CN106906232A - A kind of cloned dna molecule method and its application - Google Patents
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Abstract
The invention discloses a kind of cloned dna molecule method and its application, the described method comprises the following steps：Obtain entry vector and contain ccdB genes, ccdB genes two ends are sequentially connected XcmI restriction enzyme sites, joint sequence and SfiI restriction enzyme sites respectively；Obtain expression vector and contain ccdB genes, ccdB genes two ends are sequentially connected SfiI restriction enzyme sites and joint sequence respectively；Target dna is obtained by PCR, is cloned by TA and is inserted into the entry vector, obtain entry clones；Or corresponding joint sequence is added by primer during PCR, obtain linear PCR product；Above-mentioned entry clones or linear PCR product are mixed with expression vector, clone is completed under the effect of cloning reaction liquid.Present invention improves over cloning reaction liquid and the structure of entry vector and expression vector, realize DNA on single or multiple linear PCR products or cyclic plasmid under the effect of cloning reaction liquid Direct Cloning to one or more positions of ring target carrier, cloning efficiency is improve, various types of cloned dna molecules are can be widely used for.
Description
Technical field
The present invention relates to biology field, more particularly to a kind of cloned dna molecule method and its application.
Background technology
Cloned dna molecule technology is the basis of molecular biology research, and its innovation and development push directly on molecular biosciences
Progress.The molecular cloning method for using at present mainly has 3 classes：1. digestion-connection method.Using restriction enzyme and
DNA ligase is earliest molecular cloning method, and at present still widely using, but it is limited (bag by DNA inside corresponding site
Include the Golden Gate clones for setting up in recent years).2. overlap is cloned.Such method include Gibson splicings, In-Fusion,
LIC etc., has the advantages that not limited by DNA sequence dna, can synchronous tiled multiple fragments, but Gibson and In-Fusion kits
Costliness, and need the carrier of linearisation, it is impossible to directly use cyclic plasmid as Gateway clones.3. specific site weight
Group clone.Such method is mainly Gateway clones, DNA restructuring directly can be carried out between plasmid, easy to operate, with high pass
The advantage of amount, it has the disadvantage expensive reagents, multiple clips clone limited (can only clone 2-4 fragment and efficiency is low), specific site
Sequence can not be removed.Therefore, current molecular cloning method still suffers from some shortcomings, and shortage one kind is applied widely, receive journey
Degree method high, also has that cloning efficiency is low.Therefore it is badly in need of a kind of cloned dna molecule method, solve above-mentioned technical problem.
The content of the invention
In view of this, the invention provides a kind of cloned dna molecule method and its application, optimize cloning reaction liquid into
Point, next to that establishing the clone between 2 closed loop carriers, and the seamless clone of joint sequence is can remove, improve clone's effect
Rate, can be widely used for various types of cloned dna molecules.
The technological means that the present invention is used is as follows：A kind of cloned dna molecule method, comprises the following steps：
S1, acquisition entry vector, the entry vector contain ccdB genes, and described ccdB genes one end is sequentially connected XcmI
Restriction enzyme site, the first joint sequence and SfiI restriction enzyme sites, the other end be sequentially connected XcmI restriction enzyme sites, the second joint sequence and
SfiI restriction enzyme sites, i.e., described entry vector contains " SfiI restriction enzyme sites the-the first joint sequence-XcmI restriction enzyme site-ccdB bases
The structure of cause-XcmI restriction enzyme site the-the second joint sequence-SfiI restriction enzyme sites "；
S2, acquisition ring-type expression vector, the expression vector contain ccdB genes, and described ccdB genes one end is sequentially connected
SfiI restriction enzyme sites and the 3rd joint sequence, the other end are sequentially connected SfiI restriction enzyme sites and the 4th joint sequence；Described 3rd
Joint sequence is identical with the first joint sequence, and the 4th joint sequence is identical with the second joint sequence, i.e., described expression vector
Clone's frame knot containing " the 3rd the-the four joint sequence of joint sequence-SfiI restriction enzyme site-ccdB gene-SfiI restriction enzyme sites "
Structure；
S3, target dna is obtained by PCR, cloned by TA and be inserted into the entry vector, obtain entry clones；
Or corresponding joint sequence is added by primer during PCR, obtain linear PCR product；
S4, above-mentioned entry clones or linear PCR product are mixed with expression vector, completed under the effect of cloning reaction liquid gram
It is grand.
Further, the target dna is single or multiple DNA fragmentations, and multiple DNA are by the intersegmental joint sequence of piece
Complete clone.
Further, the cloning reaction liquid is included：SfiI restriction enzymes, T5 exonucleases and Phusion
Archaeal dna polymerase, it is circumscribed using 5 ＇ -3 ＇ 5 prime excision enzyme activities of T5 exonucleases in cloning reaction liquid and 3 ＇ -5 ＇ of Phusion enzymes
The collective effect of enzymatic activity can digest the joint sequence on removal expression vector, realize seamless clone.
Further, first joint sequence and the second joint sequence are 15-40bp.
Further, the cloning reaction condition be 50 DEG C react 15~60 minutes.
The present invention provides a kind of target dna molecule clone's recombinant vector, the carrier by above-mentioned cloned dna molecule method
And prepare.
The present invention provides a kind of kit of cloned dna molecule, and the kit is included：Above-mentioned cloning reaction liquid, enter
Door carrier and expression vector.
The present invention provides single DNA fragmentation or many DNA fragmentations clone, or the multiple location of expression vector is cloned.
Target DNA fragments in the present invention both can be cloned into ring-type by PCR adjunction heads in the form of linear PCR product
Expression vector, after also target DNA fragments being cloned into entry vector, is cloned into ring-type expression in the form of ring-type entry clones
Carrier.
In an embodiment of the invention, by " SfiI restriction enzyme sites the-the first joint sequence-XcmI restriction enzyme site-ccdB bases
Cause-XcmI restriction enzyme site the-the second joint sequence-SfiI restriction enzyme sites " structure is inserted respectively into pENTR/D-TOPO carriers
At TOPO cloning sites, the supporting entry vector of kanamycins (Kan) resistance is obtained.
In an embodiment of the invention, will " the 3rd joint sequence-SfiI restriction enzyme site-ccdB gene-SfiI digestions position
The joint sequence of point-the four " structure replaces the lacZ gene on pUC19 plasmids, obtains the supporting table of ampicillin (Amp) resistance
Up to carrier.
In an embodiment of the invention, from lacZ gene, compare the cloning system belt lacing sequence clone and
The efficiency that seamless clone clones with Gibson.
In an embodiment of the invention, entry clones are built from lacZ gene, complete between 2 closed loop carriers gram
It is grand.
In an embodiment of the invention, from papaya eIF4E genes and the VPg bases of PRSV (PRSV)
Cause, completes 2 gene clonings to 2 positions of expression vector.
In an embodiment of the invention, there is provided a kind of kit of cloned dna molecule.
The technical scheme of offer of the invention compared with prior art, has the following advantages that：
Present invention optimizes the composition and entry vector and the structure of expression vector of cloning reaction liquid, establish 2 and close
Clone between ring carrier, it was found that can remove the seamless clone of joint sequence, realize single or multiple linear PCR products or ring-type
DNA on the plasmid Direct Cloning under the effect of cloning reaction liquid, to one or more positions of ring target carrier, improves gram
Grand efficiency.Wherein, DNA fragmentation can be designed in joint sequence with the overlap of destination carrier SfiI-ccdB-SfiI structure flanks
Periphery, nonoverlapping joint sequence can be by 3 ＇ -5 ＇ of 5 ＇ -3 ＇ 5 prime excision enzyme activities of T5 exonucleases and Phusion enzymes outside
Enzyme cutting activity digestion removal so that the method can be not only used for the standardization clone of general overlap (joint), it can also be used to special
The seamless clone of the removal joint sequence that needs are asked, improves cloning efficiency.Cloned dna molecule method of the invention can be used extensively
In various types of cloned dna molecules.
Brief description of the drawings
Fig. 1 is the application schematic diagram of cloned dna molecule method of the invention, can flexibly be used for single slice, multiple clips, many grams
The clone of the diversified forms such as grand site；
Fig. 2 is the structural representation of supporting entry vector, contains " SfiI restriction enzyme sites the-the first joint sequence-XcmI digestions
The structure of site-ccdB gene-XcmI restriction enzyme site the-the second joint sequence-SfiI restriction enzyme site "；
Fig. 3 is the structural representation of supporting expression vector, contains " the 3rd joint sequence-SfiI restriction enzyme site-ccdB bases
Clone's mount structure of the-the four joint sequence of cause-SfiI restriction enzyme sites "；
Fig. 4 is the efficiency comparison of belt lacing sequence clone and seamless clone with Gibson clones of cloning process of the present invention；
Fig. 5 is that 2 genes are cloned into 2 identifications of the recon at position of expression vector in the form of linear PCR product, its
Middle M：DL2000Marker (Dalian is precious biological), 1-10 are random 10 clones for selecting, and upper and lower 2 band is respectively papaya
The VPg genes of eIF4E genes and PRSV.
Fig. 6 is that 2 genes are cloned into 2 identifications of the recon at position of expression vector in the form of entry clones, wherein
M：DL2000Marker (Dalian is precious biological), 1-10 are random 10 clones for selecting, and upper and lower 2 band is respectively papaya
The VPg genes of eIF4E genes and PRSV.
Specific embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from start to finish
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
It is exemplary to scheme the embodiment of description, is only used for explaining the present invention, and is not considered as limiting the invention.In embodiment
Unreceipted actual conditions person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production of instrument
Manufacturer person, be can by city available from conventional products.Scheme and formula that embodiment is not described in detail, refer to《Molecule gram
Grand experiment guide》, the third edition, Science Press.
Embodiment 1
(1) structure of entry vector
Clone's " SfiI restriction enzyme sites the-the first joint sequence-XcmI restriction enzyme site-ccdB gene-XcmI digestions first
Point the-the second joint sequence-SfiI restriction enzyme sites " structure.Nucleotide sequence (GenBank database logins number according to ccdB genes：
JQ085427), pair of primers, sense primer FP (SEQ ID NO are designed:1)：
TC AGCTGTCTCGTT ATTCTCGACTAA
GTTGGCAG, and anti-sense primer RP (SEQ ID NO:2)：
TC AACAAACGCAGA CTGTGTATAAG
The sequence of GGAGCCTG, wherein italic is respectively SfiI restriction enzyme sites and XcmI restriction enzyme sites, and underlined sequences are joint sequence.
With plasmid pRNAi-GG (the GenBank database logins number containing ccdB genes：JQ085427 it is) template, is drawn using above-mentioned two
Thing enters performing PCR amplification, and PCR amplification programs are：94 DEG C 4 minutes；94 DEG C 30 seconds again, 55 DEG C 30 seconds, 72 DEG C 60 seconds, 30 circulations；
Last 72 DEG C extend 5 minutes.Amplification obtains the PCR primer of ccdB genes, mixed with pENTR/D-TOPO carriers after being reclaimed through electrophoresis
Close, be inserted on carrier by TOPO cloning reactions, obtain supporting kanamycins (Kan) the resistance entry vector of present invention clone
(as shown in Figure 2).The structure of the supporting entry vector of other types (different resistances, different joint sequences) is with reference to the method.
(2) structure of expression vector
" the 3rd joint sequence-SfiI restriction enzyme site-ccdB gene-SfiI the-the four joint sequences of restriction enzyme site are cloned first
Clone's mount structure of row ".Design pair of primers, sense primer FP (SEQ ID NO:3)：AGCTGTCTCGTTCCATCTCT ATTCTCGACTAAGTTGGCAG, and anti-sense primer RP (SEQ ID NO:4)：AACAAAC GCAGACCACAAGT The sequence of CTGTGTATAAGGGAGCCTG, wherein italic is SfiI enzymes
Enzyme site, the sequence of underscore is joint sequence, and joint sequence is consistent with the joint sequence in above-mentioned entry vector respectively.To contain
The plasmid pRNAi-GG of ccdB genes is template, and entering performing PCR using this pair of primer expands, and PCR amplification programs are：94 DEG C 4 minutes；
94 DEG C 30 seconds again, 55 DEG C 30 seconds, 72 DEG C 60 seconds, 30 circulations；Last 72 DEG C extend 5 minutes.Amplification obtains the PCR of ccdB genes
Product, electrophoresis is reclaimed.
Then pUC19 carrier frames are expanded, pair of primers is designed,
Sense primer FP (SEQ ID NO:5)：
AGAGATGGAACGACAGCTTCACTGCCCGCTTTCCAGTC,
And anti-sense primer RP (SEQ ID NO:6)：
ACTTGTGGTCTGCGTTTGTTTGTGACCGTCTCCGGGAGCT, wherein underlined sequences connect with above-mentioned respectively
Header sequence reverse complemental.It is template with carrier containing pUC19, entering performing PCR using this pair of primer expands, and PCR amplification programs are：94℃
4 minutes；94 DEG C 30 seconds again, 55 DEG C 30 seconds, 72 DEG C 90 seconds, 30 circulations；Last 72 DEG C extend 5 minutes.Amplification obtains pUC19 loads
The PCR primer of body framework, electrophoresis is reclaimed.
Clone's mount structure after recovery mixes with pUC19 carrier frames, is cloned by Gibson, obtains present invention clone and matches somebody with somebody
The expression vector (as shown in Figure 3) of ampicillin (Amp) resistance of set, is named as pUC19-NC.(difference is anti-for other types
Property, different joint sequence) supporting expression vector structure with reference to the method.
(3) cloning reaction：The cloning efficiency that cloning process of the present invention is cloned with Gibson compares
It is target gene from lacZ gene, designs pair of primers,
Sense primer FP (SEQ ID NO:7)：
AGCTGTCTCGTTCCATCTCTGCGCAACGCAATTAATGTGAG, and anti-sense primer RP (SEQ ID NO:
8)：
AACAAACGCAGACCACAAGTACAGCTTGTCTGTAAGCGGA, underlined sequences respectively with supporting expression vector
On joint sequence it is identical, for belt lacing sequence of the invention clone and Gibson clone；
Design is another to lacZ gene primer, sense primer FP (SEQ ID NO:9)：
GACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG, and anti-sense primer RP (SEQ ID NO:
10)：
AGCTCCCGGAGACGGTCACAACAGCTTGTCTGTAAGCGGA, underlined sequences not with supporting expression vector
On joint sequence it is identical, but with joint sequence periphery carrier sequence reverse complemental, for it is of the invention removal joint sequence
The seamless clone of row.It is template with carrier containing pUC19, entering performing PCR respectively to primer using this 2 points expands, PCR amplification programs
For：94 DEG C 4 minutes；94 DEG C 30 seconds again, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 30 circulations；Last 72 DEG C extend 5 minutes.Amplification is obtained
2 PCR primers of lacZ gene (containing prokaryotic promoter), electrophoresis is reclaimed, and is respectively designated as lacZ and lacZ-sl
(seamless clone).
Cyclic plasmid by lacZ and lacZ-sl respectively with supporting expression vector pUC19-NC mixes, using containing SfiI
The cloning reaction liquid of restriction enzyme, T5 exonucleases and Phusion archaeal dna polymerases, 50 DEG C are reacted 1 hour, carry out band
Joint sequence clone and seamless clone, wherein, in cloning reaction liquid 5 ＇ -3 ＇ 5 prime excision enzyme activities of T5 exonucleases and
The collective effect of 3 ＇ -5 ＇ 5 prime excision enzyme activities of Phusion enzymes can digest the joint sequence on removal expression vector, and then realize nothing
Seam clone；The SfiI digestions of pUC19-NC cyclic plasmids, the linear carrier of recovery are mixed with lacZ, Gibson clones are carried out,
Reaction condition is similarly 50 DEG C and reacts 1 hour.Blue colonies are counted after conversion Escherichia coli, as a result as shown in figure 4, the present invention gram
The belt lacing sequence clone of grand method and the efficiency of seamless clone are cloned high than Gibson.
Embodiment 2：2 gene clonings are to 2 positions of carrier
(1) 2 gene is cloned into 2 positions of expression vector in linear PCR product form
From papaya eIF4E genes and the VPg genes of PRSV (PRSV), using clone side of the invention
This 2 genes are synchronously cloned into method 2 positions of integrated plant BiFC carriers (pBiFC-GG).
The primer of pair for amplification papaya eIF4E genes is designed,
Sense primer FP (SEQ ID NO:11)：
GTCTGGAGACCTAGGTTCTG ATGGTAGTAGAAGGAACCC,
And anti-sense primer RP (SEQ ID NO:12)：
CTGAACCACCTGAGACCGAA TACCGAGTAGCGATTCTTAG,
As template, amplification obtains target gene to cDNA with papaya.
The primer of the VPg genes of design pair for amplification PRSV,
Sense primer FP (SEQ ID NO:13)：
TAGTGGCGGAGACCATGGAG ATGGGTTTCTCCGCGCGACAG, and anti-sense primer RP (SEQ ID NO:
14)：
TACTACCTCCTGAAGAGACC TTCATGGTGAACAGATTTTGTT, be with the papaya cDNA for infecting PRSV
Template, amplification obtains target gene.2 genes mix after being reclaimed through electrophoresis with pBiFC-GG carriers, are limited by containing SfiI
Property restriction endonuclease, T5 exonucleases and Phusion archaeal dna polymerases cloning reaction liquid effect, obtain recombinant vector.Restructuring is carried
Body identifies that as a result see Fig. 5, the VPg genes containing papaya eIF4E genes and PRSV are cloned in 10 for selecting at random through PCR.
(2) 2 genes are cloned into 2 positions design of expression vector without joint sequence in the form of entry clones
PCR primer, wherein the primer of amplification papaya eIF4E genes is, sense primer FP (SEQ ID NO:15)：
ATGGTAGTAGAAGGAACCC, and anti-sense primer RP (SEQ ID NO:16)：TACCGAGTAGCGATTCTTAG, with a kind wood
The cDNA of melon is template, and amplification obtains target gene.
The primer of VPg genes for expanding PRSV is,
Sense primer FP (SEQ ID NO:17)：ATGGGTTTCTCCGCGCGACAG,
And anti-sense primer RP (SEQ ID NO:18)：
TTCATGGTGAACAGATTTTGTT, is template with the papaya cDNA for infecting PRSV, and amplification obtains target gene.
2 genes are cloned by TA after being reclaimed through PCR and electrophoresis and are inserted into the entry vector of ammonia benzyl resistance, and with target gene 2
Individual entry clones mix with pBiFC-GG carriers, by containing SfiI restriction enzymes, T5 exonucleases and Phusion
The cloning reaction liquid effect of archaeal dna polymerase, obtains recombinant vector.As a result recombinant vector is shown in Fig. 6 through PCR identifications, selects at random
10 VPg genes cloned containing papaya eIF4E genes and PRSV.
Embodiment 3
A kind of kit of cloned dna molecule, the kit is included：Cloning reaction liquid, entry vector and expression vector,
Wherein, the cloning reaction liquid is included：SfiI restriction enzymes, T5 exonucleases and Phusion archaeal dna polymerases；It is described
Entry vector contains ccdB genes, and described ccdB genes one end is sequentially connected XcmI restriction enzyme sites, the first joint sequence and SfiI
Restriction enzyme site, the other end is sequentially connected XcmI restriction enzyme sites, the second joint sequence and SfiI restriction enzyme sites, i.e., described entry vector
Contain " SfiI restriction enzyme sites the-the first joint sequence-XcmI restriction enzyme site-ccdB gene-XcmI restriction enzyme site the-the second joint sequences
The structure of row-SfiI restriction enzyme sites "；The expression vector contains ccdB genes, and described ccdB genes one end is sequentially connected SfiI
Restriction enzyme site and the 3rd joint sequence, the other end are sequentially connected SfiI restriction enzyme sites and the 4th joint sequence；3rd joint
Sequence is identical with the first joint sequence, and the 4th joint sequence is identical with the second joint sequence, i.e., described expression vector contains
Clone's mount structure of " the 3rd the-the four joint sequence of joint sequence-SfiI restriction enzyme site-ccdB gene-SfiI restriction enzyme sites ".
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Within god and principle, any modification, equivalent substitution and improvements done etc. should be included within the scope of protection of the invention.
Claims (7)
1. a kind of cloned dna molecule method, it is characterised in that：Comprise the following steps：
S1, acquisition entry vector, the entry vector contain ccdB genes, and described ccdB genes one end is sequentially connected XcmI digestions
Site, the first joint sequence and SfiI restriction enzyme sites, the other end are sequentially connected XcmI restriction enzyme sites, the second joint sequence and SfiI
Restriction enzyme site；
S2, acquisition ring-type expression vector, the expression vector contain ccdB genes, and described ccdB genes one end is sequentially connected SfiI
Restriction enzyme site and the 3rd joint sequence, the other end are sequentially connected SfiI restriction enzyme sites and the 4th joint sequence；3rd joint
Sequence is identical with the first joint sequence, and the 4th joint sequence is identical with the second joint sequence；
S3, target dna is obtained by PCR, cloned by TA and be inserted into the entry vector, obtain entry clones；
Or corresponding joint sequence is added by primer during PCR, obtain linear PCR product；
S4, above-mentioned entry clones or linear PCR product are mixed with expression vector, clone is completed under the effect of cloning reaction liquid.
2. cloned dna molecule method according to claim 1, it is characterised in that：The target dna is single or multiple
DNA fragmentation, multiple DNA complete clone by the intersegmental joint sequence of piece.
3. cloned dna molecule method according to claim 1, it is characterised in that：The cloning reaction liquid is included：SfiI is limited
Property restriction endonuclease processed, T5 exonucleases and Phusion archaeal dna polymerases.
4. cloned dna molecule method according to claim 1, it is characterised in that：The joint sequence is 15-40bp.
5. cloned dna molecule method according to claim 1, it is characterised in that：The cloning reaction condition is 50 DEG C anti-
Answer 15~60 minutes.
6. the cloned dna molecule recombinant vector that prepared by the claim according to any one of Claims 1 to 5.
7. a kind of kit of cloned dna molecule, it is characterised in that：The kit is included：1. cloned described in claim 3
Reaction solution；2. entry vector described in claim 1；3. expression vector described in claim 1.
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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 |
| CN108588101B (en) * | 2018-04-27 | 2022-03-04 | 苏丹 | Molecular cloning method for constructing different expression vectors of same gene |
| CN110863004A (en) * | 2019-12-06 | 2020-03-06 | 中国热带农业科学院热带生物技术研究所 | Yeast two-hybrid vector, construction method and application thereof in protein interaction |
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