CN1165622C - Zero-background high-flux directed cloning expression method - Google Patents
Zero-background high-flux directed cloning expression method Download PDFInfo
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Abstract
The present invention relates to a method for directed cloning and expression with zero background and high flux, which is a method based on the dual enzyme cleavage expression vector of a specific restriction enzyme, using a T4DNA pclymerase to digest a PCR product amplified by a primer specially designed and cloning the PCR product to an expression vector in a directed mode. The specific restriction enzyme can be any two (homogeneous or heterogeneous) of CpoI, Not, EarI, SapI and StyI. The method can be compatible with all the reported methods based on PCR library construction and can realize directed cloning with zero background and high flux. The method has the advantages of short experimental period and low cost; the method is suitable for high-flux cloning and expression for bacteria, fungi, animal, plants, etc. as parasitifers.
Description
Technical field
What the present invention relates to is the method for biological gene clonal expression technology, particularly a kind of Zero-background high-flux directed cloning expression.Be particularly suitable for utilizing the direct construction expression of PCR product library.
Background information
Gene order-checking provides a large amount of new genes, press for the new technology of development and study gene and proteinic function on a large scale, thereby a lot of company all is devoted to develop high-throughput clonal expression technology.
At present existing several companies have released high-throughout clonal expression system, as the Echo of Invitrogen
TMThe Gateway of system, Gibco/Life Technologies
TMSystem, the cloning system of Novagen pTriEx-1, the technological line that these several systems adopt is similar, after being the product of clone PCR, homologous recombination construction expression vector with Cre enzyme mediation LoxP site, do not need through restriction enzyme mediation carrying out subclone, can the multiple expression vector of high flux construction.This has improved conventional efficient to a certain extent, has reduced experimental cost.But all there is deficiency in this several method, mainly shows following three aspects:
One, experimental period long, cloning efficiency is low, can not zero background, directed cloning.What above high-throughput clonal expression system carried out that the first step of gene clone adopts is conventional pcr amplification, the method that the ligase enzyme mediation connects.In order to improve joint efficiency, often introduce lethal gene in the carrier and eliminate carrier from connecting background.With T carrier cloning PCR product is an efficient and simple method, but utilize the T carrier, can not use the archaeal dna polymerase amplifying target genes of high-fidelity, because have only to lack 3 '---5 ' archaeal dna polymerase of proofreading and correct enzymic activity could add a base A at pcr amplification product 3 ' end, with the T complementation that T carrier 3 ' distal process goes out, caused PCR to mix wrong increasing like this.In addition, goal gene can not directed cloning to expression vector, so these methods all need just can obtain correct clone through complicated screening.The cloning process that connected fast in five minutes based on topoisomerase I (Topoisomerase I) mediation of Invitrogen company is considered to present state-of-the-art cloning process, but it does not also solve clone's fidelity and directional problems.After the goal gene of pcr amplification is cloned into the carrier of topoisomeraseization, need to find out the clone that gene inserts with correct direction through PCR, random choose is several through the sudden change of sequencing analysis to determine that clone gene inside has or not PCR to introduce from these correct clones then; As everyone knows, lack 3 '--5 ' archaeal dna polymerase of proofreading and correct enzymic activity be 0.1% in every error rate of taking turns in the pcr amplification process (and the fidelity of the archaeal dna polymerase of high-fidelity (as Pfu) be lack 3 '---5 ' proofread and correct more than 10 times of archaeal dna polymerase of enzymic activity), thereby sequencing analysis is inevitable, correct from amplifying target genes like this to filtering out direction of insertion, the correct clone of frame needs three days time at least, in addition, because the base sequence of topoisomerase identification is CCCTT, and it is catalytic to be a reversible reaction, so if cloned sequence is bigger, clone's efficient can reduce greatly.When people such as John AHeyman utilized the extensive clone of this method and expressed 6035 open reading frame of cereuisiae fermentum in 1999, have only 75% open reading frame to obtain inserting the clone with correct direction, wherein have only 41% to be cloned in successful expression in the yeast, thereby the success ratio of whole experiment finally have only 30%.
Two, the method for the homologous recombination construction expression vector in Cre enzyme mediation loxP site, can be widely used in making up with bacterium, fungi, animal, plant etc. is host's expression vector, be a high-throughout method, but it also has defective:
1, mainly be fit to amalgamation and expression, the general C end that merges in the loxP site of goal gene, the target protein N end of expression can have more more than ten amino acid, must have certain difference on the conformation of this and natural protein and the function.
2, the loxP site is an inverted repeats, can form a loop-stem structure, reduces the efficient of translation, is difficult to adapt to non-fusion expression.
3, be difficult to parallel structure prokaryotic expression carrier and the non-fusion vector of eukaryotic expression, introduce the loxP site in the initiation codon upstream, certainly will thirties extra Nucleotide to be introduced, the required distance of protokaryon SD sequence and the coherence request of eucaryon Kozak sequence can not be satisfied simultaneously.
Three, experimental cost is higher, and is also uneconomical concerning large-scale research.The carrier costliness of the topoisomeraseization that gene clone is used (19 dollars/reaction), the Cre enzyme of mediation homologous recombination also more expensive (10 dollars/reaction) is identified clone's direction and is selected correct open reading frame all to increase research cost greatly by sequencing analysis with PCR.
(referring to publication " 1999 the 9th volumes of Genome Research 383-392 page or leaf ";
" Protein Expression and Purification calendar year 2001 the 22nd volume 159--164 page or leaf ")
Summary of the invention
The technology that the purpose of this invention is to provide a kind of biological gene cloning by expression really can be accomplished zero background, and high-flux directed clone, and can make up prokaryotic expression carrier and eukaryotic expression carrying agent simultaneously can amalgamation and expression, also can non-fusion expression.
The present invention realizes like this.Target call by the clone is selected an expression vector and target gene.Two particular restriction endonuclease digestion site sequences and a lethal gene expression unit in expression vector, have been introduced; Introduced 3----5 specific base respectively at 5 of target gene one couple of PCR amplimer ' end, under the condition that dATP (or dGTP or dCTP or dTTP) exists, amplified production is through T
4The sticky end that archaeal dna polymerase digestion back produces mates with the sticky end that the carrier double digestion obtains, thus can directed cloning on the carrier of two special restriction enzymes double zyme cuttings.
Specific practice is to select an expression vector and target gene.At first design the joint of polynucleotide on expression vector, its two ends are introduced a Cpo I and Not I restriction enzyme site respectively, and the expression unit of a lethal gene of indirect is introduced this joint on the expression vector.Carrier produces such two sticky ends through Cpo I and Not I double digestion:
3′ GC——————CGCCGG?5′
5′GTCCG——————GC 3′；
5 ' end of the one couple of PCR primers of amplification target gene is introduced 5 ' GACT3 ' and 5 ' GGCCT3 ' 4--5 Nucleotide so respectively, and the product that pcr amplification obtains adds dATP and T
4The archaeal dna polymerase effect produces such sticky end:
5′GACT——————A 3′
3′ A——————TCCGG?5′；
This sticky end just in time can with the carrier double digestion produce the sticky end coupling, thereby make PCR product directed cloning to carrier.
The restriction enzyme at polynucleotide joint two ends can be any two kinds or Cpo I, Ear I, among Sap I, the Sty I any among Cpo I, Ear I, Not I, Sap I, the Sty I.That any enzyme is cut the back is getable 5 ' and restriction enzyme that the base kind of outstanding sticky end is no more than 3 kinds (containing 3 kinds) all can use.
The present invention compared with prior art has remarkable advantages:
One, can accomplish zero background, high-flux directed clone.At first PCR primer and cloning vector have all passed through special design, and every one couple of PCR primers all can be at the extra individual specific nucleic acid of 2--5 of introducing of its 5 ' end, and the PCR product that obtains is through T
4After archaeal dna polymerase is handled, can produce can not mate mutually sticky end, and joint of introducing on the carrier, just can mate with the sticky end of the PCR product of handling with the sticky end that produces behind the special digestion with restriction enzyme, make external source fragment directed cloning to carrier, need not judge the direction of insertion of gene again, simplify experimental procedure.Because all pcr amplification and vector constructions all can adopt this thinking, thereby this method is applicable to clone's all archaeal dna polymerase amplification PCR products at present, especially be fit to carry out pcr amplification with the best polysaccharase of present fidelity (Pfu), minimum is reduced in the sudden change that PCR introduces, can save the trouble of order-checking.From the background interference that connects, on carrier, also introduce the expression unit of a lethal gene for fear of carrier, really accomplished zero background clone.Can carry out connecting fast in 5 minutes, transform fast in 30 minutes, improve conventional efficient greatly, whole gene clone is operated in one day and can finishes, and clone's recombination efficiency can reach more than 99% theoretically.
Two, to build the method in storehouse compatible with present all PCR-based of report, can utilize the direct construction expression of PCR product library, both be suitable for cloning and expressing as host's high-throughput, also be applicable to high-throughput clone and the expression as the host such as animal, plant with bacterium, fungi.
Three, experimental cost is low.At first this technology has been saved through pcr amplification and has been judged the direction of insertion of gene and this two step of exactness of sequencing analysis gene, has significantly reduced experimental expenses; Secondly do not use the carrier of Cre enzyme and topoisomeraseization, only use conventional T
4Archaeal dna polymerase and ligase enzyme, the cost of each reaction are very economical methods about 1 yuan of Renminbi.If carry out high-throughout clone and expression, its superiority is more outstanding.
Embodiment
The present invention is further described with embodiment below:
Embodiment 1:
Utilize the oligomerization-1 of this method, the 6-glucuroide the expression in escherichia coli subtilis.
Select expression vector pBV220 and target gene oligomerization-1, the 6-glucuroide.The joint of polynucleotide of design, its two ends are introduced a Cpo I and Not I restriction enzyme site respectively, the expression unit of a lethal gene of indirect, this joint is introduced on the coli expression carrier pBV220, promptly obtain Zero-background high-flux directed clone's expression vector, this carrier with Cpo I and Not I double digestion, is reclaimed big fragment, and its two ends respectively have a 5 ' distal process to go out the sticky end of 3 or 4 bases.According to subtilis oligomerization-1, the gene order of 6-glucuroide designs a pair of primer, and its 5 ' end has been introduced these several extra Nucleotide of 5 ' GACT3 ', 5 ' GGCCT3 ' respectively; Genomic dna with subtilis is a template, and (Pfu) carries out pcr amplification with high-fidelity DNA polymerase, and the product that obtains is under there is situation in dATP, through T
4After archaeal dna polymerase is handled, its PCR product two ends respectively have a 5 ' distal process to go out the sticky end of 3 or 4 bases, this product energy directed cloning is on the carrier of Cpo I, Not I double digestion, transformed into escherichia coli again, the transformant that obtains all can correctly be expressed oligomerization-1,6-glucuroide more than 99%.
Embodiment 2:
The expression of aspergillus niger endoinulase in pichia yeast.
Design a same joint by embodiment 1 method, introduce on the pichia yeast expression vector pIC3.5K, the primer of design amplification aspergillus niger endoinulase gene is used Pfu polymeric enzymatic amplification aspergillus niger endoinulase gene in the same way, amplified production is used T equally under the condition that d ATP exists
4Archaeal dna polymerase is handled the back directed cloning on the carrier of Cpo I, Not I double digestion, transforms pichia yeast, and the transformant that obtains all can correctly be expressed endoinulase more than 90%.
Claims (5)
1, the method for a kind of zero background, high-throughput, directed cloning expression, mainly comprise an expression vector and through the goal gene of PCR primer amplification, it is characterized in that in expression vector, having introduced two particular restriction endonuclease digestion site sequences and a lethal gene is expressed the unit; 5 ' end in amplification target gene one couple of PCR primers has been introduced 2～5 specific deoxynucleotides respectively, and under the condition of dATP or dGTP or dCTP or dTTP existence, amplified production is through T
4The sticky end that archaeal dna polymerase digestion back produces mates with the sticky end that the carrier double digestion obtains, thus can directed cloning on the carrier of two special restriction enzymes double zyme cuttings.
2, the method for zero background according to claim 1, high-throughput, directed cloning expression, it is characterized in that on expression vector, at first designing the joint of polynucleotide, its two ends are introduced a CpoI and NotI restriction enzyme site respectively, the expression unit of a lethal gene of indirect, this joint is introduced on the expression vector, carrier produces such two sticky ends through CpoI and NotI double digestion:
3′GC——————CGCCGG 5′
5′GTCCG——————GC 3′；
5 of goal gene one couple of PCR primers ' end is introduced 5 ' GACT3 ' and 5 ' GGCCT3 ' 3--4 Nucleotide so respectively, and the product that pcr amplification obtains adds dATP and T
4The archaeal dna polymerase effect produces such sticky end:
5′GACT————A 3′
3′A——————TCCGG?5′；
This sticky end just in time can with the carrier double digestion produce the sticky end coupling, thereby make PCR product directed cloning to carrier.
3, the method for zero background according to claim 1, high-throughput, directed cloning expression, two special restriction enzymes that it is characterized in that polynucleotide joint two ends, can be enzyme cut the back getable 5 ' the base kind of outstanding sticky end is no more than 3 kinds restriction enzyme, concrete restriction enzyme can be any two kinds or CpoI, EarI, among SapI, the StyI any among CpoI, EarI, NotI, SapI, the StyI.
4, the method expressed of zero background according to claim 1, high-throughput, directed cloning, it is characterized in that can to build the method in storehouse compatible with all PCR-based.
5, the method expressed of zero background according to claim 1, high-throughput, directed cloning is characterized in that can be used for high-throughout clone and the expression as the host of bacterium, fungi, animal, plant.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011335181A CN1165622C (en) | 2001-09-30 | 2001-09-30 | Zero-background high-flux directed cloning expression method |
| PCT/CN2002/000686 WO2003029472A1 (en) | 2001-09-30 | 2002-09-27 | A method for directional expression cloning with zero background and high throughput |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011335181A CN1165622C (en) | 2001-09-30 | 2001-09-30 | Zero-background high-flux directed cloning expression method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1341748A CN1341748A (en) | 2002-03-27 |
| CN1165622C true CN1165622C (en) | 2004-09-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011335181A Expired - Fee Related CN1165622C (en) | 2001-09-30 | 2001-09-30 | Zero-background high-flux directed cloning expression method |
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| Country | Link |
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| CN (1) | CN1165622C (en) |
| WO (1) | WO2003029472A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307321B (en) * | 2008-06-12 | 2010-12-22 | 湖北大学 | High throughput directional T carrier cloning process |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5691140A (en) * | 1995-05-18 | 1997-11-25 | New England Biolabs, Inc. | Bidirectional in vitro transcription vectors utilizing a single RNA polymerase for both directions |
| US6184000B1 (en) * | 1999-07-23 | 2001-02-06 | The United States Of America As Represented By The Secretary Of Agriculture | System for the sequential, directional cloning of multiple DNA sequences |
-
2001
- 2001-09-30 CN CNB011335181A patent/CN1165622C/en not_active Expired - Fee Related
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2002
- 2002-09-27 WO PCT/CN2002/000686 patent/WO2003029472A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| CN1341748A (en) | 2002-03-27 |
| WO2003029472A1 (en) | 2003-04-10 |
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