CN110218733A - Utilize the method for ccdB lethal gene rapid build recombinant plasmid - Google Patents
Utilize the method for ccdB lethal gene rapid build recombinant plasmid Download PDFInfo
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- CN110218733A CN110218733A CN201910382063.0A CN201910382063A CN110218733A CN 110218733 A CN110218733 A CN 110218733A CN 201910382063 A CN201910382063 A CN 201910382063A CN 110218733 A CN110218733 A CN 110218733A
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- 101150102092 ccdB gene Proteins 0.000 title claims abstract description 70
- 239000013612 plasmid Substances 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 34
- 108700005090 Lethal Genes Proteins 0.000 title claims abstract description 20
- 239000013598 vector Substances 0.000 claims abstract description 25
- 241000894006 Bacteria Species 0.000 claims abstract description 13
- 230000006801 homologous recombination Effects 0.000 claims abstract description 13
- 238000002744 homologous recombination Methods 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 53
- 230000014509 gene expression Effects 0.000 claims description 48
- 108020004414 DNA Proteins 0.000 claims description 30
- 239000013604 expression vector Substances 0.000 claims description 25
- 230000003321 amplification Effects 0.000 claims description 15
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 15
- YRMCBQLZVBXOSJ-PCFSSPOYSA-N (e)-3-[(6r,6as)-4-hydroxy-6-methoxy-3-methyl-11-oxo-5,6,6a,7-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-8-yl]prop-2-enamide Chemical compound CO[C@H]1NC2=C(O)C(C)=CC=C2C(=O)N2C=C(\C=C\C(N)=O)C[C@@H]12 YRMCBQLZVBXOSJ-PCFSSPOYSA-N 0.000 claims description 8
- 230000003115 biocidal effect Effects 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 229960005091 chloramphenicol Drugs 0.000 claims description 5
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 238000012217 deletion Methods 0.000 claims description 2
- 230000037430 deletion Effects 0.000 claims description 2
- 239000013613 expression plasmid Substances 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 238000003259 recombinant expression Methods 0.000 claims description 2
- 238000010276 construction Methods 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 3
- 230000002255 enzymatic effect Effects 0.000 abstract 1
- 238000001727 in vivo Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 27
- 101150094267 mqo gene Proteins 0.000 description 12
- 125000004122 cyclic group Chemical group 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 7
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- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
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- 238000000338 in vitro Methods 0.000 description 3
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- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
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- 229910052799 carbon Inorganic materials 0.000 description 2
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- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
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- 239000012498 ultrapure water Substances 0.000 description 2
- 238000011238 DNA vaccination Methods 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
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- 239000013067 intermediate product Substances 0.000 description 1
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- 229930182823 kanamycin A Natural products 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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Abstract
The present invention relates to molecular biology fields, and in particular to utilizes the method for ccdB lethal gene rapid build recombinant plasmid.The present invention is combined using the mechanism of PCR and bacterium In vivo homologous recombination, gets rid of the dependence cut and connected to DNA enzymatic in vector construction；CcdB lethal gene is introduced, the screening of positive colony is simplified.The present invention can be used for high-throughput vector construction, have the advantages that time saving, laborsaving, inexpensive.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to utilizes the side of ccdB lethal gene rapid build recombinant plasmid
Method.
Background technique
Plasmid is the basic tool of modern biology research, has been applied to life science and medical treatment & health development
Various aspects, such as library foundation, protein expression, gene therapy, gene editing and DNA vaccination every aspect.Molecular cloning skill
Art is that the target DNA fragment of amplification is inserted into the process of purpose plasmid vector, is the core technology of modern biomedical research
One of, plasmid construction almost becomes the primary work of all biomedical basic research.With third generation gene sequencing technology
It promotes, a large amount of gene informations that digitize need to be converted into various recombinant plasmids necessary to biomedical basic research.Fast run-up
The multipurpose carrier library of vertical individual gene, becomes the urgent need of Many researchers.
Conventional molecular clone technology relies on a variety of bioengineered enzymes, and needs Ligation in vitro to react, and there are bacterium colony verifyings
The problem of false positive, workflow is cumbersome, complicated, time-consuming, laborious, has been not suitable with the need of modern biomedical basic research
It asks.
It can be avoided the endonuclease and ligase that conventional molecular clone technology is relied on by the method for homologous recombination.
The basic principle is that distinguishing PCR amplification plasmid vector and target gene by 2 pairs of primers with homologous sequence, it is respectively provided with
(wherein linear plasmid carrier and linear target gene have homologous sequence to the PCR product of linear plasmid carrier and linear target gene
Column, which introduced by PCR primer), then aforementioned 2 kinds of PCR products are transferred in competent cell simultaneously, make it
In linear plasmid carrier and linear target gene occur homologous recombination, then by antibiotic-screening (in plasmid vector with should
The resistant gene of antibiotic), positive recombinant vector can be obtained.But this method has the shortcomings that false positive: in PCR product
Still with the cyclic annular empty carrier that a part does not expand, cyclic annular empty carrier will not be recombinated with linear target gene, and having should
The competent cell of cyclic annular empty carrier still can form clone under antibiotic-screening, thus produce false positive.
Summary of the invention
During preventing construction of recombinant plasmid, false positive caused by empty carrier, the present invention provides following technologies
Scheme:
Utilize the method for ccdB lethal gene rapid build recombinant plasmid, comprising the following steps:
A, PCR amplification ccdB gene expression cassettes, expression vector and target gene are obtained containing linear ccdB expression casette
PCR product, the PCR product containing linear expression vector and the PCR product containing linear target gene of son；Used primer are as follows:
(1) the amplimer ccdB-HF and ccdB-HR of ccdB gene expression cassettes；(2) vector amplification primer HF and HR；(3) mesh is expanded
Gene primer gene-HF and gene-HR；
B, the PCR product containing linear ccdB gene expression cassettes for obtaining step a is produced with the PCR containing linear expression vector
It is transferred to the competent cell of tolerance ccdB albumen after object mixing, is cultivated in the culture medium containing antibiotic A and antibiotic B, line
Property ccdB gene expression cassettes and linear expression vector homologous recombination occurs, obtain cyclic annular load with ccdB gene expression cassettes
Body；Antibiotic A is different with antibiotic B；
C, the circular vectors with ccdB gene expression cassettes obtained using vector amplification primer amplification step b, are contained
There is the PCR product for the linear carrier for deleting ccdB gene expression cassettes；
D, the PCR product containing linear target gene for obtaining step a contains deletion ccdB gene table with what step c was obtained
It is transferred to the competent cell for not tolerating ccdB albumen after up to the PCR product mixing of the linear carrier of box, is containing antibiotic B's
It is cultivated in culture medium, it is purposeful to obtain band for linear target gene and the linear carrier homologous recombination for deleting ccdB gene expression cassettes
The circular vectors of gene；
CcdB gene expression cassettes described in step a are the resistant genes containing promoter, ccdB gene and antibiotic A
DNA molecular；
The amplimer sequence of ccdB gene expression cassettes and the primer sequence of amplifying target genes include in step a
2 segments: (I) can expand the segment of ccdB gene expression cassettes or target gene, and (II) can be homologous heavy with expression vector
The segment of group；Segment (I) is located at 3 ' sides of segment (II)；
The segment of the homologous recombination is some or all of vector amplification primer and expression vector pairing segment.
The method of recombinant plasmid as the aforementioned, the length of the segment (II) are 18nt.
The method of recombinant plasmid as the aforementioned, the segment of the homologous recombination are that vector amplification primer and expression vector match
Whole segments.
The method of recombinant plasmid as the aforementioned, competent cell described in step b are that the competence of Escherichia coli DB3.1 is thin
Born of the same parents.
The method of recombinant plasmid as the aforementioned, the antibiotic A is chloramphenicol.
The method of recombinant plasmid as the aforementioned, the ccdB gene expression cassettes DNA sequence dna is as shown in SEQ ID NO:14.
The quantity of the method for recombinant plasmid as the aforementioned, expression vector described in step a is 2 or more, and expression vector is taken
Fixed sequence program with one section of 36bp or more, the fixed sequence program can be matched the overall length for amplifying carrier by primer HF and HR.
The method of recombinant plasmid as the aforementioned, the competence that competent cell described in step d is coli strain DH5d
Cell.
The method of recombinant plasmid as the aforementioned, in step d, " PCR product containing linear target gene that step a is obtained " with
The molar ratio of " PCR product containing the linear carrier for deleting ccdB gene expression cassettes that step c is obtained " is 2: 1.
The method of recombinant plasmid as the aforementioned, further includes step e: selecting monoclonal colonies and carries out bacterium solution PCR verifying, extracts
Simultaneously identification is sequenced in plasmid, obtains the recombinant expression plasmid containing mutually isogenic different function purposes.
The prior art and comparison of the invention are as follows:
Existent technique route are as follows:
Using cyclic annular empty carrier as template, PCR obtains linear empty carrier A, then linearisation empty carrier A and target gene is homologous heavy
Group obtains the cyclic annular recombinant vector with target gene.
And the technology of the present invention route is then divided into 2 parts:
1) using cyclic annular empty carrier as template, PCR obtains linear empty carrier A, then will linearisation empty carrier A and ccdB gene expression
Box recombination, obtains the recombinant vector with ccdB gene expression cassettes；
2) using the recombinant vector with ccdB gene expression cassettes as template, PCR must linearize empty carrier B and (delete ccdB
That Duan Xulie of gene expression cassettes), then empty carrier B and target gene homologous recombination will be linearized, obtain band target gene
Cyclic annular recombinant vector.
Linearisation empty carrier A above-mentioned is consistent with the main component of B, is all the product that empty carrier passes through PCR linearisation.But
It is impurities difference, linearizing the impurity in empty carrier A is (not being amplified) cyclic annular empty carrier, is linearized in empty carrier B
Impurity be (not being amplified) have ccdB gene expression cassettes recombinant vector.Cyclic annular empty carrier can be such that host cell has
Antibiotic resistance, and generate false positive；But the recombinant vector with ccdB gene expression cassettes can press down due to having lethal gene
Host cell growth processed, and avoid aforementioned false positive.
In short, compared with prior art, the additional mostly intermediate steps of the present invention, the main component of obtained intermediate product
It is constant, but since the impurity of generation changes, greatly reduce false positive rate (theoretically false positive rate is 0).
The invention has the following beneficial effects:
1) present invention is independent of any restrictions restriction endonuclease, be not necessarily to Ligation in vitro reaction process, it can be achieved that it is high-throughput from
Dynamicization operation, with efficient low-consume, the advantages such as random error is low.
2) present invention reversely screens quick clone using ccdB lethal gene, has prevented the false positive of empty carrier generation, can
The step of saving traditional verifying positive bacterium colony, it is time saving and energy saving.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Above content of the invention is described in further detail again below by way of specific embodiment.But it should not be by this
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below.All technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Detailed description of the invention
Fig. 1, the method design diagram of ccdB lethal gene rapid build recombinant plasmid is utilized；The insert is insertion
Target gene.
Fig. 2, obtained PCR product (Mqo) and Mqo-HF using primer Mqo-HF and Mqo-HR amplifying target genes Mqo
And Mqo-HR2The agarose gel electrophoresis figure for the PCR product (Mqo-dTAA) that amplifying target genes Mqo is obtained.
Fig. 3, the different expression vectors of 12 kinds of carrying ccdB gene expression cassettes are expanded using primer HF and HR linearisation
PCR product agarose gel electrophoresis figure.
Fig. 4, the flat of conversion coated is mixed from Mqo gene with 12 kinds of carrying ccdB gene expression cassettes difference expression vectors
Plate, random 3 monoclonals of each picking carry out bacterium solution PCR and verify product agarose gel electrophoresis figure.
Specific embodiment
Embodiment 1 carries out the quick clone Mqo gene multichip carrier reversely screened using ccdB gene using the method for the present invention
The carrier that the present embodiment uses has plasmid vector respectively: pFastV1~V12 is that commercial carrier is inserted into a Duan Gongtong
What sequence obtained.Corresponding commercial carrier is successively: pET28a pET29a, pET30b, pET33b, pET28aMBP,
pET28aMBPC,pGEX6p-1,pGEX4T-1,pETDuet1,pCold1,pET15b,pET32a.Each carrier carries kanamycins
Resistant gene.
The common sequence: ctggtgccgc gcggcagcgg aggaggaatc atcatc (SEQ ID NO:1).
In the present embodiment, HF and HR primer is to be somebody's turn to do " common sequence " by matching to be expanded.
The universal primer for verifying foregoing plasmid carrier sequence is as shown in Table 1 and Table 2.
Various reagents and carrier are conventional commercial.Wherein, MCLab DNA Polymerase Mix holds up section from Beijing
Chengdu branch company, Biotechnology Co., Ltd, it is limited that Utaq DNA Polymerase Mix carrys out self-contained Beijing Zhuan Meng biotechnology
Company, Dpn I restriction enzyme come from precious bioengineering (Dalian) Co., Ltd, and DH5 α, DB3.1 competent cell are from north
The trans-oceanic biological Co., Ltd in capital.
1 12 kinds of prokaryotic expression carrier insertion verifying universal primer tables of table
2 universal primer sequence information of table
The antibiotics resistance gene that ccdB gene expression cassettes in the present embodiment carry is chloramphenicol resistance gene, entirely
The DNA sequence dna for expressing box is as shown in table 3.
3 ccdB gene expression cassettes DNA sequence dna table of table
Primer sequence used in the present embodiment is shown in Table 4, and target gene information is shown in Table 5.
The various primer sequences that 4 embodiment 1 of table uses
Note: the sequence for drawing solid line is identical, and the sequence for drawing dotted line is identical.
The target gene information that 5 embodiment 1 of table uses
Specific process are as follows:
1, building carries the high-throughput expression vector pFastB1-B12 of ccdB gene expression cassettes.
According to the DNA sequence dna design amplifying target genes ccdB gene expression cassettes of ccdB gene expression cassettes in table 3
Primer (ccdB-HF and ccdb-HR) and vector amplification primer (HF and HR), are specifically shown in Table 4.Using corresponding primer, contained
There are the PCR product of ccdB gene expression cassettes and the PCR product respectively containing pFastV1-V12 linearized vector.
Aforementioned two kinds of products are mixed respectively, are transferred to the competent cell of the Escherichia coli DB3.1 of tolerance ccdB albumen,
It is cultivated in culture medium containing kanamycin and chloramphenicol, picking positive monoclonal, culture, upgrading grain, purifying is carried
The pFastB1-B12 carrier of lethal ccdB gene expression cassettes.Simplified process is as shown in Figure 1.All recombinant plasmids pass through DNA
Sequence verification, ccdB expression casette subsequence are inserted into successfully.
2, according to the sequence of Mqo gene in table 5, design synthesis amplifying target genes primer is specifically shown in Table 4.Wherein, Mqo-
HF and Mqo-HR amplification DNA fragmentation for be inserted into containing carbon teminal affinity tag carrier pFastB1, B3, B5, B7, B9, B10,
B11,B12.Mqo-HF and Mqo-HR2The DNA fragmentation of amplification for be inserted into containing carbon teminal affinity tag carrier pFastB2, B4,
B6、B8。
3, using Mqo-HF and Mqo-HR/HR2Amplifying target genes carry ccdB expression casette using HR and HF amplification
The high-throughput expression vector (pFastB1-B12 carrier) of son, obtains the PCR product containing target gene and the PCR containing linear carrier
Product.PCR reaction system is as follows: (1) 1 μ L forward primer (20 μM) and 1 μ L reverse primer (20 μM)；(2) 6ng, which contains, purpose base
Because of the DNA profiling of (or pFastB1-B12 carrier)；(3)24μL MCLabDNAPolymerase Mix；(4) 24 μ L are ultrapure
Water.
PCR reaction condition are as follows: 98 DEG C of initial denaturation 2min；98 DEG C of denaturation 10s, 55 DEG C of annealing 15s, elongating temperature is 72 DEG C,
Duration is set by 15s/bp；Recurring number is 18~25.
Mqo containing target gene or PCR product agarose gel electrophoresis results such as Fig. 2 and Fig. 3 containing pFastB1-B12, are obtained
The single DNA fragmentation of band was obtained, can be used for next step Molecular Cloning: A Laboratory.
4, the PCR product of the aforementioned Mqo containing target gene is pressed with the PCR product containing pFastB1-B12 to 2: 1 mole respectively
Than mixing, mixture is obtained.Specifically, the quality that linearized vector is added is according to formula " carrier base pairs × 0.01=load
The quality (unit: ng) that body is added ", and the quality that Linearized genes are added is according to formula " gene base pairs × 0.02=
The quality (unit: ng) that gene is added ".The volume that carrier and gene are added is respectively according to addition quality needed for calculating divided by pure
PCR product concentration after change is eventually adding sterile ultrapure water and complements to 10 μ L.
5, DH5 α competent cell is converted with the mixture of previous step, in the culture medium culture containing chloramphenicol.
6, second day, conventional method picking monoclonal colonies were dissolved in 10 μ L ultrapure waters as bacterium solution pcr template, each plate
3 bacterium colonies are selected, high throughput bacterium solution PCR verifying is carried out.General positive and negative draw is added in 10 μ L UTaq-Mix enzyme systems of premix
Each 0.1 μ L of object (20 μM), then it is separately added into 0.5 μ L bacterium solution template, extension of time is set according to genetic fragment size, recurring number is set
30 are set to, PCR amplification is carried out.PCR product is subjected to 1% agarose gel electrophoresis after PCR, for big in theoretical molecular weight
There is single DNA band and is considered as positive bacterium colony in small position.For the positive bacterium colony that PCR is proved to be successful, it is inoculated in 5mL LB training
It supports in base, plasmid is extracted after 37 DEG C of 12~18h of 220rpm shake culture and send company's sequencing identification, recombination is obtained and successfully contains
The different expression vector plasmids of Mqo gene, as shown in Figure 4.
For bacterium solution PCR as a result, clone's positive rate is very high, cloned by the random picking of each plate 3, it is disposable to obtain
9 different prokaryotic expression carriers containing Mqo gene, are pFastB2, B3, B4, B5, B7, B8, B9, B10, B12 respectively, can
It is screened for the subsequent protein expression to Mqo, and then studies Mqo protein function and structure.It is subsequent to confirm by sequencing, pass through one
Time cloning realizes high flux construction and contains same gene by the recombinant plasmid of 9 kinds of same gene difference expression vectors of acquisition
Different expression vectors.
To sum up, the present invention can get rid of the dependence to restriction enzyme, and react without Ligation in vitro, false positive rate
It is low, efficiently and rapidly by gene constructed into multiple carriers.
SEQUENCE LISTING
<110>Sichuan University
<120>method of ccdB lethal gene rapid build recombinant plasmid is utilized
<130> GYKH1227-2019P016054CC
<160> 22
<170> PatentIn version 3.5
<210> 1
<211> 36
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ctggtgccgc gcggcagcgg aggaggaatc atcatc 36
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<400> 2
taatacgact cactataggg 20
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gctagttatt gctcagcgg 19
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atcgccgtta atccagatta 20
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acaacaacct cgggatcg 18
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gggctggcaa gccacgtttg gtg 23
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ttctgaaatg agctgttgac 20
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atgcgtccgg cgtaga 16
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gattatgcgg ccgtgtacaa 20
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acgccatatc gccgaaagg 19
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ggcagggatc ttagattctg 20
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ggcgaaaatg agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa 60
gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa 120
tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac 180
attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata 240
ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc 300
acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg 360
agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa 420
cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt 480
cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga 540
atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg 600
ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg 660
acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg 720
tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa 780
cgccgcgtgg atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt 840
tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc 900
tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata 960
tatgatgtca atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg 1020
cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt 1080
gaaatgaacg gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta 1140
cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga 1200
cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt 1260
ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac 1320
cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg 1380
cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataa 1429
<210> 15
<211> 18
<212> DNA
<213>artificial sequence
<400> 15
ggaggaggaa tcatcatc 18
<210> 16
<211> 18
<212> DNA
<213>artificial sequence
<400> 16
gctgccgcgc ggcaccag 18
<210> 17
<211> 38
<212> DNA
<213>artificial sequence
<400> 17
ctggtgccgc gcggcagcgg cgaaaatgag acgttgat 38
<210> 18
<211> 38
<212> DNA
<213>artificial sequence
<400> 18
gatgatgatt cctcctcctt atattcccca gaacatca 38
<210> 19
<211> 36
<212> DNA
<213>artificial sequence
<400> 19
ctggtgccgc gcggcagcat gtcagaccta gccaga 36
<210> 20
<211> 38
<212> DNA
<213>artificial sequence
<400> 20
gatgatgatt cctcctcctc atgcggcacc taacttca 38
<210> 21
<211> 38
<212> DNA
<213>artificial sequence
<400> 21
gatgatgatt cctcctcctg cggcacctaa cttcagcg 38
<210> 22
<211> 1482
<212> DNA
<213>artificial sequence
<400> 22
atgtcagacc tagccagaac cgacgtcgtg ctgatcggtg cgggcatcat gagcgccacg 60
ctgggggtgc tgctgcgtcg gctcgaaccg aactggtcaa tcaccctgat cgaacggctg 120
gacgcggtag ccgccgaaag cagcggtccc tggaacaacg ccggcaccgg gcactccgcg 180
ctgtgcgaga tgaactacac cccagaaatg ccggacggct cgatcgacat caccaaagcg 240
gtgcgtgtca acgagcaatt ccaggtcacc cgccagttct gggcatacgc ggccgaaaac 300
ggcatcctca ccgacgtgcg cagcttcctc aaccctgtgc cgcacgtgag tttcgtccat 360
ggatcgcggg gcgtcgagta tctacggcgc cgccaaaagg cgttggccgg caacccgctg 420
ttcgccggca ccgagttcat cgagagtccc gacgaattcg cccgccggct gccgttcatg 480
gccgctaaac gggccttctc cgagccggtg gcgctcaact gggccgccga cggcaccgac 540
gtcgacttcg gtgccctcgc caaacaactc atcggctatt gcgtgcaaaa tggcactacc 600
gcgttgttcg ggcacgaggt tcgcaacctc tcgcggcaat ccgacggcag ctggacggtc 660
accatgtgca accgccggac cggcgaaaag cgcaagttga acaccaagtt cgtctttgtc 720
ggggccgggg gtgacacctt gccggtgctg cagaaatccg ggatcaaaga ggtcaaaggc 780
ttcgccggct tcccgattgg cggtcggttc ctgcgcgccg ggaacccggc gctcaccgcc 840
tcgcatcggg caaaggtata tggcttcccg gcgccgggcg ccccgccgtt gggcgccttg 900
catctggatc tgcggtttgt caacggcaag tcgtggctgg tgttcgggcc atacgccggc 960
tggtcgccga agttcttgaa acacgggcag atcagcgacc tgccccggtc gatcaggccg 1020
gacaatctgt tgtccgtgct cggcgtgggc ctcaccgagc ggagactgct gaactacttg 1080
atcagccagc tgcgtctctc tgaacccgag cgggtcagtg cgctgcgcga attcgcccct 1140
agcgcaatcg attcggactg ggagttgacg atagccggtc agcgggtaca ggtgatccgg 1200
cgagatgaac gcaacggcgg ggtgctcgag ttcggcacga cggtcatcgg cgatgctgac 1260
ggtagtattg ccggactact gggcggctcc ccaggggctt cgaccgcggt ggcgatcatg 1320
ctggacgtgc tgcagaaatg ctttgccaac cgctatcaat cctggctgcc cacgctcaaa 1380
gaaatggtgc cgtcgctggg ggtgcagctg tcgaatgaac ccgcgctgtt cgacgaggtg 1440
tggtcatgga gcaccaaggc gctgaagtta ggtgccgcat ga 1482
Claims (10)
1. utilizing the method for ccdB lethal gene rapid build recombinant plasmid, which comprises the following steps:
A, PCR amplification ccdB gene expression cassettes, expression vector and target gene are obtained containing linear ccdB gene expression cassettes
PCR product, the PCR product containing linear expression vector and the PCR product containing linear target gene；Used primer are as follows: (1)
The amplimer ccdB-HF and ccdB-HR of ccdB gene expression cassettes；(2) vector amplification primer HF and HR；(3) purpose is expanded
The primer gene-HF and gene-HR of gene；
B, the PCR product containing linear ccdB gene expression cassettes for obtaining step a is mixed with the PCR product containing linear expression vector
It is transferred to the competent cell of tolerance ccdB albumen after conjunction, is cultivated in the culture medium containing antibiotic A and antibiotic B, linearly
Homologous recombination occurs for ccdB gene expression cassettes and linear expression vector, obtains the circular vectors with ccdB gene expression cassettes；
Antibiotic A is different with antibiotic B；
C, the circular vectors with ccdB gene expression cassettes obtained using vector amplification primer amplification step b are obtained containing deleting
Except the PCR product of the linear carrier of ccdB gene expression cassettes；
D, the PCR product containing linear target gene for obtaining step a contains deletion ccdB expression casette with what step c was obtained
It is transferred to the competent cell for not tolerating ccdB albumen after the PCR product mixing of the linear carrier of son, in the culture containing antibiotic B
It is cultivated in base, linear target gene and the linear carrier homologous recombination for deleting ccdB gene expression cassettes are obtained with target gene
Circular vectors；
CcdB gene expression cassettes described in step a are the DNA of the resistant gene containing promoter, ccdB gene and antibiotic A
Molecule；
The amplimer sequence of ccdB gene expression cassettes and the primer sequence of amplifying target genes include 2 in step a
Segment: (I) can expand the segment of ccdB gene expression cassettes or target gene, and (II) can be with expression vector homologous recombination
Segment；Segment (I) is located at 3 ' sides of segment (II)；
The segment of the homologous recombination is some or all of vector amplification primer and expression vector pairing segment.
2. the method according to claim 1 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that: institute
The length for stating segment (II) is 18nt.
3. the method according to claim 3 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that: institute
The segment for stating homologous recombination is whole segments of vector amplification primer and expression vector pairing.
4. the method according to claim 1 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that: step
The competent cell that competent cell described in rapid b is Escherichia coli DB3.1.
5. the method according to claim 1 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that: institute
Stating antibiotic A is chloramphenicol.
6. the method according to claim 5 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that: institute
CcdB gene expression cassettes DNA sequence dna is stated as shown in SEQ ID NO:14.
7. the method according to claim 1 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that: step
The quantity of expression vector described in rapid a is 2 or more, and expression vector carries the fixed sequence program of one section of 36bp or more, the fixation sequence
Column can be matched the overall length for amplifying carrier by primer HF and HR.
8. the method according to claim 1 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that: step
The competent cell that competent cell described in rapid d is coli strain DH5 α.
9. the method according to claim 1 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that: step
In rapid d, " PCR product containing linear target gene that step a is obtained " with " step c obtain containing deleting ccdB gene expression
The molar ratio of the PCR product of the linear carrier of box " is 2: 1.
10. the method according to claim 1 using ccdB lethal gene rapid build recombinant plasmid, it is characterised in that:
Further include step e: selecting monoclonal colonies and carry out bacterium solution PCR verifying, extracts plasmid and identification is sequenced, obtain containing mutually homogenic
Different function purposes recombinant expression plasmid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108588101A (en) * | 2018-04-27 | 2018-09-28 | 四川大学 | Build the molecular cloning method of same gene difference expression vector |
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Non-Patent Citations (2)
| Title |
|---|
| HAILONG WANG: "Improved seamless mutagenesis by recombineering using ccdB for counterselection", 《NUCLEIC ACIDS RESEARCH》 * |
| JUDITH SCHOLZ: "A new method to customize protein expression vectors for fast, efficient and background free parallel cloning", 《BMC BIOTECHNOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588101A (en) * | 2018-04-27 | 2018-09-28 | 四川大学 | Build the molecular cloning method of same gene difference expression vector |
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