CN102311968A

CN102311968A - T vector and its construction method, and pre-T vector thereof

Info

Publication number: CN102311968A
Application number: CN201110242359A
Authority: CN
Inventors: 李威
Original assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Current assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Priority date: 2011-08-22
Filing date: 2011-08-22
Publication date: 2012-01-11

Abstract

The invention provides a T vector and its construction method, and a pre-T vector thereof. Two terminals of the T vector in the invention both carry a 3'-dT protruding linear vector, flanking sequences of the two 3'-dT protruding terminals can meet the demand that when the T vector is connected with a PCR product whose two terminals carry 3'-dA, two restrictive endonuclease enzyme cutting sites can be formed at two terminals of an inserted segment, wherein, the two restrictive endonuclease enzyme cutting sites are enzyme cutting sites of a same enzyme. The invention further provides a pre-T vector capable of generating the T vector. The T vector constructed in the invention can be used for TA clone and enables a high clone efficiency to be obtained, and positive transformant can be identified by using the method of single enzyme digestion.

Description

A kind of T carrier and construction process thereof and its pre-T carrier

Technical field

The invention belongs to molecular biology and technical field of bioengineering, relate to the dna fragmentation cloning process in a kind of genetically engineered operation.Particularly, the present invention relates to a kind of T carrier and construction process thereof and its pre-T carrier.

Background technology

The recombinant technology of DNA, just gene clone technology is one of most important achievement in the modern molecular biology development, also is engineered core technology.In broad terms; The recombinant technology of DNA is meant the genetic material that utilizes donor biological; Or the dna molecular of synthetic; Couple together the new recombinant DNA molecules of formation through processing such as external separation and purification, pcr amplification, restriction enzyme cutting back and appropriate carriers, then recombinant DNA molecules is imported in the receptor biological, thereby realization is to the operations such as preservation, amplification and analysis of target dna molecule.

What can be used as dna vector has plasmid, clay, phage, an artificial chromosome etc., and wherein plasmid vector is to use molecular cloning vector the most widely.Complete genetically engineered plasmid vector comprises that replicon (be used to control plasmid molecule the host is intravital duplicate and increase), screening-gene are (like resistant gene, reporter gene; Or the both has, be used to confirm, the host of tracking or decoupled band target plasmid), MCS (cutting of being convenient to dna molecular be connected) and be used to control plasmid behavior (like copy number) or some special dna sequence dnas of being used to analyze (as be used for plasmid order-checking some primer complementary sequences etc.).

The cutting of dna molecular be connected and can accomplish through restriction enzyme and ligase enzyme; Promptly respectively with corresponding restriction enzyme cutting target dna molecule and vector dna molecule; Make its two ends obtain to contain the outstanding end (sticky end) of part strand respectively, the end (being obtained by same enzyme or isocaudarner cutting) that contains complementary sequence forms a complete DNA chain under the effect of dna ligase.

The generation of round pcr and development make and to utilize round pcr to obtain that dna molecular to be cloned becomes a kind of convenience and means efficiently; But for the fragment of utilizing PCR method to obtain; Adopt the method for restriction enzyme cutting to obtain inconvenience is arranged: at first with the end of carrier coupling; Need introduce corresponding restriction enzyme site at PCR product two ends, increase the cost of primer; Secondly, when the restriction enzyme enzyme recognition site was positioned at dna fragmentation terminal, its cutting efficiency was influenced; Once more, after the restriction enzyme processing, correctly cutting or uncut dna molecular are difficult to separate, and this will influence the joint efficiency of follow-up dna fragmentation and carrier.Therefore, a kind of developed and be widely used to the segmental clone technology of PCR---TA clone---.

The TA clone technology is meant that a PCR fragment and one have the method that the outstanding vector dna molecule of single 3 '-dT couples together.Its principle is; Employed in the PCR reaction have the terminal activity that shifts like archaeal dna polymerases such as Taq; This activity makes it to add an outstanding dA at 3 ' terminal end of the dna molecular that pcr amplification obtains, and is connected thereby vector dna molecule that can be outstanding with having 3 '-dT is complementary.Cut latter linked method with restriction enzyme and compare, the TA clone makes can directly be cloned in the carrier through the dna fragmentation of PCR acquisition, has simplified clone's process greatly, has improved efficient.The two ends of special design of process and processing and preparing have the outstanding linear DNA carrier of 3 '-dT and then are called the T carrier.

The T carrier is the core of TA clone technology; T preparing carriers method commonly used at present has two kinds: a kind of is that flat end adds the T method, promptly cuts with suitable restriction enzyme (like EcoR V, Sma I etc.) in cyclic plasmid vector (T precursor carrier) predetermined position; Form two ends and be flat terminal linear DNA molecule; Add 3 '-dT with having the active enzyme of terminal enzyme (DNA) two ends at linear DNA molecule under appropriate condition subsequently,, obtain the T carrier for preparing through being further purified;

The another kind of method that prepare the T carrier is an enzyme cutting method, this method utilize some restriction enzyme (like Xcm I, Eam1 105 I etc.) cutting afterwards 3 ' stay single Nucleotide characteristic; Through well-designed; Make that the Nucleotide of this position is T, therefore, the recognition site of two placed in-line these enzymes of design on carrier; Then the cutting back obtains a 3 ' outstanding-dT respectively at the two ends of carrier, reclaims this linear DNA molecule and is the T carrier.

Summary of the invention

The object of the present invention is to provide a kind of T carrier and construction process thereof and its pre-T carrier.

The present invention at first discloses a kind of T carrier; Be that two ends all have the outstanding linear carrier of 3 '-dT; The flanking sequence of said two 3 '-dT protruding terminuses satisfy when through said T carrier with after the PCR product of two ends band 3 '-dA links to each other, can form two restriction enzyme digestion sites at insertion fragment two ends; Said two restriction enzyme digestion sites are the restriction enzyme site with a kind of restriction enzyme.

Said restriction enzyme digestion sites can be, but be not limited to Hind III restriction enzyme site.

Also should have other T carriers necessary element commonly used in the said T carrier.

The necessary element that said T carrier is commonly used comprises: replicon, resistant gene (like the penbritin expression casette) and selection markers (like lacZ ' marker gene etc.).Also optionally comprise following elements in the T carrier: be used to control plasmid behavior (like copy number) or some special dna sequence dnas of being used to analyze etc.

Preferable, said T carrier except that the restriction enzyme digestion sites that the PCR product carries, only contains two restriction enzyme enzyme recognition sites with after the PCR product of two ends band 3 '-dA links to each other.Do not have other restriction enzyme enzyme recognition sites commonly used in the T carrier, can carry out determined dna sequence with universal primer.

Arbitrary method prepared below T carrier of the present invention can adopt:

Method one: after using the enzyme enzyme that produces flat end to cut pre-T carrier, produce to have and put down terminal linear molecule, after the end of said linear molecule adds T, obtain subsequently.

Producing flat terminal enzyme can be, but is not limited to EcoR V.

The enzyme that the linear molecule end adds T can be, but be not limited to Taq DNA polymerase.

Concrete, the linear molecule end add T method can for: under the situation that dTTP exists, utilize the effect of Taq DNApolymerase to add the 3 ' end that a dT is projected into DNA down.

Method two: obtain after using the enzyme enzyme that produces 3 '-dT protruding terminus to cut pre-T carrier.

The outstanding enzyme of said generation 3 '-dT can be, but be not limited to Xcm I.

The present invention also further discloses a kind of pre-T carrier, can be used for preparing said T carrier.

Pre-T carrier of the present invention is the cyclic plasmid vector; Comprise MCS; Wherein: said MCS comprises the dna fragmentation of one section total following five restriction enzyme site; Two restriction enzyme sites that produce 3 '-dT protruding terminus, two evaluations are with restriction enzyme site and a restriction enzyme site that produces flat end; The flat terminal restriction enzyme site of said generation is positioned between the restriction enzyme site of two generation 3 '-dT protruding terminuses; The restriction enzyme site of said generation 3 '-dT protruding terminus can produce two 3 '-dT protruding terminuses on carrier after the enzyme enzyme that produces 3 '-dT protruding terminus is cut; The flanking sequence of said two 3 '-dT protruding terminuses satisfies said carrier when cutting through the enzyme enzyme that produces 3 '-dT protruding terminus after with after PCR product with 3 '-dA links to each other, and can form two evaluations at insertion fragment two ends and use restriction enzyme site; Identify and use the restriction enzyme site of restriction enzyme site for said two as the same enzyme.

The restriction enzyme site of said generation 3 '-dT protruding terminus can be, but be not limited to Xcm I restriction enzyme site.

Said evaluation with restriction enzyme site can be, but be not limited to Hind III restriction enzyme site.

The flat terminal restriction enzyme site of said generation can be, but be not limited to EcoR V restriction enzyme site.

Preferably, the restriction enzyme site of said generation 3 '-dT protruding terminus is an Xcm I restriction enzyme site, and said the evaluation uses restriction enzyme site to be Hind III restriction enzyme site, and the flat terminal restriction enzyme site of said generation is EcoR V.

In the MCS of said pre-T carrier, except that these above-mentioned five restriction enzyme sites, do not contain other common restriction enzyme digestion sites.

Further, the sequence of said dna fragmentation is: SEQ IN NO:1.

Said pre-T carrier should be the same with other plasmid vectors commonly used, comprises the element that some are necessary, like replicon, resistant gene (like the penbritin expression casette) and selection markers.Also optionally comprise: be used to control plasmid behavior (like copy number) or some special dna sequence dnas of being used to analyze etc.

Further, said pre-T carrier can be by adopt aforementioned dna fragmentation replacement to obtain for the MCS with existing plasmid vector.

Existing plasmid vector can be selected from pUC19, pUC18, pUC119, pUC118, pUC57, pBlueSciptII SK (+/-), pBlueScript II KS (+/-), pGEX-2T, pGEX-4T, pGEX-6p, pGEM-Z, pTZ19u, pTZ19r, pTZ18u, pTZ18r, pBR322 and derivative vector, pACYC and derivative vector thereof, pSC101 and derivative vector thereof etc.

Further, said pre-T carrier is for being connected into pUC19 the fragment acquisition of following sequence after Hind III and EcoR I enzyme are cut:

5′-AGCTCGCACGCCAAGCTTCGCTTGGGATATCCAAGCGAAGCTTGGATCGTACCGTCA-3′

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3′-GCGTGCGGTTCGAAGCGAACCCTATAGGTTCTCTTCGAACCTAGCATGGCAGTTTAA-5′

Above-mentioned fragment can be according to the sequence that designs, two complementary dna singles of synthetic chain molecule, and annealed back obtains.

Pre-T carrier of the present invention after treatment; Can obtain two ends and have the outstanding linear carrier (being the T carrier) of 3 '-dT; This carrier can efficiently be connected by directly outstanding with having 3 '-dA PCR product; And after connecting product and transforming, can use the method evaluation transformant of blue hickie and single endonuclease digestion, greatly simplified testing process.

The TA cloning vector (T carrier) that adopts method of the present invention to produce has following advantage:

1, compare with present commercial normally used T carrier, removed restriction enzyme site unnecessary on the carrier, the site of avoiding existing on the carrier with insert the site of designing in the fragment and disturb;

2, between two Xcm I sites, insert one section short dna sequence dna; Increase the distance between two Xcm I sites, the enzyme of avoiding excessively closely bringing owing to two Xcm I sites in the production process is cut problems such as incomplete, simultaneously; The fragment that falls down behind this Xcm I double digestion is 20bp; Can use the method for PCR product purification to remove, simplify production stage, and avoided of the damage of glue removal process DNA;

3, between two Xcm I sites, introduce an EcoR V restriction enzyme site, the introducing in this site makes this carrier all be suitable for (Xcm I enzyme cutting method and EcoR V enzyme are cut the flat end in back and added the T method) for two kinds of working methods;

4, in two Hind III sites of design, the outstanding two ends of T; Can insert son with single enzyme Hind III identifies; The design in this site makes carrier have following characteristics: A, insert segmental carrier for having, and will downcut after Hind III enzyme is cut and the consistent fragment of insertion clip size;

B, for falling T from the carrier that connects, Hind III can not cut; C, cut not exclusively from the carrier that connects for enzyme, fall down the 25bp fragment after Hind III enzyme is cut, size can be separated on sepharose;

5,, make it no matter is because Xcm I enzyme is cut not exclusively or because two make a clean sweep of the situation of T, carrier is locus coeruleus from connecting the transformant that is produced, thereby reduces false positive rate through sequence optimisation;

6, the distance of insertion both sides, site checks order as using the M13 universal primer through optimizing, and when the good sequence of acquisition read, to reduce the carrier sequence residual as far as possible.

Description of drawings

Fig. 1 is carrier core sequence and restriction enzyme site position example thereof

Fig. 2 is the duplex molecule synoptic diagram that the complementary annealing of dna single chain forms

Embodiment

The present invention has at first designed the section of DNA sequence, makes it to have following characteristics:

1, this dna fragmentation can be cloned in the MCS of destination carrier, such as pUC19;

2, this fragment does not interrupt the proteic coding region of coding lacZ α on the original carrier, thereby does not destroy the function of blue hickie screening;

3, contain two Xcm I restriction enzyme sites in this fragment, in addition two Hind III restriction enzyme sites and an EcoRV restriction enzyme site do not contain other common restriction enzyme digestion sites;

4, after two Xcm I enzymes cut, the end that stays was that 3 '-dT is outstanding;

5, the special optimization of distance between the restriction enzyme site and sequence results makes it to realize function of the present invention.

Further, the present invention has synthesized dna fragmentation according to above-mentioned thinking and has been cloned in the pUC19 carrier, through determined dna sequence, confirms that the actual vector sequence is consistent with the desired design sequence;

Then, this carrier is handled, obtained two ends and have the outstanding linear carrier of 3 '-dT.There are two kinds of methods can accomplish this step here; First method is that flat end adds the T method; At first carrier is cut, obtain two ends and have flat terminal linear DNA molecule, next adopt archaeal dna polymerase under the condition that dTTP exists, to add dT to the DNA end with EcoR V enzyme; Second method is, with Xcm I enzyme carrier cut, and it is outstanding that two Xcm I site stay two 3 '-dT respectively.

Particularly, the present invention includes following steps:

(1) according to foregoing thinking design dna sequence;

(2) adopt the method for chemosynthesis to obtain two single strand dnas and obtain dna double chain as previously mentioned through the annealed method, this two strands has the end that the end that stays after through the restriction enzyme site cutting with carrier is complementary;

(3) carrier is cut with corresponding restriction endonuclease;

(4) linearized vector after the enzyme that obtains in the dna fragmentation that obtains in the step (2) and the step (3) is cut is connected, and transformed into escherichia coli is selected positive transformant and confirmed sequence through dna sequencing;

(5) with the carrier that obtains in the step (4), the flat end of employing adds the T method or the preparation of Xcm I enzyme cutting method has the outstanding linear DNA molecule of 3 '-dT;

The joint efficiency of the carrier that (6) is obtained in the PCR product verification step (5) with different lengths and source.

Said chemosynthesis is meant, adopts the method for organic synthesis, and nucleotide monomer is connected according to desired sequence successively, forms the process with dna molecular that particular bases puts in order;

Said restriction enzyme is meant, the biological intravital one type of endonuclease that can discern and cut specific double chain DNA sequence, and the dna molecular that has this restriction endonuclease recognition sequence stays fixed, special DNA end sequence after treatment;

Said have the outstanding linear DNA molecule of 3 '-dT and be meant, a kind of linearizing carrier has one and have only an outstanding T base at 3 ' end of two ends of this carrier, can be used for the TA clone, also is called as the T carrier;

Elaborate in the face of embodiments of the invention down: present embodiment is being to implement under the prerequisite with technical scheme of the present invention; Detailed embodiment and concrete operating process have been provided; Be intended to further illustrate the present invention, but be not used for limiting invention which is intended to be protected.

The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; Sambrook etc. for example; Molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment 1

Carrier core sequences Design example and structure thereof

1.1 the design of core sequence

According to mentality of designing of the present invention, the core sequence of this carrier shown in SEQ ID NO.1, concrete restriction enzyme site with and the position as shown in Figure 1.This core sequence amino acid sequence coded is shown in SEQ ID NO:2.

1.2 the selection of skeleton carrier and preparation

For for simplicity, in the present embodiment, adopt the pUC19 carrier as skeleton carrier.The e.colistraindh5 that will have the pUC19 plasmid is inoculated in the LB substratum that contains the 50ng/ml penbritin, and shaking culture is spent the night in 37 ℃ of shaking tables." SanPrep pillar DNA is the extraction agent box in a small amount " that utilize Sangon Biotech (Shanghai) Co., Ltd. to produce extracts the pUC19 DNA according to its operation instructions.

With restriction enzyme Hind III and EcoR I the pUC19 DNA is carried out enzyme and cut, all available from safe this biotechnology (Shenzhen) of rich enzyme ltd, reaction system is following for Hind III and EcoR I:

Reaction conditions is: 37 ℃, and 3 hours.

After reaction finishes; Carry out electrophoresis with 1% sepharose; Separate to obtain size and be the fragment of 2.6kb, the method for utilizing glue to reclaim, " SanPrep pillar DNA glue reclaims test kit " of producing with Sangon Biotech (Shanghai) Co., Ltd. is according to its this band of step that provides recovery.

1.3 insert the preparation of dna fragmentation

According to the dna sequence dna that is designed, it is following to design two oligonucleotide DNA molecules:

pSGTSIF：5’-AGCTCGCACGCCAAGCTTCGCTTGGGATATCCAAGCGAAGCTTGGATCGTACCGTCA-3’(SEQ?ID?NO：3)

pSGTSIR：5’-AATTTGACGGTACGATCCAAGCTTCGCTTGGATATCCCAAGCGAAGCTTGGCGTGCG-3’(SEQ?ID?NO：4)

These two dna moleculars adopt the solid phase phosphoramidite triester method synthetic by Sangon Biotech (Shanghai) Co., Ltd., carry out purifying with the method for polyacrylamide gel electrophoresis, and with mass spectrometric detection molecular weight and purity.

Two dna moleculars are calculated according to synthetic amount and its molecular weight, and with TE buffer (pH 8.0 for 10mM Tris, 1mM EDTA) it being diluted to concentration is 100 μ mol/L, and equal proportion is carried out anneal according to following program after mixing:

Response procedures: 94 ℃ of for 2min, 0.2 ℃ of per sec of 25 ℃ of at of 94 ℃ of down to, 25 ℃ of for 8min.

After reaction finishes, obtain complementary dna double chain, this dna double chain has the structure shown in figure two, and the sequence after two ends outstanding cut respectively at Hind III and EcoR I enzyme is complementary.

1.4DNA molecule connects and transforms

With the carrier-pellet degree that obtains in the dna fragmentation and 1.2 that obtains in above-mentioned 1.3 with 5: 1 mixed of mol ratio; Under the effect of T4DNA ligase enzyme, connect; Connect product transformed into escherichia coli DH5 α bacterial strain competent cell; At the enterprising row filter of amicillin resistance LB plate culture medium, obtain positive transformant.

Picking positive transformant enlarged culturing; Extract DNA; On 3730 type dna sequence analysis appearance, identify the sequence of transformant in Sangon Biotech (Shanghai) Co., Ltd. with the Sanger method, the on all four transformant of picking and expected sequence is used for subsequent experimental, and this correct carrier also is called pre-T carrier.

Embodiment 2

T preparing carriers method one (flat end adds the T method)

2.1 the preparation of pre-T carrier

The e.colistraindh5 that will have pre-T carrier is seeded in the LB substratum that contains the 50mg/L penbritin, in 37 ℃ of shaking table shaken overnight." the Qiagen Plasmid Maxi Kit " test kit that adopts triumphant outstanding biotechnology (Shanghai) Co., Ltd. to provide extracts plasmid according to manufacturers instruction.

2.2 carrier linearizing

With EcoR V restriction enzyme carrier is carried out enzyme and cut, form two ends and be flat terminal linearized vector, reaction system is following:

The endonuclease reaction condition is: 37 ℃, and 3 hours.

Endonuclease reaction finishes the back and cuts entirely with 1% agarose gel electrophoresis detection enzyme; " the SanPrep pillar PCR product purification test kit " produced with Sangon Biotech (Shanghai) Co., Ltd. then carries out purifying to the endonuclease reaction system, obtains to be about the linearizing dna fragmentation of 2.6kb.

2.3 end adds T

At first prepare 10 and add T damping fluid (10x T-Tailing Buffer) doubly, composition is following:

Add the T reaction according to following reaction system:

Reaction conditions is: 72 ℃, and 2 hours

After reaction finished, " the SanPrep pillar PCR product purification test kit " produced with Sangon Biotech (Shanghai) Co., Ltd. carried out purifying, obtains pure dna fragmentation, and it is outstanding that this dna fragmentation end has 3 '-dT.

Embodiment 3

T preparing carriers method two (Xcm I enzyme cutting method)

3.1 the preparation of pre-T carrier

3.2Xcm the I enzyme is cut

The pre-T carrier that obtains in 3.1 is carried out enzyme cut, as previously mentioned, contain two Xcm I restriction enzyme sites in this pre-T carrier, each restriction enzyme site will stay the next one outstanding with 3 '-dT at the carrier end after the final linearizing.Xcm I is available from NEB (Beijing) ltd, and reaction conditions is following:

Reaction conditions is: 37 ℃, and 3 hours.

After endonuclease reaction finishes, detect enzyme with 1% agarose gel electrophoresis and cut whether fully.

3.3T the recovery of carrier and purifying

" the SanPrep pillar PCR product purification test kit " produced with Sangon Biotech (Shanghai) Co., Ltd. carries out purifying, obtains pure dna fragmentation, and it is outstanding that this dna fragmentation end has 3 '-dT.

Embodiment 4

The application of novel T carrier

4.1 novel T carrier connects effect measuring

Designing 3 pairs of primers, is template with Lambda DNA, and amplification length is 1kb respectively, and the dna fragmentation of 2kb and 5kb uses the Taq archaeal dna polymerase to obtain the amplified production that 3 ' terminal end of dna molecular adds an outstanding dA in the PCR reaction.

Said primer sequence is following:

Reaction system is following:

Response procedures: 94 ℃ of for 2min, (94 ℃ of for 30sec, 58 ℃ of for 30sec, 72 ℃ of for 3min) x 33 cycles, 72 ℃ of for 8min.

After reaction finishes; Agarose gel electrophoresis is presented at target bit and is equipped with tangible band; " SanPrep pillar PCR product purification test kit " this fragment of purifying with Sangon Biotech's production; Respectively with

embodiment

2 and 3 prepared T carriers with 3: 1 ratio of mol ratio, under the effect of T4DNA ligase enzyme, connect.

To connect product transformed into escherichia coli DH5 α bacterial strain competent cell; Transform the back with containing the 50mg/L penbritin; 0.1mM IPTG (Isopropyl beta-D-thiogalactopyranoside; Isopropyl-) and 40mg/L X-gal (5-bromo-4-chloro-3-indolyl-D-Galactopyranoside; 5-bromo-4-chloro-3-indoles-β-D-semi-lactosi) the enterprising row filter of LB culture medium flat plate on activated back coating of the competence after the conversion and the above-mentioned flat board, is inverted overnight cultures in 37 ℃.

Flat board is carried out blue hickie counting confirm blue hickie ratio; And with M13+/M13-primer (M13+:5 ' GTAAAACGACGGCCAGT-3 ' (SEQ ID NO:11); M13-:5 '-CAGGAAACAGCTATGACC-3 ' (SEQID NO:12)) hickie is carried out PCR and detect, measure the ratio that contains the segmental transformant of correct insertion in the hickie.The result is following:

Insert fragment length	Hickie ratio (%)	Hickie positive rate (%)
			1kb	93±2	99±1
2kb	90±3	96±2
			5kb	88±3	92±2

4.2 the application of novel T carrier in transformant screening

In above-mentioned experimental result, choose the unnegative transformant enlarged culturing of positive transformant of PCR result and PCR result respectively, extract plasmid, carry out enzyme with Hind III and cut checking.The result finds that all PCR positive transformant plasmids all can discharge the fragment consistent with inserting clip size after being cut by Hind III enzyme, and is identical with the positive transformant 100% that PCR detects, therefore, and the method Rapid identification transformant that can cut with Hind III enzyme.

Claims

1. T carrier; Be that two ends all have the outstanding linear carrier of 3 '-dT; The flanking sequence of said two 3 '-dT protruding terminuses satisfy when said T carrier with after the PCR product of two ends band 3 '-dA links to each other, can form two restriction enzyme digestion sites at insertion fragment two ends; Said two restriction enzyme digestion sites are the restriction enzyme site with a kind of restriction enzyme.

2. T carrier according to claim 1 is characterized in that said restriction enzyme digestion sites is a Hind III restriction enzyme site.

3. T carrier according to claim 1 is characterized in that, said T carrier except that the restriction enzyme digestion sites that the PCR product carries, only contains two restriction enzyme digestion sites with after the PCR product of two ends band 3 '-dA links to each other.

4. the preparation method of T carrier according to claim 1 for after producing flat terminal enzyme enzyme and cutting pre-T carrier, produces and has flat terminal linear molecule, after the end of said linear molecule adds T, obtains subsequently.

5. like the preparation method of the said T carrier of claim 4, it is characterized in that the flat terminal enzyme of said generation is EcoRV.

6. like the preparation method of the said T carrier of claim 4, it is characterized in that used enzyme was Taq DNA polymerase when said linear molecule end added T.

7. the preparation method of T carrier according to claim 1 obtains after cutting pre-T carrier with the enzyme enzyme that produces 3 '-dT protruding terminus.

8. like the preparation method of the said T carrier of claim 7, it is characterized in that the outstanding enzyme of said generation 3 '-dT is Xcm I.

9. pre-T carrier; Be the cyclic plasmid vector; Comprise MCS; Wherein: said MCS comprises the dna fragmentation of one section total following five restriction enzyme site, two restriction enzyme sites that produce 3 '-dT protruding terminus, and two evaluations are with restriction enzyme site and a restriction enzyme site that produces flat end; The flat terminal restriction enzyme site of said generation is positioned between the restriction enzyme site of two generation 3 '-dT protruding terminuses; The restriction enzyme site of said generation 3 '-dT protruding terminus can produce two 3 '-dT protruding terminuses on carrier after the enzyme enzyme that produces 3 '-dT protruding terminus is cut; The flanking sequence of said two 3 '-dT protruding terminuses satisfies said carrier when cutting through the enzyme enzyme that produces 3 '-dT protruding terminus after with after PCR product with 3 '-dA links to each other, and can form described two evaluations at insertion fragment two ends and use restriction enzyme site; Identify and use the restriction enzyme site of restriction enzyme site for said two as the same enzyme.

10. like the said pre-T carrier of claim 9, it is characterized in that the restriction enzyme site of said generation 3 '-dT protruding terminus is an Xcm I restriction enzyme site, the said evaluation uses restriction enzyme site to be Hing III restriction enzyme site, and the flat terminal restriction enzyme site of said generation is EcoR V.

11., it is characterized in that said pre-T carrier adopts said dna fragmentation replacement back to obtain by the MCS in the existing plasmid vector like the said pre-T carrier of claim 9.

12., it is characterized in that the sequence of said dna fragmentation is: SEQIN NO:1 like claim 9 or 11 said pre-T carriers.

13., it is characterized in that said pre-T carrier is for being connected into pUC19 the fragment acquisition of following sequence like the said pre-T carrier of claim 9 after Hing III and EcoR I enzyme are cut:

5′-AGCTCGCACGCCAAGCTTCGCTTGGGATATCCAAGCGAAGCTTGGATCGTACCGTCA-3′

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3′-GCGTGCGGTTCGAAGCGAACCCTATAGGTTCTCTTCGAACCTAGCATGGCAGTTTAA-5′。