CN100465278C - Method of constructing T carrier - Google Patents
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- CN100465278C CN100465278C CNB2004100479027A CN200410047902A CN100465278C CN 100465278 C CN100465278 C CN 100465278C CN B2004100479027 A CNB2004100479027 A CN B2004100479027A CN 200410047902 A CN200410047902 A CN 200410047902A CN 100465278 C CN100465278 C CN 100465278C
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Abstract
A process for configuring the T carrier with higher Ta cloning efficiency includes preparing the T carrier precursor containing XcmI box or AhdI box, and severing the T carrier precursor by XcmI (or its isoschizomer) or AhdI (or its isoschizomer) to obtain T carrier.
Description
Technical field
The present invention relates to the method for a kind of carrier construction in the genetically engineered field, particularly a kind of method that makes up the T carrier.
Background technology
PCR (polymerase chain reaction) is the normal experiment technology in the molecular biology.Need in most cases pcr amplification product is cloned on the plasmid vector, so as to the PCR product check order, operation such as in-vitro transcription, external translation.The method of clone PCR products has a variety of, but method the most direct, most convenient is the TA clone.So-called TA clone has the PCR product cloning to 3 of single A tail with 3 ' end ' end has on the T carrier of single T tail.TA clone's theoretical foundation is: in the pcr amplification process, because it is active and terminally add single deoxynucleotide at 3 ' of PCR product that the Taq polysaccharase has the terminal enzyme (DNA) of the template of not relying on, and preference is added deoxyadenylic acid (the A) (Clark in four kinds of deoxynucleotides, J.M., 1988.Novelnon-templated nucleotide addition reactions catalyzed prokaryotic andeucaryotic DNA polymerase.Nucleic Acids Res.16,9677-9686; Cha, J., Bishai, W., Chandrasegaran, S., 1993.New vectors for direct cloning of PCR products.Gene 136,369-370; Schutte, B.C., Ranade, K., Pruessner, J., Dracopoli, N., 1997.Optimized conditions for cloning PCR products into an XcmI T-vector.BioTechniques 22 40-42), makes 3 ' end of the PCR product more than 50% have single deoxyadenylic acid, promptly 3 ' hold A tail (Ichihara, Y., Kurosawa, Y., 1993.Construction of new Tvectors for direct cloning of PCR products.Gene 130,153-154).
At present, the preparation method of T carrier has 3 kinds.First method is to utilize the restriction endonuclease that produces flat end that the cyclic plasmid enzyme is cut into the wire plasmid, utilize terminal enzyme (DNA) single pair of dT acid (ddTTP) to be added to 3 ' end (Holton of wire carrier then, T.A., Graham, M.W., 1991.A simple and efficientmethod for direct cloning of PCR products using ddT-tailed vectors.NucleicAcids Res.19,1156).Second method is the 3 ' end (Marchuk that the terminal enzyme (DNA) activity that does not rely on template of utilizing the Taq enzyme is added single dT acid (dTTP) in the wire carrier, D., Drumm, M., Saulino, A., Collins, F.S., 1991.Construction of T-vectors, a rapid andgeneral system for direct cloning of unmodified PCR products.Nucleic AcidsRes.19,1154).The third method is to introduce specific restriction enzyme site in the multiple clone site of plasmid vector, produces 3 ' end with corresponding endonuclease digestion and has the outstanding linear T carrier of single T.Preceding two kinds of methods exist tailing efficient sequences lower and linear plasmid its two ends in the tailing process problem (Shuman such as to be partially removed sometimes in preparation T carrier process, S., 1994.Novel approach to molecular cloning andpolynucleotide synthesis using vaccinia DNA topoisomerase.J.Biol.Chem.265,32678-32684).Compare with preceding two kinds of methods, the third method is faster, more efficient and more stable.In theory, the restriction endonuclease that can be used for preparing the T carrier comprises: XcmI (Jo, C., Jo, S.A., 2001.A simple methodto construct T vectors using Xcm I cassettes amplified by nonspecific PCR.Plasmid 45,37-40; Arashi, N., Miwa, M., Shibata, H., 1999.XcmIsite-containing vector for direct cloning and in vitro transcription for PCRproduct.Mol.Biotechnol.12,281-283), AhdI/Eaml105I/EcIHKI/AspEI/NruGI/BspOVI (Jeung, J.U., Cho, S.K., Shim, K.S., Ok, S.H., Lim, D.S., Shin, J.S., 2002.Construction of two pGEM-7Zf (+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products.Plasmid48,160-163; Ichihara, Y., Kurosawa, Y., 1993.Construction of new T vectorsfor direct cloning of PCR products.Gene 130,153-154), BfiI/BmrI, HphI/AsuHPI (Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A., Smith, L.M., 1991.A universal method for the direct cloning of PCR amplified nucleic acid.Bio/Technology 9,657-663), MboII/NcuI, BfuI/BciVI, HpyAV/Hin4II.Wherein XcmI and AhdI and isoschizomers thereof are used widely in preparation T carrier.Other restriction endonuclease or because too expensive, or because too many recognition site is arranged on common cloning vector, thereby the application in preparation T carrier is restricted.In the common cloning vector that contains the Amp resistance, for example pUC18, pBlueScript SK carrier etc., do not have the XcmI restriction enzyme site, but the restriction enzyme site that in the Amp resistant gene, contains an AhdI and isoschizomers thereof, thereby more more convenient in the multiple clone site introducing XcmI site of these carriers, Here it is, and why more people adopt XcmI to prepare T carrier (american documentation literature US005487993A; Chinese patent literature CN1142279C, CN1344795A and CN1362521A).If introduce the restriction enzyme site of AhdI and isoschizomers thereof, then must be by corresponding restriction enzyme site in the reticent Amp resistant gene of the method for rite-directed mutagenesis.However, utilize AhdI and isoschizomers thereof to prepare the T carrier and more have superiority, show following several aspect compared with XcmI.First, AhdI has 5 kinds of isoschizomerss, 4 kinds of isoschizomers commercializations wherein, thereby add that AhdI has 5 kinds of restriction endonucleases can supply to select for use, and XcmI does not have isoschizomers (Roberts R.J., T.Vincze, J.Posfai, and D.Macelis.2003.REBASE:restriction enzymes andmethyltransferases.Nucleic Acids Res.31:418-420).Second, the performance of AhdI restriction endonuclease is better than XcmI (Jeung, J.U., Cho, S.K., Shim, K.S., Ok, S.H., Lim, D.S., Shin, J.S., 2002.Construction of two pGEM-7Zf (+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products.Plasmid48,160-163), and the sale price of market of AhdI is far below XcmI (seeing " NEB products catalogue and technical information ", 2003-2004 years).The 3rd, the sequence of AhdI and isoschizomers thereof identification 11bp, and the sequence of XcmI identification 15bp, thereby introduce (for example in the PCR primer, introducing) AhdI and the isoschizomers recognition sequence easier.
No matter be to use XcmI also to be to use AhdI and isoschizomers thereof to make up the T carrier and all have a potential problem: partially digested pre-T carrier is mingled in the too high (Mead of the background that can cause non-recombinant conversion in the T carrier, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A., Smith, L.M., 1991.A universal methodfor the direct cloning of PCR amplified nucleic acid.Bio/Technology 9,657-663; Harrison, J., Molly, P.L., Clark, S.J., 1994.Direct cloning ofpolymerase chain reaction products in an XcmI T-vector.Anal.Biochem.216,235-236).At present, address this problem effective means and be between two XcmI of pre-T carrier or two AhdI restriction enzyme sites and introduce sufficiently long spacer DNA, behind agarose gel electrophoresis, the plasmid of complete degestion and partially digested plasmid are separated (Testori, A., Sollitti, P., 1996.Cloning unmodified PCRproducts using engineered XcmI restriction sites in a portable cassette.Methods Mol.Biol.67,89-100; Jo, C., Jo, S.A., 2001.A simple method toconstruct T vectors using Xcm I cassettes amplified by nonspecific PCR.Plasmid45,37-40; Jeung, J.U., Cho, S.K., Shim, K.S., Ok, S.H., Lim, D.S., Shin, J.S., 2002.Construction of two pGEM-7Zf (+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products.Plasmid48,160-163).Yet, introducing a new problem of bringing behind the spacer DNA is: because ring-like plasmid moves soon compared with the suitable linear plasmid of molecular weight in agarose gel electrophoresis, thereby, not digested ring-like plasmid (pre-T carrier, have spacer DNA) often mix with the T carrier (not having spacer DNA) of molecular weight less than self, finally cause the background of non-recombinant conversion too high.
PBlueScript II SK (+/-) carrier (GenBank Accession No.X52330) is to be derived from and the phagemid carrier (phagemid or phasmid) that comes by the pUC carrier, except containing an Amp resistant gene and a lacZ gene as the selection markers, the promotor that has T3 and T7 phage in the both sides of multiple clone site has the replication orgin (unique difference of pBlueScript II SK (+) and pBlueScript II SK (-) is the oppositely opposite of both f1) of a single stranded phage f1 simultaneously.
CcdB gene (GenBank Accession No.AP001918) is positioned on the F plasmid, and it and ccdA gene constitute ccd (the control of cell death) site of F plasmid together.The ccd site reaches the effect of stablizing the F plasmid by killing the Bacillus coli cells that does not contain the F plasmid.CcdB albumen disturbs colibacillary dna gyrase, thereby suppress most of coli strains (DH5 α for example, JM109, DH10B, growth TOP10) (Bernard, P., and Couturier, M., 1992.Cell Killing by the F Plasmid CcdB Protein InvolvesPoisoning of DNA-Topoisomerase II Complexes.J.Mol.Biol.226,735-745).But place sudden change has taken place in the A subunit gene of gyrase in the intestinal bacteria DB3.1 bacterial strain, gyrase after the sudden change can be resisted the proteic toxic effect of CcdB, thereby the plasmid that contains the ccdB gene can be bred (Miki in the DB3.1 bacterial strain, T., Park, J.A., Nagao, K., Murayama, N., and Horiuchi, T., 1992.Control of Segregation of Chromosomal DNA by Sex Factor F in Escherichia coli.Mutants of DNA Gyrase Subunit A Suppress letD (ccdB) Product Growth Inhibition.J.Mol.Biol.225,39-52).
Summary of the invention
The purpose of this invention is to provide the T carrier method that a kind of preparation has higher TA cloning efficiency.
The method for preparing the T carrier provided by the present invention may further comprise the steps:
1) preparation contains the pre-T carrier of XcmI box or AhdI box; Described XcmI box comprises the spacer DNA sequence, closely is connected in each XcmI restriction enzyme site sequences of described spacer DNA sequence both sides, and described AhdI box comprises described spacer DNA sequence, closely is connected in each AhdI restriction enzyme site sequences of described spacer DNA sequence both sides;
2) with the isoschizomers of XcmI or XcmI, or the isoschizomers enzyme of AhdI or AhdI cuts the pre-T carrier in the step 1), obtains the T carrier;
Wherein, the spacer DNA sequence in the described step 1) is the ccdB gene order.
Described ccdB gene order can be ccdB gene complete sequence (GenBank Accession No.AP001918), also can be the open reading frame of ccdB gene.
Described XcmI box also comprises the restriction enzyme site sequence except that XcmI restriction enzyme site sequence that is connected in described each XcmI restriction enzyme site sequence; Described AhdI box also comprises the restriction enzyme site sequence except that AhdI restriction enzyme site sequence that is connected in described each AhdI restriction enzyme site sequence.
The carrier that sets out that is used for preparing described pre-T carrier can be the cyclic plasmid carrier in genetically engineered field, be preferably pBlueScript II SK or pUC18 or pUC19 or pUC118 or pUC119 or pGEM series etc., especially be preferably pBlueScript II SK (-).
When the carrier that sets out that is used to prepare described pre-T carrier was pBlueScript II SK (-), the AhdI restriction enzyme site of the Amp resistant gene among the described pBlueScript II SK (-) was by silence.Wherein, the AhdI restriction enzyme site of the Amp resistant gene among the pBlueScriptII SK (-) can pass through the mutation method silence, as the method by rite-directed mutagenesis, will sport C from 5 of Amp resistant gene ' end the 783rd bit base G.
Can be introduced described AhdI box by the multiple clone site among the pBlueScript II SK (-) of silence at the AhdI of Amp resistant gene restriction enzyme site; Wherein, introduce described AhdI box between SalI restriction enzyme site that is preferably in described multiple clone site and the XbaI enzyme cutting site or between KpnI restriction enzyme site and the SacI restriction enzyme site or between EcoRI restriction enzyme site and the EcoRI restriction enzyme site or between EagI restriction enzyme site and the EagI restriction enzyme site.Described AhdI box is preferably the nucleotide sequence that has shown in sequence in the sequence table 1, sequence 2, sequence 3 or sequence 4.
Sequence 1 in the sequence table is by 343 based compositions; Sequence 2 in the sequence table is by 343 based compositions; Sequence 3 in the sequence table is by 343 based compositions; Sequence 4 in the sequence table is by 373 based compositions.
Described pre-T carrier is preferably pSK01 or pSK02 or pSK03 or pSK04; Described T carrier is preferably pSK01-T (its physical map as shown in Figure 2) or pSK02-T (its physical map as shown in Figure 3) or pSK03-T (its physical map as shown in Figure 4) or pSK04-T (its physical map as shown in Figure 5).
The isoschizomers of described AhdI comprises Eaml 105I/EcIHKI/AspEI/NruGI/BspOVI.
XcmI box or AhdI box that the present invention prepares the pre-T carrier in the method for T carrier adopt the ccdB gene as spacer DNA, make spacer DNA have at interval two XcmL or AhdI restriction enzyme site simultaneously and as the negative dual function of selecting mark.Can in the DB3.1 bacterial strain, breed as the pre-T carrier of spacer DNA with the ccdB gene, when the linked system that T carrier and PCR product etc. are formed transforms Bacillus coli communis bacterial strain (DH5 α for example, JM109, DH10B, TOP10) time, mix pre-T carrier in the T carrier (comprising ring-like plasmid that enzyme is not cut and the partially digested ring-like plasmid of linear plasmid) and can kill Bacillus coli cells through being formed by connecting.Like this, the too high problem of the sub-background of non-recombinant conversion that is caused by pre-T carrier in the TA clone process is solved.In addition, the T tail of sub-fraction T carrier (for example exonuclease activity that AhdI and isoschizomers and ligase enzyme etc. are remaining, multigelation etc.) for various reasons can be removed, and the T carrier of removing the T tail is another source of forming the sub-background of non-recombinant conversion through self connecting the transformant that produces.
When the carrier that sets out of the present invention is pBlueScript II SK (-), makes non-recombinant conversion in above-mentioned source have complete lacZ reading frame by accurate design AhdI box, thereby can remove this non-recombinant conversion through blue hickie screening.The method of the T of preparation carrier of the present invention can reduce miscellaneous in the T the carrier not ring-like plasmid cut of enzyme and partially digested linear plasmid to greatest extent.
PSKxx series pre-T carrier (comprising pSK01, pSK02, pSK03 and pSK04) that makes up with the inventive method and the pSKxx-T series T carrier (comprising pSK01-T, pSK02-T, pSK03-T and pSK04-T) that is come by these pre-T carriers preparations are based on pBlueScript II SK (-) and make up and form.These carriers have the characteristics of following several aspects:
1, all characteristics that has pBlueScript II SK (-) carrier.Except containing an Amp resistant gene and a lacZ gene as the selection markers, there is the promotor of T3 and T7 phage in the both sides of multiple clone site, have the replication orgin of a single stranded phage f1 simultaneously.
2, utilize rite-directed mutagenesis the method silence AhdI restriction enzyme site of Amp resistant gene in pBlueScript II SK (-) carrier.The carrier called after pSK Δ Ahd of reticent AhdI restriction enzyme site.The base (base in the frame) that the rite-directed mutagenesis front and back take place in the Amp resistant gene is as follows:
Sequence before the sudden change: 5 '-
ACGGGGAGTC-3 '
Sequence after the sudden change: 5 '-
ACGGGGAGTC-3 '.
3, pSKxx series pre-T carrier is to introduce AhdI box structure to form in the multiple clone site of pSK Δ Ahd.The core sequence of AhdI box following (dash area shows the AhdI restriction enzyme site, and partly representing ccdB gene order, N in the frame is A or T or C or G, the concrete site of " ^ " expression enzyme cutting):
4, the ccdB gene in the AhdI box has spacer DNA and the negative dual function of selecting mark.CcdB is meant that as spacer DNA the ccdB gene order is spaced apart with two AhdI restriction enzyme sites, and the ccdB gene will kill Bacillus coli cells after being transformed into the Bacillus coli communis strain cell as the negative plasmid that selects marker gene to be meant to contain the ccdB gene.CcdB gene in the AhdI box can have three kinds of forms: first kind of lacZ reading frame fusion that form is ccdB gene and its front, and under the lacZ promoters driven, express to produce and merge CcdB albumen; The front that second kind of form is the ccdB gene has ribosome bind site (RBS) sequence, expresses to produce CcdB albumen under the lacZ promoters driven; The third form is that the front of ccdB gene has self promotor and RBS sequence, expresses producing CcdB albumen under the double drive of lacZ promotor and himself promotor.
5, use AhdI restriction endonuclease or its isoschizomers (Eam1105I/EcIHKI/AspEI/NruG/BspOVI) enzyme to cut pSKxx series pre-T carrier, enzyme is cut system and is separated through agarose gel electrophoresis, and the big fragment of cutting the glue recovery promptly is corresponding T carrier.The difference of 4 kinds of T carriers is the kind and the different amts of restriction enzyme site in the multiple clone site.Wherein, pSK02-T has minimum restriction enzyme site, can reduce the influence to in-vitro transcription of high GC content and palindrome, so pSK02-T is particularly suitable for carrying out in-vitro transcription to inserting fragment.PSK03-T has kept whole restriction enzyme sites of pBlueScript IISK (-) carrier, therefore can select more restriction endonuclease to discharge from the T carrier and insert fragment.In addition, owing to additionally introduced an EcoR I restriction enzyme site, therefore can discharge from the T carrier and insert fragment with EcoR I single endonuclease digestion.How paired restriction enzyme site has been introduced in the TA cloning site both sides of pSK04-T carrier, therefore can identify positive colony or discharge with more restriction endonuclease single endonuclease digestion and insert fragment, NotI restriction enzyme site wherein belongs to rare restriction enzyme site, and it is very low to insert the probability that contains this restriction enzyme site in the fragment.
6, the sub-fraction in the serial T carrier of pSKxx-T (comprising pSK01-T, pSK02-T, pSK03-T and pSK04-T) of preparation (for example exonuclease activity that AhdI and isoschizomers and ligase enzyme etc. are remaining, multigelation etc.) when its T tail is removed for various reasons, these plasmids that lose the T tail can form complete lacZ reading frame after self connects, thereby can get rid of these non-recombinant conversion by blue hickie method for screening.
When carrying out the TA clone, the T carrier that adopts method of the present invention to prepare, therefore improved the quality and the stability of T carrier greatly owing to thoroughly solved the too high problem of the sub-background of non-recombinant conversion.PSKxx-T series T carrier with the inventive method preparation has more good quality production characteristic.Experimental results show that T carrier of the present invention clones the TA cloning efficiency of PCR product of 1.8kb and 0.68kb respectively more than 95%.The method of the T of preparation carrier of the present invention will play an important role in the genetically engineered field.
Description of drawings
Figure 1A is the structural representation of AhdI box among the pre-T carrier pSK01
Figure 1B is the structural representation of AhdI box among the pre-T carrier pSK02
Fig. 1 C is the structural representation of AhdI box among the pre-T carrier pSK03
Fig. 1 D is the structural representation of AhdI box among the pre-T carrier pSK04
Fig. 2 is the physical map of pSK01-T
Fig. 3 is the physical map of pSK02-T
Fig. 4 is the physical map of pSK03-T
Fig. 5 is the physical map of pSK04-T
Embodiment
The structure of embodiment 1, pSK Δ Ahd
Utilize the AhdI restriction enzyme site of Amp resistant gene in reticent pBlueScript SK (-) carrier of method of rite-directed mutagenesis, obtain pSK Δ Ahd, its concrete construction process is as follows: the base of introducing sudden change in the PCR primer, with pBluescript II SK (-) carrier is template, carries out pcr amplification with the Pfu archaeal dna polymerase.Wherein, the pcr amplification primer is (add the mutational site (in original series be G) of C for introducing of frame, dash area is the AhdI restriction enzyme site after sudden change destroys):
AmpF：5’-CTACGATACGGGAGGGCTTACCATCTGG-3’；
AmpR：5’-TTATCTACAC
AGGCAAC-3’；
The reaction system of pcr amplification is: contain 5U Pfu in the 50 μ L reaction systems, and 0.2mmol/L dNTPs, 1 * Pfu buffer, each 10 μ mol/L of primer are about template 5ng; The cycling program of pcr amplification is: 94 ℃ of pre-sex change 5min, (94 ℃, 30S; 60 ℃, 30S; 72 ℃, 5min) * 30, the 72 ℃ last 5min that extends.Behind 1% agarose gel electrophoresis, cut glue and reclaim pcr amplification product.Afterwards, set up following ligation: contain 3U ligase enzyme (Promega) in the 10 μ L linked systems, 1 * connection Buffer, about 50ng PCR product.4 ℃ connect 16 hours.After will connecting product transformed into escherichia coli (DH10B), behind blue hickie and Amp resistance screening, obtain positive colony, at random 6 positive colonies of picking, order-checking one by one, obtain AhdI restriction enzyme site ruined pBluescript II SK (-) carrier, called after pSK Δ Ahd.Wherein, the base (base in the frame) before and after the generation rite-directed mutagenesis is as follows in the Amp resistant gene:
Sequence before the sudden change: 5 '-
ACGGGGAGTC-3 '
Sequence after the sudden change: 5 '-
ACGGGGAGTC-3 ';
Sequencing primer is: Amp18:5 '-AATAGACTGGATGGAGGC-3 '.
Embodiment 2, in the multiple clone site of pSK Δ Ahd, introduce the AhdI box and make up pre-T carrier
With plasmid pENTR1A (available from Invitrogen company) is template, carries out pcr amplification with CcdB01F and CcdB01R primer.The reaction system of pcr amplification is: contain 5U Pfu in the 50 μ L reaction systems, and 0.2mmol/L dNTPs, 1 * Pfu buffer, each 10 μ mol/L of primer are about template 5ng; The cycling program of pcr amplification is: 94 ℃ of pre-sex change 5min, (94 ℃, 30S; 60 ℃, 30S; 72 ℃, 1.5min) * 30, the 72 ℃ last 5min that extends.The PCR product is cut glue and is reclaimed behind 1% agarose gel electrophoresis, the PCR product and the pSK Δ Ahd plasmid that reclaim are used SalI and XbaI double digestion respectively, and enzyme is cut product and cut the glue recovery behind 1% agarose gel electrophoresis.PSK Δ Ahd carrier after PCR product after enzyme cut and enzyme are cut is set up following ligation system: contain 3U ligase enzyme (Promega) in the 10 μ L linked systems, 1 * connect Buffer, about 50ng plasmid, about 50ng PCR product.4 ℃ connect more than 16 hours.Connect product and transform DB3.1 bacterial strain (Invitrogen) according to ordinary method.Obtain positive bacterium colony through the Amp resistance screening, positive bacterium colony is identified through order-checking and is obtained pre-T carrier pSK01.
The building process of pSK02 is that CcdB02F and CcdB02R and resulting pcr amplification product and pSK Δ Ahd use respectively SacI and the KpnI double digestion except that used pcr amplification primer, other step and pSK01 with.
The structure of the building process of pSK03 and pSK01 and pSK02 is similar substantially, different is to carry out pcr amplification with CcdB03F and CcdB03R primer, pcr amplification product and pSK Δ Ahd plasmid are cut with the EcoRI enzyme respectively, and the pSK Δ Ahd plasmid after enzyme is cut is being removed 5 '-phosphoric acid to prevent recirculation with handling with alkaline phosphatase before PCR product after enzyme is cut is connected.Connect product and transform the DB3.1 intestinal bacteria, carry out colony PCR amplification, identify the positive colony that inserts (consistent) with forward, obtain pSK03 with lacZ reading frame direction with M13-47 and ccdBR primer according to ordinary method.
The building process of pSK04 was divided into for two steps.The first step is a template with the pENT-NL plasmid (http://deepgreen.stanford.edu/cell imaging site/html/vectors.html) that contains the EGFP gene, is that primer carries out pcr amplification with EGFPF and EGFPR.The reaction system of pcr amplification is: contain 5U Pfu in the 50 μ L reaction systems, and 0.2mmol/L dNTPs, 1 * Pfu buffer, each 10 μ mol/L of primer are about template 5ng; The cycling program of pcr amplification is: 94 ℃ of pre-sex change 5min, (94 ℃, 30S; 60 ℃, 30S; 72 ℃, 2min) * 30, the 72 ℃ last 5min that extends.PCR product after enzyme is cut and pSK Δ Ahd set up following ligation system: contain 3U ligase enzyme (Promega) in the 10 μ L linked systems, 1 * connection Buffer, about 50ng plasmid, 50ng PCR product.4 ℃ connect 16 hours.Connect product according to the ordinary method transformed into escherichia coli, obtain positive bacterium colony through the Amp resistance screening, positive bacterium colony obtains intermediate carrier pSKEGFP after order-checking is identified.Second step was a template with plasmid pENTR1A, carried out pcr amplification with CcdB04F and CcdB04R primer.The reaction system of pcr amplification is: contain 5U Pfu in the 50 μ L reaction systems, and 0.2mmol/L dNTPs, 1 * Pfu buffer, each 10 μ mol/L of primer are about template 5ng; The cycling program of pcr amplification is: 94 ℃ of pre-sex change 5min, (94 ℃, 30S; 60 ℃, 30S; 72 ℃, 1.5min) * 30, the 72 ℃ last 5min that extends.The PCR product is cut glue and is reclaimed behind 1% agarose gel electrophoresis, the PCR product and the pSKEGFP that reclaim are cut with the NotI enzyme respectively, pSK Δ Ahd carrier after PCR product after pSKEGFP after enzyme is cut handles PCR product after cut with enzyme the back enzyme is cut with alkaline phosphatase and enzyme are cut is by the reaction that connects of the DNA Ligation Kit Ver.2 specification sheets of TaKaRa company: contain 5 μ L Solution I in the 10 μ L linked systems, the 50ng plasmid, 50ng PCR product.Connect product and transform DB3.1 bacterial strain (Invitrogen), carry out bacterium colony PCR, identify the positive colony that inserts (consistent) with forward, obtain pSK04 with lacZ reading frame direction with M13-47 and ccdBR primer according to ordinary method.
Be used for making up the primer sequence following (sequence of left side square frame is a restriction enzyme site, and the sequence in the middle shade is the AhdI restriction enzyme site, and the sequence in the square frame of the right is the sequence from ccdB or EGFP gene) of above-mentioned pre-T carrier (pSK01,02,03 and 04):
CcdB01F：5’-ACTG
G
-3’
CcdB01R：5’-AGTC
-3’
CcdB02F：5’-ACTG
-3’
CcdB02R：5’-AGTC
-3’
CcdB03F：5’-ACTG
-3’
CcdB03R：5’-AGTC
-3’
EGFPF：5’-ACTG
GAATTCCCGCGGCCGC
-3’
EGFPR：5’-AGTC
GAATTCCCGCGGCCGC
-3’
CcdB04F：5’-ACTG
-3’
CcdB04R：5’-AGTC
-3’
M13-47：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’
CcdBR：5’-TCAGCCACTTCTTCCCCGATAACG-3’
The structure of the AhdI box among pre-T carrier pSK01, pSK02, pSK03 and the pSK04 is shown in Figure 1A-Fig. 1 D, among Figure 1A-Fig. 1 D, sequence in the square frame of both sides is a restriction enzyme site, sequence in the shade is respectively two AhdI restriction enzyme sites, part in the middle square frame is represented the ccdB gene order, the concrete site of " ^ " expression enzyme cutting.Sequence among Figure 1A in the square frame of both sides is respectively SalI and XbaI enzyme cutting site, sequence among Figure 1B in the square frame of both sides is respectively SacI and KpnI restriction enzyme site, sequence among Fig. 1 C in the square frame of both sides is the EcoRI restriction enzyme site, sequence among Fig. 1 D in the left frame comprises the SacI-EcoRI-SacII-NotI-EagI restriction enzyme site, and the sequence in the right-hand frame comprises the EagI-NotI-SacII-EcoRI-KpnI restriction enzyme site.First restriction enzyme site among restriction enzyme site among Figure 1A-1C in the square frame of both sides and Fig. 1 D in the square frame of the left side and last restriction enzyme site in the square frame of the right correspond respectively to the restriction enzyme site on pBlueScript SK (-) plasmid.
The preparation and the TA cloning efficiency of embodiment 3, pSKxx-T series T carrier are analyzed
1, preparation T carrier
With AhdI respectively enzyme cut pre-T carrier pSK01,02,03 and 04, the endonuclease reaction system is as follows: 20 μ L enzymes are cut and are contained 3U AhdI restriction endonuclease (available from NEB company) in the system, 1 * NEBuffer4, about 1 μ g plasmid.After 37 ℃ of enzymes are cut 3 hours, behind 1% agarose gel electrophoresis, cut glue respectively and reclaim big fragment.The big fragment of cutting glue recovery purifying promptly is corresponding T carrier.The physical map of pSK01-T, pSK02-T, pSK03-T and pSK04-T is respectively as Fig. 2, Fig. 3, Fig. 4 and shown in Figure 5.
2, test TA cloning efficiency
With the Taq archaeal dna polymerase is primer PCR amplification Arabidopis thaliana NCED3 (1.8kb with NCEDF and NCEDR respectively, GenBank Accession No.AT3G14440) with DREBF and DREBR is primer PCR amplification DREB1A (680bp, GenBank Accession No.At4g25480) gene fragment.Primer sequence is as follows:
NCEDF5’-AACGGATCCATGGCTTCTTTCACGGCAACGGC-3’
NCEDR5’-AACGAGCTCTCACACGACCTGCTTCGCCAAATC-3’
DREBF5’-AAGGATCCTTCTGATCAATGAACTCATTTTCTG-3’
DREBR5’-AACACGTGGTTTTAATAACTCCATAACGATACG-3’
Pcr amplification product is cut glue and is reclaimed behind 1% agarose gel electrophoresis.Set up 8 ligation systems, reaction system is as follows: contain 3U ligase enzyme (Promega) in the 10 μ L linked systems, 1 * connect Buffer, the T carrier of about 50ng and the NCED3 of about 92ng or the DREB1A of 35ng, 4 ℃ connect more than 16 hours.Connect product according to ordinary method transformed into escherichia coli DH10B, and carry out blue hickie screening.Each linked system 30 hickies of random choose is respectively carried out bacterium colony PCR evaluation, calculates the shared per-cent of positive colony.The result shows, pSK01,02,03 and 04 and the TA cloning efficiency of the DREB1A ligation of setting up be 100% (30/30); PSK01,02,03 and 04 and the TA cloning efficiency of the NCED3 ligation of setting up be respectively 100% (30/30), 100% (30/30), 96% (29/30) and 96% (29/30).Locus coeruleus quantity behind 8 linked system transformed into escherichia coli all is lower than 5% with the ratio of total colony number.These results show that the T carrier of the present invention's preparation has very high TA cloning efficiency.
Sequence table
<160>4
<210>1
<211>343
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
<210>2
<211>343
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
<210>3
<211>343
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
<210>4
<211>373
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
Claims (8)
1, a kind of method for preparing the T carrier may further comprise the steps:
1) preparation contains the pre-T carrier of XcmI box or AhdI box; Described XcmI box comprises the spacer DNA sequence, closely is connected in each XcmI restriction enzyme site sequences of described spacer DNA sequence both sides, and described AhdI box comprises described spacer DNA sequence, closely is connected in each AhdI restriction enzyme site sequences of described spacer DNA sequence both sides; The carrier that sets out that is used to prepare described pre-T carrier is pBlueScriptIISK (-) or pUC18 or pUC19 or pUC118 or pUC119 or pGEM serial carrier;
2) with the isoschizomers of XcmI or XcmI, or the isoschizomers enzyme of AhdI or AhdI cuts the pre-T carrier in the step 1), obtains the T carrier;
It is characterized in that: the spacer DNA sequence in the described step 1) is the ccdB gene order, and the GenBank sequence number of described ccdB gene is No.AP001918.
2, method according to claim 1 is characterized in that: described XcmI box also comprises the restriction enzyme site sequence except that XcmI restriction enzyme site sequence that is connected in described each XcmI restriction enzyme site sequence; Described AhdI box also comprises the restriction enzyme site sequence except that AhdI restriction enzyme site sequence that is connected in described each AhdI restriction enzyme site sequence.
3, method according to claim 1 and 2 is characterized in that: the described carrier that sets out is pBlueScriptII SK (-).
4, method according to claim 3 is characterized in that: the AhdI restriction enzyme site of the Amp resistant gene among the described pBlueScript II SK (-) is by silence.
5, method according to claim 4 is characterized in that: the multiple clone site in described pBlueScript II SK (-) is introduced described AhdI box.
6, method according to claim 5 is characterized in that: introducing described AhdI box between the SalI of described multiple clone site restriction enzyme site and the XbaI enzyme cutting site or between KpnI restriction enzyme site and the SacI restriction enzyme site or between EcoRI restriction enzyme site and the EcoRI restriction enzyme site or between EagI restriction enzyme site and the EagI restriction enzyme site.
7, method according to claim 6 is characterized in that: described AhdI box has the nucleotide sequence of sequence 1 in the sequence table, sequence 2, sequence 3 or sequence 4.
8, method according to claim 7 is characterized in that: described T carrier is pSK01-T or pSK02-T as shown in Figure 3 or pSK03-T as shown in Figure 4 or pSK04-T as shown in Figure 5 as shown in Figure 2.
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| CN100383248C (en) * | 2005-12-15 | 2008-04-23 | 南京大学 | Preparation method of T vector for T-A cloning |
| CN101177689B (en) * | 2006-11-09 | 2011-01-26 | 天津医科大学 | Method for high-effective construction of T-carrier based on polymerase chain reaction |
| CN101177690B (en) * | 2006-11-09 | 2012-05-30 | 天津医科大学 | T-carrier capable of directionally cloning promoter and studying its activity as well as constructing method thereof |
| CN101333538B (en) * | 2007-06-29 | 2010-12-08 | 浙江工业大学 | Pre-T carrier, preparation thereof and applications |
| CN102220363A (en) * | 2011-05-20 | 2011-10-19 | 方辉 | Method for constructing T vector |
| CN102304537A (en) * | 2011-07-28 | 2012-01-04 | 上海捷瑞生物工程有限公司 | High-molecular-weight T carrier and preparation method thereof |
| CN102311968A (en) * | 2011-08-22 | 2012-01-11 | 生工生物工程(上海)有限公司 | T vector and its construction method, and pre-T vector thereof |
| CN104911199B (en) * | 2015-06-25 | 2018-04-27 | 中国热带农业科学院热带生物技术研究所 | Cloned dna molecule method |
| CN106399348B (en) * | 2016-10-26 | 2019-05-31 | 南京师范大学 | A kind of novel gene clone's T- carrier and its construction method and application |
| CN107058357A (en) * | 2017-03-13 | 2017-08-18 | 四川农业大学 | A kind of TA cloning vectors and its construction method and application based on digestion |
| CN108588102B (en) * | 2017-12-29 | 2021-10-08 | 苏州金唯智生物科技有限公司 | Pre-T vector, T vector composed of pre-T vector and application of pre-T vector |
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