CN101921747A - Cloning method based on locus specificity recombination - Google Patents

Abstract

The invention belongs to the technical field of biotechnology and gene engineering, in particular to a cloning method based on locus specificity recombination. The invention is based on the in vitro application of serine recombinase phi BT1 integrase. The invention provides a mutational integrase recognition sequence of which the recognized and cut efficiency is equivalent to that of the wild type. On the basis, a carrier pDZM102 which contains a wild type and mutation recognition locus and is used for locus specificity recombination cloning is built, and also provides a design method of a primer sequence of a target gene during cloning. The cloning method provided by the invention has high efficiency, high speed and high specificity, and can get rid of complex enzyme digestion connection to realize the quick cloning of a target gene only by adding the integrase recognition locus on a PCR amplification primer sequence 5' end.

Description

A kind of cloning process based on the locus specificity reorganization

Technical field

The invention belongs to biotechnology and gene engineering technology field, be specifically related to a kind of recombinant clone method based on φ BT1 intergrase and sudden change recognition site thereof.

Background technology

By the DNA recombinant technology, people can arbitrarily be spliced the gene fragment of different sources, with this proterties that obtains the special genes product or change biont.The foundation of tradition recombinant technology has benefited from the discovery and the widespread use of restriction enzyme and dna ligase, and up to now, this classical way still generally uses in the molecular cloning operation of routine.But, be applicable to that the demand of the high-throughput quick clone that group is learned is urgent day by day along with further going deep into of gene and functional study thereof.Obviously, present loaded down with trivial details enzyme is cut and is connected not only complex operation of cloning process, and a large amount of reagent costs also increase thereupon, and the selection of suitable restriction enzyme site simultaneously also becomes the problem that big fragment is operated and extensive gene clone can't be avoided.

For overcoming the defective of traditional cloning process, also successively be developed and the person of being studied uses based on the cloning process of homologous recombination and locus specificity reorganization.Homologous recombination clone's principle is that recombinase can be discerned the also exchange of the chain between the catalysis homologous sequence, reaches the purpose of reorganization.This principle is applied to external, uses the recombinase of purifying, under optimized reaction conditions, the reorganization of experiment carrier and goal gene.Reorganization can not stay any unnecessary sequence after finishing on carrier, but on the one hand, homologous recombination might produce unexpected chain exchange, and specificity remains further research; On the other hand, the efficient of homologous recombination is generally lower.

Locus specificity reorganization extensively occurs in phage and is incorporated in the process on the host chromosome.A kind of site-specific recombinase of phage encoded, attP site and host chromosome attB site that can specific identification self, and mediate the cutting and the chain exchange in two sites, phage genome is integrated into host chromosome.The in vitro study of loci specificity recombinase finds that such enzymatic recombining reaction is the specificity height not only, and rapidly and efficiently.Very fast, research early and mechanism clearly the λ integration system be applied in the external recombinant clone, taken the lead in breaking through traditional clone's shortcoming.Along with going deep into of research, the recombination efficiency of finding Serine enzyme (φ C31 integrase, φ BT1 integrase) is far above tyrosine class recombinase (λ integrase).So, the cloning system based on serine recombinases may become a kind of novel method that substitutes at present existing recombinant clone system more efficiently.

Summary of the invention

The cloning system that the objective of the invention is to overcome the tyrosine recombinase needs the plurality of enzymes acting in conjunction, and the relatively low shortcoming of efficient, and a kind of cloning process based on the locus specificity reorganization is provided.

Cloning process based on the locus specificity reorganization provided by the invention is a kind of high-efficient cloning method based on serine recombinases φ BT1integrase.The invention provides the intergrase recognition sequence of a pair of sudden change, its efficient that is identified and cut is suitable with wild-type.Make up the carrier pDZM102 that is used for the locus specificity recombinant clone contain wild-type and sudden change recognition site on this basis, and the method for design of primer sequence when the goal gene clone is provided.

Cloning process provided by the invention is efficiently quick, and the specificity height only need add the intergrase recognition site at pcr amplification primer sequence 5 ' end, can break away from loaded down with trivial details enzyme and cut the quick clone that connects the realization goal gene.

The aforesaid method that the present invention proposes is specific as follows:

1, the sudden change of φ BT1 intergrase recognition sequence and recombination efficiency are determined

Design contains the primer primer 1 (SEQ No.1) and the primer 3 (SEQ No.3) of the sudden change of intergrase recognition sequence, and designs corresponding antisense primer primer 2 (SEQ No.2) and primer 4 (SEQ No.4).With plasmid pRT802 is template, amplifies the attP that contains sudden change with primer 1 and primer 2PCR ₁Fragment; With plasmid pZL5812 is template, amplifies the attB that contains sudden change with primer 3 and primer 4PCR ₁Fragment.With mutant nucleotide sequence attP ₁With attB ₁React under the effect of intergrase, wild-type sequence attP and attB reaction are in contrast.Reaction back densitometric scan assaying reaction efficient, the result shows that mutant is suitable with wild-type substrate reactions efficient.With mutant nucleotide sequence and wild-type sequence cross reaction, i.e. attP ₁With attB reaction, attP and attB ₁Reaction finds that cross reaction can not take place.Therefore mutant site and wild-type site can applied in any combination in unified system, realize that single-factor (only needing φ BT1 integrase) mediation is down to clone rapidly and efficiently.

2, make up the cloning vector pDZM102 that contains φ BT1 intergrase recognition site (wild-type and mutational site)

With pUC119 is template, contains mutant attP with primer 5 (SEQ No.5) and primer 6 (SEQ No.6) amplification one end ₁LacZ α (SEQ No.7) fragment.The restriction enzyme enzyme recognition site of NheI and EcoRV is designed at the primer two ends respectively, cuts the PCR product with these two kinds of enzyme enzymes and makes the insertion fragment; With these two kinds of enzyme digested plasmid pRT802, make the linear plasmid skeleton simultaneously.The linear plasmid skeleton is connected the back transformed into escherichia coli with lacZ α fragment with the T4 ligase enzyme, and the picking positive transformant also extracts plasmid DNA, is pDZM102 (SEQ No.8).And be designed for the universal primer ZLF (SEQ No.9) of order-checking, ZLR (SEQ No.10).

The respective element of plasmid pDZM102 comprises plasmid replication initiation site, drug resistance gene, conjugal transfer initiation site, blue hickie screening genes involved and locus specificity reorganization recognition site attP and attP ₁

3, the goal gene directed cloning is gone into carrier pDZM102

For obtaining higher recombinant clone positive rate, can be earlier pDZM102 be continued next-step operation after with restriction endonuclease HindIII or BamHI linearizing again.So that ampicillin resistance gene (bla) is cloned into pDZM102 is example: with pUC119 is template, contain the dna fragmentation (SEQ No.13) of bla gene with primer primer 9 (SEQ No.11) and primer 10 (SEQ No.12) pcr amplification, on the primer respectively design wild-type attB and mutant attB are arranged ₁Sequence, thus this PCR product can be directly and pDZM102 under the effect of intergrase, recombinate.With the recombinant products transformed into escherichia coli, positive rate is not less than 95%.

The present invention relates to plasmid DNA pRT802[1] and pZL5812[2] relevant information sees corresponding reference; Plasmid pUC119 purchases in precious biotechnology company limited; Carrier pDZM102 makes up in designing for this patent.

Description of drawings

Fig. 1: the structure iron of recombinant cloning vector pDZM102.Wherein:

Fig. 2: with bla gene example, the PCR product structure figure of acquisition.Wherein:

Fig. 3: vitro recombination reaction synoptic diagram.

Embodiment

1, the sudden change of φ BT1 intergrase recognition sequence and recombination efficiency are determined

(1) preparation contains the fragment in mutant site: the pcr amplification reaction system is (with primer 1 and primer 2 amplification attP ₁Fragment is with primer 3 and primer 4 amplification attB ₁Fragment), 10 * Taq DNA Polymerase buffer, 5 μ l, 10mM each dNTP 1 μ l, dna profiling, primer and Taq DNA Polymerase be 0.5 μ l respectively, with ddH ₂O replenishes cumulative volume to 50 μ l; The PCR condition is, 95 ℃ of pre-sex change 5min, 95 ℃ 30sec/60 ℃ 30sec/72 ℃ of 1min circulation 30 times, 72 ℃ of insulation 10min; The PCR product reclaims the test kit purifying with the GenClean of GeneRay company sepharose DNA and reclaims behind 0.7% agarose gel electrophoresis, earlier the gel piece that downcuts is mixed with the long-pending Binding Solution of triploid, transfer to after the heating for dissolving in the centrifugal post with the centrifugal 30sec of 3000rpm, with 500 μ l Wash Solution washed twice, add 50 μ l ddH ₂The O wash-out is collected.

(2) vitro recombination reaction: add two kinds of substrates and (be respectively attP ₁And attB ₁, attP and attB) and in little centrifuge tube, keep the mol ratio of two kinds of substrates is 1: 1 as far as possible, adds 1 μ l Recombination Buffer, 0.5 μ l φ BT1 integrase, with ddH ₂O replenishes cumulative volume to 10 μ l; 30 ℃ of incubation 0.5-2hrs, agarose gel electrophoresis analysis behind 75 ℃ of high-temperature inactivation 10min.

(3) determining of recombination efficiency and cross reaction: the densitometric scan analysis is carried out in the vitro recombination reaction behind agarose gel electrophoresis, three results confirm that mutant site and wild-type site reaction efficiency do not have marked difference; Respectively with attP ₁And attB, attP and attB ₁For substrate carries out the cross reaction experiment, vitro recombination reaction system such as above-mentioned, product carries out the densitometric scan analysis behind agarose gel electrophoresis, do not see that cross reaction takes place.

2, make up the cloning vector pDZM102 that contains φ BT1 intergrase recognition site (wild-type and mutational site)

(1) primer design is obtained with lacZ α is segmental: cut the method that is connected with enzyme earlier and will have attP ₁LacZ α gene be inserted into carrier and get on, plasmid pRT802 itself has the attP site, therefore only need add mutant nucleotide sequence attP at an other end ₁Get final product.5 ' the end of primer primer 5 has the NheI recognition site, and the 5 ' end of primer primer 6 has mutant nucleotide sequence attP ₁With the EcoRV recognition site.PCR reaction (system such as above-mentioned) afterwards with these two kinds of digestion with restriction enzyme (system: an amount of PCR product, NEB " 10 * buffer 2 " 5 μ l, 10 * BSA, 5 μ l, NheI and EcoRV be 1 μ l respectively, with ddH ₂O replenishes cumulative volume to 50 μ l), enzyme is cut the back agarose gel electrophoresis, reclaims the test kit purifying with GenClean sepharose DNA and reclaims (as above-mentioned).

(2) preparation of linear plasmid skeleton: obtain the plasmid skeleton with NheI and EcoRV digested plasmid pRT802 (system is gone into above-mentioned), reclaim the test kit purifying with GenClean sepharose DNA and reclaim (as above-mentioned).

(3) connect and transform: with the plasmid skeleton for preparing with contain attP ₁LacZ α connect (system is: plasmid skeleton 1.5 μ l, PCR fragment 7 μ l, 10 * ligation buffer, 1 μ l, T4ligase 0.5 μ l) with the T4 ligase enzyme; After spending the night, connection will connect product with CaCl ₂Method transforms DH5 α, and resistance is a kantlex, and adds IPTG and X-gal in substratum, 37 ℃ of incubated overnight.

(4) plasmid extracts and checking: the picking blue colonies is cultivated, and extracts plasmid DNA with the described alkaline process of molecular cloning experiment guide, and restriction enzyme KpnI enzyme is cut (system such as above-mentioned) checking, and screening obtains positive colony and is pDZM102 (SEQNo.8).

3, the goal gene directed cloning is gone into carrier pDZM102

(1) primer design with contain obtaining of target gene fragment: recombinant cloning vector pDZM102 has contained attP and attP ₁Sequence so only need add attB and attB in the goal gene both sides when pcr amplification ₁Sequence can realize recombinant clone.Hold the attB sequence (part in the SEQ No.11, frame) of introducing wild-type at 5 ' of primer primer 9, the 5 ' end of primer primer10 is introduced the attB of wild-type ₁Sequence (part in the SEQ No.12, frame).With pUC119 is template, with this two primer PCRs amplification target DNA fragment (is example with the bla gene), reclaims the test kit purifying with GenClean sepharose DNA behind the product electrophoresis and reclaims (as above-mentioned).

(2) vitro recombination reaction: add target DNA fragment and pDZM102 (system such as above-mentioned) in little centrifuge tube and carry out recombining reaction, the incubation time is 0.5-2 hour.For guaranteeing higher recombination efficiency, the mole number of target DNA fragment is that the carrier mole number is advisable for three to ten times; Be to guarantee the high positive cloning efficiency, can be in advance pDZM102 be continued next-step operation after with restriction endonuclease HindIII or BamHI (being single point of contact) linearizing again.

(3) screening of recombinant clone and evaluation: recombinant products transforms DH5 α, and resistance is a kantlex, and adds IPTG and X-gal in substratum, 37 ℃ of incubated overnight.White colony is positive colony, and linearizing carrier will obtain high positive rate, be not less than 95%.All right ordinary method is extracted the exactness that plasmid enzyme restriction is further verified positive colony; Universal primer sequence verification that also can be designed.

Reference:

1.Gregory，M.A.，Till，R.and?Smith，M.C.M.(2003)Integration?site?for?StreptomycesphageφBT1?and?development?of?site-specific?integrating?vectors.J?Bacteriol.，185，5320-5323.

2.Zhang，L.，Ou，X.J.，Zhao，G.P.and?Ding，X.M.(2008)Highly?efficient?in?vitrosite-specific?recombination?system?based?on?Streptomyces?phageφBT1?integrase.J?Bacteriol.，190，6392-6397.

Sequence table

SEQ?No.1：

5′-TGCTGAGTAGTTTCCCATGGATCTCTGTCCAGAGAC-3′

SEQ?No.2：

5′-AGCAGCCCTTGCGCCCTG-3′

SEQ?No.3：

5′-CCAGGTTTTTGACGAAAGAGATCCAGATG-3′

SEQ?No.4：

5′-ATAGGCGTATCACGAGGC-3′

SEQ?No.5：

5′-GCTAGCAGCGCCCAATACGCAAAC-3′

SEQ?No.6：

5′-GATATCGTGCTGGGTTGTTGTCTCTGGACACTGATCCATGGGAAACTACTCAGCAC

ACGTGGCGAGAAAGGAAG-3′

SEQ?No.7：

GCTAGCAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG

CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTA

ATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGT

ATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATG

ATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGC

TCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACC

CAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGG

CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGA

TGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAAC

CATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGGGTGGTTACGCGCAG

CGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCC

TTTCTCGCCACGTGTGCTGAGTAGTTTCCCATGGATCAGTGTCCAGAGACAACAACC

CAGCACGATATC

SEQ?No.8：

TGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGT

CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCA

GCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCT

TCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTA

CGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGT

AGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT

GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTGTG

CTGAGTAGTTTCCCATGGATCAGTGTCCAGAGACAACAACCCAGCACGATATCGAATT

CGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA

ACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAAC

TCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCA

GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTC

TTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTA

TCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA

AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT

TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC

AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG

AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC

TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT

CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCG

ACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTT

ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG

TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTT

GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGAT

CCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTA

CGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG

CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGA

TCTTCACCTAGATCCTTTTGGTTCATGTGCAGCTCCATCAGCAAAAGGGGATGATAAG

TTTATCACCACCGACTATTTGCAACAGTGCCGTTGATCGTGCTATGATCGACTGATGTC

ATCAGCGGTGGAGTGCAATGTCGTGCAATACGAATGGCGAAAAGCCGAGCTCGGTAC

CCGGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGATCATCCAGCCG

GCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAATCG

AAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTCATTTCGAACCCCAGAGTC

CCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGA

GCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCA

GCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGC

CACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGG

CATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGG

CGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGAC

AAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCG

AATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATT

GCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCC

TGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCG

AGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTC

GTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGC

GCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTG

CCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATC

CATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATCTTGATCCC

CTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCC

CAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAA

CCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGC

GCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGGGTCAGCAC

CGTTTCTGCGGACTGGCTTTCTACGTGTTCCGCTTCCTTTAGCAGCCCTTGCGCCCTG

AGTGCTTGCGGCAGCGTCGCCATTGATGCGGGCCAGCTCGCGGACGTGCTCATAGTC

CACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCT

CATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTAC

ACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGC

CCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGA

GCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGG

CCTACTTCACCTATCCTGCCCGGCTGACGCCGTTGGATACACCAAGGAAAGTCTACAC

GAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAAT

CGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGATCCGTCGACCTGCAGG

CATGCTGGCGCCGGACGGGGCTTCAGACGTTTCGGGTGCTGGGTTGTTGTCTCTGGA

CAGTGATCCATGGGAAACTACTCAGCACCACCAATGTTCCCAAAAGAAAGCGCAGGT

CA

GCGCCCATGAGCCAAGATCTAGGCATGTCGCCCTTCATCGCTCCCGACGTCCCTGAGC

ACCTTCTAGACACTGTTCGCGTCTTCCTGTACGCGCGTCAGTCTAAGGGCCGGTCCGA

CGGCTCAGACGTGTCGACCGAAGCACAGCTAGCAGCGCCCAATACGCAAACCGCCT

CTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGG

AAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCC

CAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAAC

AATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCA

SEQ?No.9：

5′-TGGCGCCGGACGGGGCTTC-3′

SEQ?No.10：

5′-ACAGCTATGACATGATTAC-3′

SEQ?No.11：

5′- CCGGGAGCTGCATGTGTC-3′

SEQ?No.12：

5′-

AACTTGGTCTGACAGTTAC-3

′

SEQ?No.13：

CCAGGTTTTTGACGAAAGTGATCCAGATGATCCAGCCCGGGAGCTGCATGTGTCAGA

GGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTAT

TTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGG

GGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG

CTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGA

GTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTT

TTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCAC

GAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCC

CCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATT

ATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAAT

GACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTA

AGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTC

TGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC

ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACG

AGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTG

GCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA

AGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA

TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGT

AAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAAC

GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA

CCAAGTTGCTGGATCATCTGGATCAGTTTCGTCAAAAACCTGG

Claims (6)

1. the recombination site sequence of the φ BT1 intergrase of a pair of sudden change identification is characterized in that being attP ₁And attB ₁Site, its sequence are respectively shown in SEQ No.1 and the SEQ No.3.

2. one kind is carried out the method for corresponding substrate to reorganization and cross reaction efficiency test based on site according to claim 1, it is characterized in that segmental vitro recombination of PCR and densitometric scan are combined.

3. a recombinant clone plasmid is characterized in that sequence is designated as pDZM102 shown in SEQ No.8 by constructed based on the described sudden change integration site of claim 1 sequence.

4. as the respective element of recombinant clone plasmid as described in the claim 3, it is characterized in that comprising plasmid replication initiation site, drug resistance gene, conjugal transfer initiation site, blue hickie screening genes involved and locus specificity reorganization recognition site attP and attP ₁

5. as the construction process of recombinant clone plasmid as described in the claim 3, it is characterized in that concrete steps are as follows:

With pUC119 is template, contains mutant attP with primer 5 and primer 6 amplifications one end ₁LacZ α fragment; The restriction enzyme enzyme recognition site of NheI and EcoRV is designed at the primer two ends respectively, cuts the PCR product with these two kinds of enzyme enzymes and makes the insertion fragment; With these two kinds of enzyme digested plasmid pRT802, make the linear plasmid skeleton simultaneously; The linear plasmid skeleton is connected the back transformed into escherichia coli with lacZ α fragment with the T4 ligase enzyme, and the picking positive transformant also extracts plasmid DNA, is pDZM102; Wherein: primer 5 sequences are SEQ No.5, and primer 6 sequences are SEQ No.6, and lacZ α sequence is SEQNo.7.

6. one kind is cloned into method as pDZM102 as described in the claim 3 with goal gene by recombining, orienting, it is characterized in that concrete steps are as follows:

Earlier pDZM102 is continued next-step operation after with restriction endonuclease HindIII or BamHI linearizing again; The goal gene and the pDZM102 that are obtained are recombinated under the effect of intergrase; With the recombinant products transformed into escherichia coli, use blue white screening, positive rate is not less than 95%.

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