（1）In, the pACYC184 expression vectors for preparing M.AluI are carried out according to below scheme：
    PACYC184 plasmids are with LightningTMNdeI and LightningTMAfter XhoI restriction enzyme double digestions, then with Alkaline Phosphatase (Fast) carries out dephosphorylation process, and digestion products enter row agarose gel electrophoresis, and recovery of tapping rubber is linear The pACYC184 carrier segments of change, are dissolved in TE Buffer；
    Enter performing PCR amplification to M.AluI genes using KOD-Pure+ DNA Polymerase, fine jade is carried out to the PCR primer of acquisition Sepharose electrophoresis, confirm after obtaining specific amplification fragment, use LightningTMNdeI and LightningTMXhoI is limited It is connected after enzyme double digestion with the pACYC184 carrier segments of linearisation, according to molal weight than 1:3 concentration, through T4 DNA Ligase (Fast) is entered in DH5 α competent cells after 1h is reacted under 22 °C using chemical transformation conversion；In chloramphenicol Cell culture is carried out under screening conditions, obtains M.AluI recombinant expression plasmid pACYC184-M.AluI；Plasmid The correctness of pACYC184-M.AluI sequences confirms by sequencing.
  4. A kind of 4. screening side of protection bacterial strain that methylates for being used to express restriction enzyme SacI according to claim 1 Method, it is characterised in that step（1）In, the screening of M.AluI protection bacterial strains is carried out according to below scheme：Correct matter will be sequenced Grain pACYC184-M.AluI enters coli strain ER2566 by chemical transformation conversion, under chloramphenicol screening conditions Carry out cell culture；Monoclonal is selected, is rule on the agar plate containing 35 μ g/ml chloramphenicol antibiotics, and is carried out corresponding For numbering after 37 °C, 200rpm cultivates 16 h, collects bacterium solution and proposes plasmid again；In 10 μ l reaction systems, 0.2 μ l are taken Restriction enzyme SacI-HF, under Cutsmart buffer conditions, with 100 ng recombinant plasmids pACYC184-M.AluI 1 h, situation about then being digested using 0.8% agarose gel electrophoresis detection substrate by SacI-HF are incubated in 37 C；Confirming should Recombinant plasmid equally may be used in host bacterial by the complete protection of M.AluI transmethylases, acquiescence Host genomic DNA Protected by the M.AluI transmethylases expressed by pACYC184-M.AluI；By phase on flat board after verifying protectiveness again The bacterial strain that should be numbered uses CaCl2Processing, is prepared into competent cell [pACYC184-M.AluI, ER2566].
  5. A kind of 5. screening side of protection bacterial strain that methylates for being used to express restriction enzyme SacI according to claim 1 Method, it is characterised in that step（1）In, the PCR amplification method of restriction enzyme SacI genes is as follows：
    Bacterial strainStreptomyces achromogenesAfter flat board culture bacterium colony is resuspended with deionized water, 95 C incubate 10 min, As pcr template；Sense primer 5 ' of the primer sequence based on the restriction enzyme SacI genes-terminal sequence and anti-sense primer of PCR amplifications 3 '-end reverse complementary sequence, beneficial to insertion vector；
    The primer sequence of synthesis is as follows:
    Forward primer：5’-GAACCATGGGCATTACC-3’
    NcoI
    Reverse primer：5’-GCCAAGCTTAGGTTTCC-3’
    HindIII
    This pair of primers is used for specific amplification R.SacI genes, and introduces NcoI, HindIII restriction enzyme site, facilitates follow-up enzyme Cut-connect；Pcr amplification reaction uses KOD-Pure+ DNA Polymerase, is carried out according to standard PCR reaction process, obtains With theoretical value R.SacI gene fragment orders 2 of the same size；Above-mentioned pcr amplification product by way of digestion-connection with PBAD carriers form recombinant expression plasmid.
  6. A kind of 6. screening side of protection bacterial strain that methylates for being used to express restriction enzyme SacI according to claim 1 Method, it is characterised in that step（1）In, the pBAD expression vectors for preparing SacI are carried out according to below scheme：
    PBAD plasmids are with LightningTMNcoI and LightningTMAfter HindIII restriction enzyme double digestions, then with Alkaline Phosphatase (Fast) carries out dephosphorylation process；Digestion products enter row agarose gel electrophoresis, and recovery of tapping rubber is linear The pBAD carrier segments of change, are dissolved in TE Buffer；
    Enter performing PCR amplification to R.SacI genes using KOD-Pure+ DNA Polymerase, fine jade is carried out to the PCR primer of acquisition Sepharose electrophoresis, confirm after obtaining specific amplification fragment, use LightningTMNcoI and LightningTMHindIII is limited It is connected after enzyme double digestion processed with the pBAD carrier segments of linearisation, according to molal weight than 1:3 concentration, through T4 DNA Ligase (Fast) is entered in DH5 α competent cells after 1h is reacted under 22 °C using chemical transformation conversion；It is mould in ammonia benzyl Cell culture is carried out under the conditions of plain resistance screening, obtains R.SacI recombinant expression plasmid pBAD-R.SacI；Plasmid pBAD- The correctness of R.SacI sequences confirms by sequencing.
  7. A kind of 7. screening side of protection bacterial strain that methylates for being used to express restriction enzyme SacI according to claim 1 Method, it is characterised in that step（2）In, restriction enzyme SacI recombinant expression method is as follows：Sequencing is correctly recombinantly expressed into matter Grain pBAD-R.SacI conversion [pACYC184-M.SacI, ER2566] competent cells, are coated on the mycin of benzyl containing ammonia and chloramphenicol Screening and culturing on the flat board of antibiotic, obtain restriction enzyme SacI recombinant strains；In LB inoculation of medium restriction enzymes After SacI recombinant strains, cultivated in 37 C shaking tables with 200rpm to OD 0.8, it is whole then to add derivant arabinose Concentration 0.2%, and cultivate 16 h in 16 C shaking tables with 200 rpm.
  8. A kind of 8. screening side of protection bacterial strain that methylates for being used to express restriction enzyme SacI according to claim 1 Method, it is characterised in that step（2）In, restriction enzyme SacI vigor testing methods are as follows：To the limit obtained after purified After enzyme SacI processed carries out gradient dilution, in 1X CutOneTMUnder Buffer buffer conditions, in 20 μ l reaction systems, with substrate λ DNA incubates 15 min in 37 C, then with the digestion situation of 1% agarose gel electrophoresis detection substrate；Restriction enzyme SacI enzyme activity is defined as：Under 37 °C, in 20 μ l reaction systems, 1 μ l can digest 1 μ g λ completely in 15min DNA。
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CN110438104A (en) * 2019-08-28 2019-11-12 莫纳(武汉)生物科技有限公司 A kind of restriction enzyme MnlI and its expression and purification method
CN110438102A (en) * 2019-09-03 2019-11-12 莫纳(武汉)生物科技有限公司 A kind of methylation protection host strain and its construction method and application
CN110577923A (en) * 2019-10-12 2019-12-17 莫纳(连云港)生物科技有限公司 Screening method of methylation protection strain for expressing restriction enzyme ApaLI
CN110643560A (en) * 2019-10-24 2020-01-03 江苏海洋大学 Engineering strain for recombinant expression of restriction endonuclease and construction method thereof
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CN110438211A (en) * 2019-08-20 2019-11-12 福建晨欣科生物科技有限公司 Specific gene segment enrichment detection kit and method based on digestion PCR
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CN110438102A (en) * 2019-09-03 2019-11-12 莫纳(武汉)生物科技有限公司 A kind of methylation protection host strain and its construction method and application
CN110577923A (en) * 2019-10-12 2019-12-17 莫纳(连云港)生物科技有限公司 Screening method of methylation protection strain for expressing restriction enzyme ApaLI
CN110643558A (en) * 2019-10-12 2020-01-03 莫纳(连云港)生物科技有限公司 Screening method of methylation protection strain for expressing restriction enzyme KasI
CN110643560A (en) * 2019-10-24 2020-01-03 江苏海洋大学 Engineering strain for recombinant expression of restriction endonuclease and construction method thereof

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