CN102732546A - Vector and buffer for gene engineering, and use methods of vector and buffer - Google Patents

Abstract

The present invention discloses a novel vector, a buffer for supporting a plurality of tool enzymes to individually or jointly work, and use methods of the vector and the buffer. Compared with the conventional methods, the method of the present invention has the following advantages that: the process of linkage of a target DNA fragment and the vector by adopting the vector and the buffer is simple, efficient and reliable.

Description

Be used for engineered carrier, damping fluid and method of use thereof

Technical field

The present invention relates to biology field, more specifically, relate to and be used for engineered a kind of novel carriers, a kind of new buffer liquid and their method of use.

Background technology

Genetically engineered (genetic engineering) is claimed gene splicing technology and DNA recombinant technology again; Be to be theoretical basis with the molecular genetics; With molecular biology and microbiological modernism is means, the gene of different sources is pressed design in advance, at the external structure recombinant DNA molecules; Import recipient cell then, to change biological original hereditary property, obtain new variety, to produce product innovation etc.

In addition; Genetically engineered also provides the most basic means for studying gene and genome structure and function; Become indispensable basic research method in the life science; Important application is all arranged in many aspects, include but not limited to: genomic organization structural research, gene expression research, recombinant protein production, genetically modified organism, gene therapy, drug screening, vegeto-animal resistance etc.

In the genetically engineered experiment of classics, generally include following key step:

1. select recipient cell and carrier

Although have many kinds of recipient cells and carrier using, most experiments are from E.coli experimental strain and plasmid cloning vector starting.Owing to complete, the highly versatile of its technology, obtain easily, E.coli and plasmid vector become the first-selection of this area already.But, if clone's DNA is big especially, for example hundreds thousand of base pairs or bigger; Use bacterial artificial chromosome or yeast artificial chromosome (hizuya H at needs; Birren B, people such as Kim UJ, Proc.Natl.Acad.Sci.U.S.A.89 (18): 8794-7).

2. prepare carrier and purpose sheet segment DNA

In the genetically engineered research of classics, for the purpose fragment cloning to carrier, carrier DNA and target DNA should use suitable restriction enzyme (calling restriction endonuclease in the following text) thus enzyme cut produce coupling end so that be connected to target DNA on the carrier.The selection of restriction endonuclease will consider that carrier and purpose fragment can produce the end that matches each other.In classical genetically engineered experiment, this process is to make up difference cut vector DNA and the realization of purpose fragment with identical restriction endonuclease or restriction endonuclease.If handle carrier, also need handle carrier, to prevent that carrier is from connecting with SEAP with flush end restriction endonuclease, single sticky end enzyme or isocaudarner.

No matter be to cut enzyme with the sticky end enzyme or with the flush end enzyme, the carrier of handling all will pass through purifying to remove the follow-up influential composition of processes such as connection.

The fragment end that two non-isocaudarners (for example BamHI and EcoRI) cutting DNA molecule produces is not complementary, therefore can not connect certainly.In classical genetically engineered operation, have two can not connect certainly non-and the most generally use with the terminal carrier of tail.

The DNA that cuts above-mentioned source with restriction endonuclease makes its generation or put down the end that terminal carrier be complementary with aforementioned ready band sticky end.During the site that not is not complementary with carrier on the purpose fragment, can be through adding joint (Brown, Terry (2006) .Gene cloning and DNA analys is:an introduction (gene clone and DNA analysis: introduce).Cambridge, the Massachusetts; ISBN 1-4051-1121-6 of Blackwell press and Russell, David W.; Sambrook, Joseph (2001) .Molecular cloning:a laboratory manual (molecular cloning: laboratory manual).The cold spring port, New York; Cold spring harbor laboratory) or through PCR make suitable restriction enzyme site on its two ends band.

3. preparation recombinant DNA molecules

Accomplish through a plurality of simple molecular biosciences chemical steps with above-mentioned pretreated carrier and purpose produced in fragments recombinant DNA molecules.Wherein, most importantly ligation.Promptly mix the qualified carrier and the target DNA fragment of above-mentioned preparation by a certain percentage, add dna ligase and corresponding damping fluid, two dna fragmentations that have compatible termini behind the insulation certain hour just can connect into recombinant DNA molecules.

In genetically engineered research, dna ligase is a kind of common tool enzyme, and it can connect into recombinant DNA molecules to two dna moleculars with compatible termini.T4DNA ligase enzyme commonly used in the genetically engineered experiment.

4. import recipient cell to recombinant DNA molecules

The method that imports recipient cell to recombinant DNA molecules has multiple, and its title is different because of used experimentation.These methods include but not limited to (Brown, Terry (2006) .Gene cloning and DNA analysis:an introduction (gene clone and DNA analysis: introduce) such as conversion well known to those skilled in the art, transduction, transfection and electric shock.Cambridge, the Massachusetts; ISBN 1-4051-1121-6 of Blackwell press and Russell, David W.; Sambrook, Joseph (2001) .Molecular cloning:a laboratory manual (molecular cloning: laboratory manual).The cold spring port, New York; Cold spring harbor laboratory).

5. screening has the cell of carrier sequence

No matter how to import, have only sub-fraction to absorb DNA in the recipient cell.In order from the recipient cell colony of conversion or transfection, to have filtered out the very low picked-up of frequency the cell of DNA (being called transformant); Scientist uses methods such as antiviral antibiotic gene that those cells that do not absorb DNA are killed dexterously; (Brown, Terry (2006) .Gene cloning and DNA analysis:an introduction (gene clone and DNA analysis: introduce) but the cell that has only self-replacation and have a carrier of selectable marker gene could be survived.Cambridge, the Massachusetts; ISBN 1-4051-1121-6 of Blackwell press and Russell, David W.; Sambrook, Joseph (2001) .Molecular cloning:a laboratory manual (molecular cloning: laboratory manual).The cold spring port, New York; Cold spring harbor laboratory).

No matter recipient cell is bacterium, animal and plant cells or other recipient cells, all accomplishes screening through resistant gene.In bacterium, usually use the antiviral antibiotic gene, like anti-penbritin gene bla etc.

6. screening has the clone of purpose fragment and biological characteristics

The popular carrier has promotor and preceding 146 the amino acid whose coding regions from e.coli (lacZ).In this coding region, inserted a MCS (MCS).This carrier constitutes highly effective screening system with the recipient cell (having codified beta-galactosidase enzymes C end parts sequence) of supporting α-Hu Bu.Though recipient cell and plasmid-encoded fragment all do not have enzymic activity, but they can produce catalysis X-Gal when existing simultaneously and produce color reaction and make bacterium colony become blue bacterial plaque.If inserted the purpose fragment at MCS, then the function of LacZ is destroyed, and color reaction can not take place, and bacterium colony is a white.Whereby, can discern at an easy rate and inserted the segmental clone of purpose.

In genetically engineered research, can insert the foreign DNA and the dna structure that can in recipient cell, duplicate and be referred to as carrier.Usually, carrier is plasmid, phage or virus, also can be with the artificial hereditary unit that rebuilds of some element that comes from plasmid, phage or virus.Carrier be at least one can self-replacation minimum hereditary unit, so it must have at least one replication initiation sequence.

Carrier can be divided into cloning vector and expression vector two big classes.

Cloning vector is the key tool of gene clone.Some commercial companies develop multiple simple in structure, can clone easily and include but not limited to structure gene and the segmental cloning vector of other function DNAs, for genetically engineered research provides widely used instrument.These carriers include but not limited to pUC series, pBlueScript series etc.

Expression vector is engineered another key tool, and can be used for making destination gene expression is albumen.The same with cloning vector, it has an effective replication initiation sequence and an effective antiviral antibiotic gene in intestinal bacteria in intestinal bacteria, and it is bred in intestinal bacteria and available its preparation DNA.

Compare with cloning vector, expression vector also has one by promotor and zero load (the not having structure gene) expression cassette that terminator sequence is formed.A MCS that can be used for inserting goal gene is arranged between promotor and terminator sequence, be used to insert the purpose fragment in various sources.Go down to posterity for expression vector can be stablized in recipient cell (like yeast, zooblast, vegetable cell etc.), also have in recipient cell effective replication initiation sequence (as in mammalian cell with the replication initiation sequence of SV40) and resistant gene (like Neo, Hygromycin etc.) such as antiviral antibiotic gene.Had at present to be applicable to the expression vector that includes but not limited to that zooblast, vegetable cell, bacterium or fungal cell's expressing gene product are used, for example the expression vector of commercial offers such as pET series, pcDNA series.

As well-known to those skilled in the art, no matter be cloning vector or expression vector, all at least one MCS must be arranged so that insert the purpose fragment.MCS is the substruction of carrier, and it has determined the needed experimental implementation of clone's purpose fragment.

Widely used carrier is a lot.When effective research tool is provided, the structures shape of the MCS of these carriers can't avoid before the cloned DNA fragment, carrier being carried out pre-treatment during with their clone's purpose fragments with suitable instruments such as restriction endonuclease.Therefore, in the experiment of the genetically engineered of classics, before connection, need carry out that enzyme is cut to carrier and purpose fragment, enzyme is cut afterwards purifying, carrier connects red tape such as rate calibrating certainly.This be one tediously long and do not allow manageable program, usually cause the failure of an experiment or not ideal enough.This is to need improved importance in the existing cloning process.

Simultaneously, owing to cut and always have when being connected some molecules can not be cut and produce carrier DNA being carried out enzyme, transform the back and always grow the clone that some have initial carrier from connecting product (being referred to as) from connecting product.This clone appears at too to have and inserts in the segmental connection product.If it is too high to connect clone's ratio of generation certainly, then can bring serious interference to experiment.Therefore, each batch enzyme carrier of cutting processing all will pass through from connecting rate and detect to estimate the quality of pre-treatment carrier.If it is too high to connect rate certainly, then can not be used for subsequent experimental.In real work, connecting rate certainly too high is the normal factor of sending out that causes preparing the carrier failure, might cause continuous failure.This situation also is that existing carrier needs improved importance.

Summary of the invention

One object of the present invention is that the end according to some restriction endonuclease or restriction endonuclease combination results " recognition site is reticent " this phenomenon can occur a kind of novel carrier is provided, so that the process that target DNA fragment is connected with carrier in the genetically engineered is simple more, effective and reliably.

Another object of the present invention is to provide a kind of support plurality of enzymes independent or cooperative damping fluid.

Another object of the present invention is to provide a kind of method that target DNA fragment is connected with carrier.

For reaching this purpose, the present invention adopts following technical scheme:

Aspect first; The invention provides a kind of carrier; It is characterized in that said carrier comprises one or more MCSs, said MCS is connected generation not by the recombinant molecule of said restriction enzyme identification with other dna fragmentations behind digestion with restriction enzyme.

In carrier provided by the invention, said restriction enzyme can be restriction enzyme or its combination outside recognition sequence of isocaudarner, restriction enzyme site.

In carrier provided by the invention, said restriction enzyme can comprise but not limit: XbaI, StyI, NcoI, NheI, SpeI, BamHI, BglII, BclI, SalI, HincII, XhoI, MluI, PauI, EcoRI, MunI, PstI, NsiI, SmaI, SmiI, PmlI, StuI, EcoRV, PvuII, Alw26I, BseGI, BseMI, BseNI, Eco31I, Eam1104I, MnlI, BseXI, MboII, SchI, BoxI, BsaBI, BseJI, BseLI, DraIII, Eco91I, SfiI, Van91I, PpiI, AjuI, AlfI, AloI, BdaI, BplI, Hin4I, TscaAI and TstI.

In carrier provided by the invention, the MCS sequence of said carrier can comprise SEQ ID NO:1 or 5.

Aspect second, the invention provides the application of the described carrier of first aspect in genetically engineered.

In the application of said carrier provided by the invention, said genetically engineered can be directed cloning or non-directional clone.

Aspect the 3rd, the invention provides the independent or cooperative damping fluid of the multiple toolenzyme of a kind of support, it is characterized in that comprising 50mM Tris-acetate; The 100mM potassium acetate, 15mM magnesium acetate, 1mM DTT, 0.1%BSA, 0.5mM ATP, pH are 7.5-8.3; Preferably; Being 7.7,7.9,8.0,8.1,8.3, more preferably is 8.3, and said toolenzyme includes but not limited to T4DNA ligase enzyme, restriction enzyme, polynueleotide kinase and Klenow fragment.

Aspect the 4th, the invention provides the application of the described damping fluid of the third aspect in genetically engineered.

Aspect the 5th, the invention provides a kind of method that target DNA fragment is connected with carrier, it is characterized in that using the described carrier of first aspect in same step, to accomplish said target DNA fragment and cut with the enzyme of said carrier and be connected.

Method provided by the invention is characterized in that using the described damping fluid of the third aspect.

Among this paper, term " target DNA fragment " is called for short " purpose fragment " and is meant the dna molecular that all need be studied.The purpose fragment can be from genomic dna, also can be from the DNA of cDNA, synthetic, and current maximum be PCR product (comprising the RT-PCR product).

Term " carrier " is meant in genetically engineered research, the dna structure that can insert foreign DNA and can in recipient cell, duplicate.

Term " MCS " is meant one section sequence of synthetic on the dna vector sequence, contains a plurality of restriction restriction endonuclease recognition sites, can multiple insertable position or interleaved plan be provided for foreign DNA.

Term " restriction enzyme " is called for short " restriction endonuclease ", is one type of DNA lytic enzyme that specific recognition site and point of contact are arranged on dna molecular.At the beginning of 2005, find 3681 restriction endonucleases altogether, 221 specific specificity are arranged.Restriction endonuclease is one of engineered key tool enzyme.

Term " isocaudarner " is meant the restriction endonuclease that has some recognition sequences differences in the restriction endonuclease but can produce the identical sequence sticky end.Some restriction endonuclease produces flat terminal.Because all flat ends can be interconnection, similar with above-mentioned isocaudarner relation, also be classified as isocaudarner to the flush end restriction endonuclease among the present invention

Term " point of contact is reticent " is meant that recognition sequence originally will not exist after the dna fragmentation connection of two kinds of isocaudarner cuttings, thereby can not be by original restriction endonuclease identification and cutting.

Term " reticent point of contact " is meant the reticent sequence that is produced in point of contact

Term " reticent presequence " be meant can through endonuclease digestion be connected the sequence that produces reticent point of contact.

Term " genetically engineered " is meant the gene of different sources and at external structure hybrid DNA molecule, imports viable cell then, to change biological original hereditary property, obtain new variety, to produce product innovation by design in advance.

Description of drawings

Fig. 1. MCS that designs among the present invention (A) and sequence thereof.

The sequencing result of Fig. 2 .pUC57MC MCS.Standard sequence is seen SEQ ID NO:1.

Fig. 3 .Buffer X supports the experiment of multiple restriction endonuclease.Figure A is that 19 kinds of main restriction endonucleases of the present invention are cut the result to the enzyme of pUC57MC, and figure B is that 19 kinds of coupling site restriction endonucleases are cut the result to the enzyme of the pUC57M that has single coupling site among the figure A.

Fig. 4. with pUC57MC carrier, the recombinant DNA that obtains with two sticky ends of two enzymes and the two sticky ends of the single enzyme basket/hickie situation after transforming.A. the two sticky ends of two enzymes, the two sticky ends of the single enzyme of B.

Fig. 5. the situation of emitting fluorescence behind the constructed pCi-Neo/II-GFPq transfection HEK293 cell.

Embodiment

The design of embodiment 1. novel carriers MCSs

One, the utilization of isocaudarner

When designing new MCS, mainly considered two principles: the isocaudarner that comprises in the flush end restriction endonuclease is as often as possible inserted in (1) makes carrier have more isocaudarner to use, thereby satisfies the demand of carrier to MCS.(2) preferentially select for use those cheap restriction enzymes to reduce use cost.

Be used for the design (* is the flush end restriction endonuclease) of MCS according to the selected following restriction endonuclease of above-mentioned two principles:

Restriction endonuclease that is used for cut vector that table 1 is selected and the restriction endonuclease that matches

According to top selected, form the MCS of carrier with main site, the coupling site is used for the purpose fragment, thus the sticky end that can obtain easily to mate or the end of other structures.Intersperse among in the main site with several flush ends and other restriction enzyme sites commonly used, can when being difficult to find other coupling sites, use.

For conveniently cloned sequence is carried out enzyme cuts evaluation, insert two PstI sites respectively at the proximal end of MCS, so that identify inserting segmental size with single enzyme.In addition, two BsaI sites in the opposite direction also in this MCS, have been inserted, so that obtain different sticky ends with single endonuclease digestion.If insert the site that matches for segmental two sections, can only accomplish directed cloning with ligase enzyme with a kind of restriction endonuclease in purpose.

After arranging the encoding sequence of above-mentioned selected restriction endonuclease in certain sequence, can obtain the dna sequence dna (Fig. 1 and SEQ ID NO:1) of MCS.

Two, the utilization of special restriction endonuclease

Some special restriction endonucleases are arranged, and its cleavage site is outside recognition site.This fermentoid has two types, and one type is that positive minus strand respectively has a point of contact and in the same side of recognition sequence, Alw26I for example, and one type is that point of contact, for example a PpiI are all arranged on the positive minus strand of recognition sequence both sides.

With Alw26I is its application of example explanation.

This enzyme point of contact sequence on carrier should be:

The forward sequence is: GTCTCN ↓ NNNN reverse sequence is: ↓ NNNN-NGAGAC

GAGGAN-NNNN↑ NNNN↑NCTCTG

If there are two back-to-back reverse Alw26I sites as follows on the carrier simultaneously; It is (convenient for narrating to produce sticky end as shown in the figure when then cutting; Represent that with A ~ H in two sites some be appointed as the particular bases in the carrier sequence, capitalization is represented complementary base with corresponding lowercase.Down together.）

-----↓abcd-ngagac?gtctcn↓EFGH------

------ABCD↑nctctgcagagn-efgh↑-----

After the above-mentioned site of carrier was cut, the lowercase part was moved from carrier, and two ends stay 3 '-end sticky end ABCD and EFGH respectively.

The point of contact of also respectively placing an Alw26 in the segmental both sides of purpose, just on direction and the carrier on the contrary.Its four terminal N are appointed as four terminal base ABCD/abcd of purpose fragment or EFGH/efgh.

GTCTCN↓abcd-XXXXXXXXXXXXXXXXXXX↓EFGH-NGAGAC

GAGGAN-ABCD↑xxxxxxxxxxxxxxxxxxx -efgh↑NCTCTG

With after the Alw26I cutting, the capitalization part is removed, both sides respectively stay with carrier on sticky end complementary 3 '-sticky end.The segmental sticky end of this carrier and purpose is a complementary, can connect easily.

Should be noted in the discussion above that the sticky end that (1) stay is different with the sticky end that recognition site reversing multiple restriction endonuclease stays here, it is not the reversing multiple; And (2) enzyme cut the both sides sticky end sequence that stays can be different; (3) the same as the isocaudarner site of paired use, because of the recognition site of this enzyme in the part of cutting away is lost, in case respective segments is connected to this site; Then can not be again by the Alw26I cutting, therefore use can realize directed cloning separately.The small segment that is cut off might be connected to the home position again according to original direction, but can be cut again; The small segment that is cut off can not be connected to an other end with opposite direction.Therefore, this method can not cut carrier pre-treatment single tube completion enzyme and be connected yet.

According to these zymoid characteristics, in MCS of the present invention, inserted the opposite Alw26I site of both direction.

Have several kinds of special restriction endonucleases in point of contact that significant application value is arranged in the clone, its title, recognition site and cutting position are listed in the table below.Wherein, Alw26I, BsaI and SchI are the most suitable.

Table 2 one-sided positive minus strand outside recognition site all has restriction endonuclease, recognition sequence and the cut point at point of contact

Behind the endonuclease digestion that the point of contact is all arranged on the positive minus strand in both sides outside the recognition sequence, all produce sticky end by the degeneracy based composition at two ends; Therefore; Through suitable design; Use a point of contact can obtain two ends and be the incomplementarity sticky end, its method of use all has the restriction endonuclease (like Alw26I) at point of contact similar on the one-sided positive minus strand of recognition sequence with above-mentioned paired use.This fermentoid includes but not limited to the enzyme shown in the table 3.

The positive minus strand of table 3 bilateral outside recognition site all has restriction endonuclease, recognition sequence and the cut point at point of contact

Method of use and the effect that has a carrier of above-mentioned two types of special restriction enzyme sites all is equivalent to two pairs and unites use with tail sticky end restriction endonuclease, and operation easily.With BsaI is example, requires the purpose fragment to have that two ends produce CGTG and CTGC sticky end respectively after BsaI cutting.In case of necessity, the carrier after this enzyme enzyme is cut can be used with the purpose fragment that has one or two 5 '-four bases sticky ends, like BamHI, and BglII, BclI, EcoRI, HindIII, Kpn2I, NcoI, NheI, NotI, XbaI, XhoI etc.

Can consider to use other specific enzymes of above-mentioned strategy to include but not limited to:

Operational other restriction endonucleases of table 4 (* is the flush end restriction endonuclease)

The structure of embodiment 2. novel carriers

Transform for the prototype carrier with pUC57, can obtain to have the novel cloning vector of above-mentioned design.Concrete grammar is:

1.pUC57 sequential analysis and transformation:

Restriction enzyme site to pUC57 is analyzed discovery, at 1786 the BsmAI site is arranged, and at 309 and 655 a PvuII site is arranged respectively.In order in new MCS, to use PvuII and BsaI site, design following mutant primer and be used to eliminate three sites:

309PvuF：5’-ctattacgccaActggcgaaagggggatgtgctg-3’

309PvuR：5’-ctttcgccagTtggcgtaatagcgaagaggc-3’

655PvuF：ggccaacgcgcggggag

655PvuR：gattcattaatgcaActggcacgacagg

1786BsmF：cggctccagatttatcagcaataaaccag

1786BsmR：gtgagcgtggCtctcgcgg

With pUC57 is template, and 309PvuF/309PvuR is a primer, carries out PCR with KOD high-fidelity enzyme (providing for oneself, down together) routine.After 2700bp left and right sides PCR product glue reclaimed, with Polynucleotide Kinae phosphorylation, conventional again T4DNA Ligase connected the back and transforms DH5 α cell.Inoculate 12 single spots, 37 spend night cultivates back extracting and purifying DNA.The DNA of pUC57 with not sudden change is contrast, cuts evaluation with PvuII enzyme (all toolenzymes are the MBI product among the present invention, down with) enzyme, obtains correctly to clone.

DNA with the correct clone of above-mentioned acquisition is a template, uses with quadrat method and accomplishes successively 655 and 1,786 two site mutation, promptly obtains the clone that eliminated above-mentioned 3 restriction enzyme sites, and note is made pUC57M.

2. obtain the MCS coding region:

The sequence of the MCS of design is shown in SEQ ID NO:1, and its length is 111bp, and two ends are that EcoRI and HindIII mate with the end site with the pUC57 MCS.According to design, but this fragment in-frame is inserted into the EcoRI/HindIII site of pUC57.Require design oligonucleotides such as SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 according to Over-lapping PCR.5 ' of SEQ ID NO:2 and SEQ ID NO:4-end adds 3 protection bases during synthetic primer.With three primers template each other, adopt KOD to carry out conventional PCR.After obtaining 117bp, ordinary method is inserted into the EcoRI/HindIII site of pUC57.

Transformed into escherichia coli DH5 α bacterial strain is coated with penbritin-agar plate.After 37 degree overnight cultures are cultivated, get 12 single spots and be inoculated into 3 milliliters of LB substratum that contain penbritin respectively, 37 degree overnight cultures, isopropanol precipitating method extracting and purifying DNA.

Cut the correct clone of screening with PvuII, BsaI and BamHI enzyme, altogether 6 three kinds of enzymes all have the clone at a point of contact.Get wherein 3 with the order-checking of M13-primer, obtains two and correctly clone.The order-checking collection of illustrative plates is seen Fig. 2.Should correct clone's note make p57MC.

3. blue hickie Function Identification:

For whether the pUC57MC that checks above-mentioned structure has normal blue hickie screening function, behind its conversion DH5 α, coat the LB agar plate that contains penbritin and X-Gal or X-Gal+IPTG, 37 degree overnight cultures.Have only on the flat board of X-Gal total 200 bacterial plaques, do not see that any blue bacterial plaque occurs, and explains that the LacZ in this carrier does not have " leakage ".But containing on the LB agar plate of X-Gal+IPTG 250 of all bacterial plaques (>) be mazarine.This explanation, the pUC57MC of structure can induce the activity of LacZ through IPTG, explains that the IPTG inducing function is normal.

The design and the checking of embodiment 3 general damping fluids

Experiment one, general buffer design and optimization

After analyzing with reference to key elements such as the composition of company's restriction endonuclease reaction buffers such as New England Biolabs, Fermentas, Promega and dna ligase, T4DNA polymerase, Klenow Fragment reaction buffer etc., pH; Confirm with T4DNA ligase enzyme and NEB 4# restriction endonuclease reaction buffer to be that a kind of reaction buffer that can support multiple toolenzymes such as restriction endonuclease according to the invention, T4DNA ligase enzyme is simultaneously set up on the basis, to reduce the operation steps of experiment.The damping fluid composition of confirming is: the ATP that Tris-Acetate damping fluid, mg ion, Repone K, BSA, DTT and dna ligase need.

In above-mentioned reaction buffer; The all existing clearer and more definite working range of the concentration of ATP, BSA and DTT; Can confirm three's working concentration according to the data of the T4DNA ligase enzyme of NEB or other producers and restriction endonuclease reaction buffer, have only the composition of Tris-Acetate and KAc need optimize to adapt to multiple toolenzyme.For this reason, represent less salt, middle salt, high salt and high pH restriction endonuclease to adopt three repetition orthogonal tests to be optimized respectively, attempt to obtain a kind of damping fluid that can support multiple restriction endonuclease simultaneously two compositions with ApaI, PvuII, BglII, HidIII.

For optimizing the optimum concn of KAc and Tris-Acetate, under the pH8.0 condition, carry out orthogonal test, reaction system 37 degree that adopt 20 μ l to contain 500ng pUC57MC DNA, 1U restriction endonuclease carried out the inspection of 1% agarose electrophoresis 90 minutes.Experimental result is following:

The general damping fluid of table 5 is optimized orthogonal experiments (pH8.0) *

*+about 25%DNA is digested in expression, ++ represent that about 50%DNA is digested, ++ about 75%DNA is digested in+expression, ++ ++ expressed enzyme cuts entirely.

According to The above results, when pH8.0, Tris-Acetate is 50mM, four types of restriction endonucleases all reached active more than 50% when KAc was 100mM, and wherein middle salt and high salt type can reach 100%.For further optimizing this damping fluid, constant in other conditions, under pH7.5,7.7,7.9,8.1 and 8.3 conditions, above-mentioned four kinds of endonuclease activities are tested, the result sees table 6.It is thus clear that, shown in the scope, the activity of all enzymes can satisfy requirement of experiment all more than 50%.Particularly, under the pH8.3 condition do except that the ApaI activity +++, all the other restriction endonucleases are ++ ++.Confirm that in view of the above optimal components is combined as: pH is 50mM Tris-Acetate (8.3,25 ° of C of pH), 100mM potassium acetate, 15mM magnesium acetate, 1mM DTT, 0.1%BSA and 0.5mMATP.This damping fluid note is made Buffer X.

With all status of a sovereign points of embodiment two said pUC57MC designs and the restriction endonuclease in coupling site Buffer X is tested; Reaction system is: 2 μ l, 10 * Buffer X, 200ng pUC57MC DNA or pUC57M (being used to mate the site), 1U restriction endonuclease, reaction system is 20 μ l.Remove the BclI heated culture temperature and be 55 and be outside one's consideration, remaining enzyme is 37 degree.Reaction is carried out after 90 minutes containing on 1% sepharose of EB electrophoresis and observing enzyme and cut situation.The result sees Fig. 3, can find out from figure A and B, and Buffe X can support all 38 kinds to supply the examination restriction endonuclease.Scheme A:19 status of a sovereign point restriction enzyme mapping.Restriction endonuclease is in proper order: EcoRI, PstI, XbaI, MluI, Eco31I (BsaI), PmlI*, ApaI, BamHI, KpnI, SmiI (SwaI) *, SacI, EcoICRI*, EcoRV (Eco32I) *, SalI, HincII*, PvuII*, StuI*, SmaI*, HindIII.Be the flush end restriction endonuclease wherein with *.#20 is the pUC57MC that does not cut.Scheme B:19 coupling site endonuclease digestion collection of illustrative plates, the enzyme tangent condition is the same.Restriction endonuclease is in proper order: MunI, NsiI, StyI, NcoI, NheI, SpeI, PauI, BglII, BclI, XhoI, BsaBI*, PmeI*, NruI*, DpnI*, DraI*, MslI*, SchI*, HaeIII* and HpaI*.Wherein, 11～19th, the flush end restriction endonuclease.#20 is the control plasmid DNA that does not cut.This result proves that the supported restriction endonuclease of this damping fluid includes but not limited to those that carrier of the present invention is related.

Experiment two, to the support of T4DNA ligase enzyme and PNK

Whether can support dna modification enzymes such as T4DNA ligase enzyme, polynueleotide kinase in order to test this damping fluid; Be template with pUC57MC, be that primer sets is carried out PCR with Pfu with 655PvuF/655PvuR by the condition among the embodiment 2, the product glue of acquisition is set up following ligation: H after reclaiming ₂O 9 μ l, 10 * Buffer X, 2 μ l, PCR product 5 μ l, T4DNA ligase enzyme 2U, T4PNK 2U transform the DH5a competent cell after 22 degree connect 30 minutes behind the mixing, are coated with IPTG/X-Gal ammonia benzyl plate, and 37 degree culturing bacterium spend the night.

Test shows that the bacterial plaque number surpasses 300/9 centimetres flat boards.This explanation has added phosphate group at flush end PCR product 5 '-end, and under the effect of T4DNA ligase enzyme, connection has taken place under the PNK effect.Prove that thus BufferX can support PNK and T4DNA ligase enzyme activity.

Experiment three, to the support of Klenow Fragment

Cut with the BamHI/EcoRI enzyme and to carry out glue behind the pUC57MC and reclaim, reclaim product and be used for following test: H ₂O 14 μ l, Klenow Fragment 5 μ l, T4DNA ligase enzyme 5 μ l, glue reclaims product 5 μ l.Do not add T4 ligase enzyme and the negative contrast of Klenow fragment.22 ℃ of reactions transformed DH5a after 30 minutes, were coated with IPTG/X-Gal ammonia benzyl plate and 37 degree culturing bacterium spend the night.

The result shows that the bacterial plaque number is 227/9 centimetres flat boards, and wherein hickie is 186,41 of locus coeruleus.Negative control has only 11 locus coeruleus.This explanation, Buffer X can support Klenow fragment and T4DNA ligase enzyme co-operation.

Should be noted that other damping fluids that can support multiple toolenzyme also possibly satisfy needs of the present invention to a certain extent.For example, the standard Taq DNA Polymerase reaction buffer of NEB company, the Tango Buffer of Fermentas company etc. also can support toolenzymes such as multiple restriction endonuclease under certain condition.The checking and the method for use of embodiment 4. novel carriers

Experiment one: the reorganization of isocaudarner endonuclease bamhi

PUC57/44B is one and has the 976bp fragment and insert segmental recombinant plasmid; Inserting segmental primer is: 44Fx (ctaCTCGAGgc tctctgattactgccataatccaggg); Wherein cta is the protection base, and CTCGAG is XhoI point of contact (with the same tail in SalI site on the carrier pUC57MC); 44Rx (ctaAGATCTctgtagcctgcctctgctctctc), wherein cta is the protection base, AGATCT is BglII point of contact (with the same tail in BamHI site on the carrier pUC57MC).

The sticky end that table 7 isocaudarner produces, coupling and the signal of connection product structure

With 44Fx and 44Rx is primer sets, should insert fragment with KOD from the pUC57/44B amplification, and glue reclaims the back is connected to the pUC57MC carrier with following method SalI/BamHI site:

In 1.5ml EP pipe, add 10 * Buffer X, 2 μ l, PCR product 1 μ l (300ng), pUC57MC1 μ l (100ng), T4DNApolymerase 1U, BamHI 2U, BglII 2U, SalI 2U, XhoI 2U add water to 20 μ l.22 degree insulations transform DH5 α behind the mixing after 30 minutes, and it is dull and stereotyped that routine is coated with the IPTG/X-Gal LB that contains penbritin, and 37 spend night cultivates.Inferior daily inspection is dull and stereotyped and choose 12 on single spot and be inoculated in 3ml respectively and contain in the LB substratum of penbritin, and extracting and purifying DNA after the 37 degree overnight cultures, PstI enzyme are cut and identified and insert segmental size.

In this experiment, SalI on XhoI on the PCR product and BglII and the carrier and BamHI site can produce " reticent point of contact " respectively.

The result: flat board has 273 bacterial plaques, and wherein locus coeruleus is 12,261 of hickies, and the hickie rate is 95.6%.The insertion clip size of 12 bacterial strains that (Fig. 4 A) PstI enzyme is cut evaluation is all correct.

Experiment two, one end is a sticky end, and an end is the connection of flat terminal fragment

Reclaim product to the glue of above-mentioned 44Rx/44Fx and be connected with pUC57MC, the restriction endonuclease combined adds PEG4000 and after final concentration 10%, 22 degree is incubated 30 minutes, transforms DH5 α with BamHI 2U, BglII 2U, PvuII 2U.Other operations are identical with experiment one.

In this connection combination, the BglII point of contact on BamHI on the carrier and the purpose fragment is isocaudarner each other, can produce " reticent point of contact ".Do not add XhoI in the reaction system, so the segmental XhoI end of PCR is not digested, for flat terminal.Should flat end can be cut flat end that the back produces by the PvuII enzyme with carrier is connected and produces " reticent point of contact ".

The result: flat board has 246, and wherein locus coeruleus is 11,235 of hickies, and the hickie rate is 95.5%.Experiment three, other can produce the use of " point of contact reticent " restriction endonuclease

Cut the site to the XhoI of 44Fx primer in the experiment one and change Alw26I (GTCTCacgtg into;); The BglII of 44Rx primer changes reverse BsaI site (ctgcaGAGAC) synthetic primer into; Method with reference to test one is carried out PCR, obtains to be connected with pUC57MC after PCR product and glue reclaim, and the restriction endonuclease combined is with the single enzyme of BsaI.Other operations are all identical with test one with condition.

The result: flat board has 187, and wherein locus coeruleus is 4,183 of hickies, and the hickie rate is 97.9% (Fig. 4 B).

Above-mentioned all experimental results are all supported as drawing a conclusion:

(1) characteristic of the pUC57MC cloning vector isocaudarner capable of using of the present invention's structure is simplified the process of experimental implementation greatly; Must carry out loaded down with trivial details pre-treatment (include but not limited to that enzyme is cut, glue reclaims, connect loaded down with trivial details process such as rate mensuration certainly) to carrier and purpose fragment when having avoided conventional carrier cloning purpose fragment, and the positive colony ratio is very high.

Its principle is following: BamHI and BglII, SalI and XhoI be isocaudarner each other, and cutting produces the sticky end (example is seen table 8) that can match each other.BglII on the PCR product and XhoI cutting produce sticky end can be respectively with carrier on BamHI mate with the sticky end that SalI produces.When T4DNA Ligase existed and function is arranged, the end of coupling can connect and produce recombinant molecule.Accomplish the recombinant molecule that connects back formation and will lose original restriction enzyme site, restriction endonuclease originally can not cut again, promptly produces at " reticent point of contact ".When inserting the point of contact equal " silence " at fragment two ends, this recombinant molecule just can not be cut by original any enzyme or enzyme combination.

The vector dna molecule that is cut might recover original structure from connecting, but can heavily cut and participate in and insert segmental the connection.Along with enzyme is cut the carrying out with ligation, the ratio of the recombinant molecule that can not be cut rises gradually, and the carrier ratio of primary formation reduces gradually.The product that reacts after about 30 minutes transforms, and can obtain a high proportion of white reorganization bacterial plaque.

Though some existing carrier also might adopt this method, because of they without specially designed, very big limitation is arranged.The MCS of cloning vector pUC57MC of the present invention the is elaborately planned point of contact of 6 groups of relatively inexpensive isocaudarners, 8 kinds of flush end restriction endonucleases, two the back-to-back Alw26I of recognition sequence site I and 4 kinds of other restriction endonucleases; Make this carrier can make full use of the characteristics and the specific purposes fragments sequence characteristic of these enzymes, need not carry out accomplishing the process that makes up recombinant molecule with method of the present invention under any pretreated situation carrier and purpose fragment.

(2) the Buffer X of the present invention toolenzymes such as all restriction endonucleases and T4DNA Ligase, PNK, Klenow fragment that can support to discern above-mentioned novel vector MCS, the site that matches are worked simultaneously, therefore can in a reaction, accomplish enzyme simultaneously and cut and be connected.

(3) MCS of the present invention and the Buffer X structure that also can be used for new expression vector waits all need prepare the experiment of recombinant DNA molecules with toolenzyme.

Structure, checking and the method for use of embodiment 5. new expression vectors

For testing said restriction enzyme site in structure possibility of its application in expression vector, with the mammalian expression vector pCI-NEO of Promega company (catalog number (Cat.No.): be that model is transformed and verified E1841), main operation as follows:

1. the transformation of MCS

According to the sequence data that producer provides, the polyclone sequence of this carrier comprises following site: NheI, XhoI, EcoRI, MluI, KpnI, XbaI, SalI, SmaI, NotI (EagI).To the analysis and the usage policy of isocaudarner " silence " type restriction endonuclease combination, the following new MCS of design is so that utilize principle of the present invention to simplify the experimental implementation of clone's process according to the present invention:

In view of the above, design following dna sequence dna:

Gctagcgagaccacgcgtgaattcggtctccagctggtcgaccgccggcg, SEQ ID NO:5 and synthetic following oligonucleotide fragment:

gatgctagcgagaccacgcgtgaattcggtctccagctggtcgaccgccggcgatg，SEQ?ID?NO:6catgcggccgcgtcgaccagctggagaccgaattcacgcgtggtctcgctagcatc，SEQ?ID?NO:7

Respectively get 0.5 μ g with sterilized water according to after the 1 μ g/ μ l dissolving, sex change 15 minutes in the boiling water behind the mixing, naturally cooling is annealed to room temperature.

PCi-Neo DNA 1 μ g is connected with above-mentioned annealing product respectively with NheI/NotI 37 degree enzymes cut 3 hours, glue reclaims the back and connects with ordinary method, has obtained the clone through screening, and sequence verification is errorless.The carrier note that is obtained is made pCi-NEO/II.

2. utilize new MCS to insert goal gene GFPq

With primer GFPqF (5 '-CAC TCTAGACCGCCATGGCTAGCAAAGGAG-3 ', XbaI) and GFPqR (CTA CTCGAGGCGGCCGCGGATCCTCAGTTGTACAGTTC XhoI) is primer, and from pGP5/GFPq carrier (providing for oneself) amplification GFPq coding region, it is subsequent use that amplified production glue reclaims the back.In a 1.5ml EP pipe, add following composition: 0.1 μ g pCi-Neo/II DNA, 0.3 μ g glue reclaim product, 2 μ l Buffer X, 2U NheI, 2U SalI, 2U XbaI, 2U XhoI, 3U T4DNALigase; Add water to 20 μ l, 22 ℃ of insulations were carried out enzyme in 30 minutes and are cut/ligation behind the mixing.Connect product with 8 μ l and transform DH5 α, get 12 single spots with reference to preceding method extracting and purifying DNA.Through identifying that wherein 10 clones' DNA is not all cut by NheI, SalI enzyme, can not be by NheI/SalI.Obtain correct clone.

Above-mentioned description of test successfully has been inserted into the GFPq fragment of amplification on the NheI/SalI carrier of said carrier through method of the present invention.

3. the functional verification of recombinant vectors

Produce Endotoxin-free test kit (catalog number (Cat.No.): 12362) the reorganization pCi-Neo DNA of the above-mentioned band GFPq gene of preparation with Qiagen; With the Lipo2000 transfection HEK293 cell of Invitrogen, cultivate 24 and as a child under inverted fluorescence microscope, observed and photograph.The result shows that this recombinant molecule can correctly be expressed the GFPq GFP.This explanation, GFPq gene and the carrier pCi-Neo/II function of the present invention inserted with this method are normal.

Provide in the preceding text specific descriptions of the present invention are set forth and explained being used to.But do not really want exhaustive or limit the invention to disclosed precise forms.According to above explanation, can realize a lot of the modification and modification.The foregoing description is selected and is used for explaining principle of the present invention and practical application thereof best, thereby makes those skilled in the art and to utilize the different modification that are suitable for specific intended purpose to utilize best the present invention with various embodiment.Scope of the present invention will be defined by accompanying claims.

Claims (10)

1. a carrier is characterized in that said carrier comprises one or more MCSs, and said MCS is connected generation not by the recombinant molecule of said restriction enzyme identification with other dna fragmentations behind digestion with restriction enzyme.

2. carrier according to claim 1, wherein said restriction enzyme are restriction enzyme or its combinations outside recognition sequence of isocaudarner, restriction enzyme site.

3. carrier according to claim 2, wherein said restriction enzyme includes but not limited to: XbaI, StyI, NcoI, NheI, SpeI, BamHI, BglII, BclI, SalI, HincII, XhoI, MluI, PauI, EcoRI, MunI, PstI, NsiI, SmaI, SmiI, PmlI, StuI, EcoRV, PvuII, Alw26I, BseGI, BseMI, BseNI, Eco31I, Eam1104I, MnlI, BseXI, MboII, SchI, BoxI, BsaBI, BseJI, BseLI, DraIII, Eco91I, SfiI, Van91I, PpiI, AjuI, AlfI, AloI, BdaI, BplI, Hin4I, TscaAI and TstI.

4. carrier according to claim 1, the MCS sequence of wherein said carrier comprises SEQ ID NO:1 or 5.

5. according to each described carrier application in genetically engineered in the claim 1 to 4.

6. application according to claim 5, wherein said genetically engineered are directed cloning or non-directional clone.

7. support the independent or cooperative damping fluid of multiple toolenzyme for one kind, it is characterized in that comprising 50mMTris-acetate, the 100mM potassium acetate; 15mM magnesium acetate, 1mM DTT, 0.1%BSA, 0.5mM ATP; PH is 7.5-8.3, preferably, is 7.7,7.9,8.0,8.1,8.3; More preferably be 8.3, said toolenzyme includes but not limited to T4DNA ligase enzyme, restriction enzyme, polynueleotide kinase and Klenow fragment.

8. the application of damping fluid according to claim 7 in genetically engineered.

9. method that target DNA fragment is connected with carrier is characterized in that using and in same step, accomplishes said target DNA fragment according to each described carrier in the claim 1 to 4 and cut with the enzyme of said carrier and be connected.

10. method according to claim 9 is characterized in that using the damping fluid according to described in the claim 7.

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