WO2003091440A1 - Methode de construction d'un vecteur d'expression lineaire sans clonage - Google Patents

Methode de construction d'un vecteur d'expression lineaire sans clonage Download PDF

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Publication number
WO2003091440A1
WO2003091440A1 PCT/CN2002/000291 CN0200291W WO03091440A1 WO 2003091440 A1 WO2003091440 A1 WO 2003091440A1 CN 0200291 W CN0200291 W CN 0200291W WO 03091440 A1 WO03091440 A1 WO 03091440A1
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WIPO (PCT)
Prior art keywords
expression vector
linear expression
primer
constructing
fragment
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PCT/CN2002/000291
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English (en)
Chinese (zh)
Inventor
Dawei Huang
Wen Xin
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Institute Of Zoology, Chinese Academy Of Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Institute Of Zoology, Chinese Academy Of Sciences filed Critical Institute Of Zoology, Chinese Academy Of Sciences
Priority to AU2002257486A priority Critical patent/AU2002257486A1/en
Priority to PCT/CN2002/000291 priority patent/WO2003091440A1/fr
Publication of WO2003091440A1 publication Critical patent/WO2003091440A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Definitions

  • the present invention relates to a method for constructing an expression vector, and in particular, to a method for constructing a linear expression vector without cloning. Background technique
  • the protein expression method is to link the target gene to be cloned with the vector, transform the recombinant into the recipient bacteria, select and identify the correct clone for amplification, and then transfer it to the host cell for expression.
  • This is the most widely used method in protein expression research. However, this method involves many steps and is tedious and time-consuming.
  • the vector contains a promoter, a target gene and a terminator fragment (Invitrogen Company TOP0 Tools Product Introduction);
  • the recognition site of I is the CCCTT 'site; in the design of the primer, the recognition site of the enzyme was added to the reverse primer of the promoter fragment, the primers at both ends of the target gene and the forward primer sequence of the terminator fragment, After DNA fragment amplification, DNA topology
  • the resulting promoter fragment is: ,,--GGGAAGCCTTG, the target gene fragment in the middle is The terminal terminator fragment is:
  • the vector obtained by this method can directly express the target protein by transfecting the recipient cell, and does not need to go through the tedious steps of cloning identification and amplification of transformants; however, when the DNA fragment to be ligated contains DNA topoisomerase I This method cannot be applied when the recognition site CCCTT 'is used, so using this method to construct a linear expression vector has certain limitations.
  • the purpose of the present invention is to provide a method for constructing a linear expression vector without cloning, so as to overcome the shortcomings of the above-mentioned method for constructing a linear expression vector by DNA topoisomerase I.
  • a (DNA + RNA) hybrid is used as a primer to amplify a gene fragment, and RNA is digested through subsequent steps to achieve the connection between the fragments, thereby achieving the purpose of constructing a linear expression vector.
  • RNA is digested through subsequent steps to achieve the connection between the fragments, thereby achieving the purpose of constructing a linear expression vector.
  • the invention provides a method for constructing a linear expression vector.
  • the steps include:
  • Primer 1 and primer 2 contain a DNA sequence and an RNA sequence complementary to the to-be-ligated fragment, and RNA sequence complementary pairing;
  • the promoter forward primer and the terminator reverse primer include only the DNA sequence complementary to the amplified fragment
  • the method for constructing a linear expression vector provided by the present invention includes purifying the digested fragments to be ligated; after step 4), including PCR amplification of the obtained linear expression vector to increase the linear expression vector.
  • the amount also includes purification after step 3), and after step 4), the obtained linear expression vector is subjected to PCR amplification.
  • RNA fragment was digested with ribonuclease H in step 3) of each of the above methods; step 4) the ligase used in the ligation reaction was T4 DNA ligase; ribonucleotides contained in the RNA sequence of the primer in step 1) of each of the above methods
  • the number is 2-10; the primer design is preferably an RNA sequence consisting of 3-6 ribonucleotides.
  • the method for constructing a linear expression vector provided by the present invention has the following characteristics: It has a wide range of applications and can be applied regardless of the DNA sequence contained in the target gene. It is a new method for constructing a linear expression vector. Brief description of the drawings
  • Figure 1 is a schematic diagram of the construction of a linear expression vector using the method of the present invention
  • A is the promoter fragment
  • B is the target gene fragment
  • C is a terminator fragment
  • D is a linear expression vector fragment
  • a J is the amplification product of A, where the box is the RNA fragment (the same below)
  • B ' is the amplification product of B
  • C' is the amplification product of C
  • Taq enzyme DNA polymerase
  • Huamei Bioengineering Company Huamei Bioengineering Company
  • plasmid pCMV / myc / cyto / GFP, pEF / myc / Lan was purchased from Invitrogen Company (USA)
  • T4DNA ligase (3unit / w 1) was purchased from Promega Company.
  • US Huamei Bioengineering Company
  • plasmid pCMV / myc / cyto / GFP pEF / myc / Lan was purchased from Invitrogen Company (USA)
  • T4DNA ligase (3unit / w 1) was purchased from Promega Company.
  • US RNaseH (60uni t / ⁇ 1) was purchased from Takara (Japan)
  • primers were synthesized by TAGC (Denmark)
  • QIAGENE Germany
  • Construction of a linear expression vector containing CMV (cytomegalovirus) promoter fragment A, GFP (green fluorescent protein) fragment B, BGHpA (polyadenylic acid) fragment (including terminator) C Using the plasmid pCMV / myc / cyto / GFP as a template, the CMV (cytomegalovirus) promoter fragment A ', GFP (green fluorescent protein) fragment B', BGHpA (polyadenylation) fragment (including termination D) C ′, which is ligated after enzymatic hydrolysis to obtain a linear expression vector D containing the above fragments.
  • the construction process is shown in FIG. 1:
  • primer 1 is the reverse primer of the promoter CMV fragment
  • primer 2 is the forward primer of the target gene fragment GFP.
  • They both include a DNA sequence and an RNA sequence, where the DNA sequence is complementary to the fragment to be amplified, the RNA sequence is shown in the box (the same below), and the RNA sequences of primer 1 and primer 2 are complementary paired.
  • Primer ⁇ is the GFP reverse primer of the target gene fragment
  • primer 2' is the forward primer of terminator BGHpA. They all include a DNA sequence and an RNA sequence, where the DNA sequence is complementary to the fragment to be amplified, the RNA sequence is shown in the box in the figure, and the RNA sequences of primer 1 'and primer 2' are complementary paired.
  • GFP forward primer (2) 5 CUGGAGatggctagcaaaggagaagaact3 '
  • the uppercase letter indicates the RNA sequence containing 6 ribonucleotides
  • PCR buffer 500mM KCl. 10mM Tris-HCl, (pH 9.0), the same below.
  • the above mixture was initially denatured at 94 ° C for 5 'minutes, followed by 30 cycles at 94 ⁇ 30 seconds, 6CTC for 30 seconds, 72 ⁇ 1 minute, and finally extended at 72 ° C for 10 minutes.
  • a promoter amplification product A ', a target gene fragment amplification product B, and a terminator amplification product C' are obtained.
  • the RNaseH was used to digest the PCR products A,, B,, C, respectively.
  • the reaction conditions were as follows: PCR product 2 g, RNaseH 0.5 ⁇ 1 (60 ⁇ m / ⁇ 1), and reacted at 37 ° C for 30 minutes. After digestion, a promoter to be ligated A ", a target gene to be ligated B", and a terminator to be ligated C "are obtained.
  • the UV spectrophotometer was used to determine the value of 0D260.
  • the above mixture was initially denatured at 94 ° C for 5 minutes, followed by 30 cycles at 94 ⁇ 30 seconds, 6CTC for 30 seconds, and 72 ⁇ 1 minute, and finally extended at 72 ° C for 10 minutes to obtain the amplified linear expression vector D.
  • the purification step (4) of the enzymatic hydrolysis product is not necessary, but the degraded ribonucleotide fragments in the solution and the impurity components in the original system are removed after purification, making the ligation reaction easier.
  • Example 2 Construction of a linear expression vector containing CMV (cytomegalovirus) promoter fragment A, GFP (green fluorescent protein) fragment B, BGHpA (polyadenylic acid) fragment (including terminator) C:
  • RNA sequence in the primer contains 4 ribonucleotides, and a linear expression vector can be obtained without a second PCR amplification.
  • the construction process is shown in Figure 1: (1) Design primers
  • primers 1, 2, ⁇ , V, and primers 3, 4 are the same as in Example 1.
  • the primer sequences are shown in the table below.
  • Example 2 The method is the same as in Example 1. After enzymolysis, the value of 0D260 was measured with a UV spectrophotometer.
  • a "500ng, B” 578.75 ng, C "275 ng (ie, molar ratio 1: 1: 1) use DNA ligation kit (Takara) to ligate at room temperature, first add equal volume solution II and mix, then add 2 times The volume of solution I was mixed for 5-10 minutes (Solution I and Solution II are solutions in the kit), and a linear expression vector was obtained, which can be directly used to transfect recipient cells.
  • Example 3 Construction of EF-1 ⁇ Linear expression vectors of promoter fragment A, GFP fragment B, BGHpA fragment (including terminator) C: The plasmid pEF / myc / nuc was used as a template to obtain the promoter EF-1 ⁇ sequence, and the plasmid pCMV / myc / cyto / GFP was used as a template to obtain the GFP fragment and the BGHpA fragment, respectively. The products A ", B", and C "were used in the ligation reaction without purification. The construction process is shown in Figure 1.
  • primers 1, 2, primers 2 ', and primers 3, 4 are the same as in Example 1.
  • the primer sequences are shown in the table below.
  • the linear expression vector constructed by this method has a wide range of applications and is not affected by the target DNA fragment. The effect of sequence changes.
  • the constructed linear expression vector can be directly used to transform host cells to express the target protein, and can provide a technical platform for gene expression regulation, antibody and vaccine production, and protein function research.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
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  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un méthode de construction d'un vecteur d'expression linéaire. En particulier, la présente invention concerne une méthode de construction d'un vecteur d'expression linéaire consistant: à amplifier des fragments de gênes à l'aide d'un hybride (ADN + ARN) en tant qu'amorce, et à lier chaque fragment par une étape ultérieure de digestion d'ARN. La présente invention n'est pas affectée par le changement de séquence du fragment d'ADN cible. Le vecteur d'expression linéaire construit tel que ci-dessus peut être utilisé pour transformer des cellules hôtes afin d'exprimer une protéine cible, et pour obtenir une plate-forme technique destinée aux domaines de l'expression de gênes, de la production d'anticorps et de vaccin ainsi que l'investigation de la fonction protéinique.
PCT/CN2002/000291 2002-04-25 2002-04-25 Methode de construction d'un vecteur d'expression lineaire sans clonage WO2003091440A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002257486A AU2002257486A1 (en) 2002-04-25 2002-04-25 Method for constructing a linear expression vector without cloning
PCT/CN2002/000291 WO2003091440A1 (fr) 2002-04-25 2002-04-25 Methode de construction d'un vecteur d'expression lineaire sans clonage

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011027808A1 (fr) * 2009-09-04 2011-03-10 国立大学法人富山大学 Méthode spécifique pour la préparation de fragments d'adn assemblés comprenant des séquences dérivées de gènes cibles

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006189A1 (fr) * 1990-09-27 1992-04-16 Invitrogen Corporation Clonage direct d'acides nucleiques amplifies par reaction en chaine de la polymerase
WO1999021977A1 (fr) * 1997-10-24 1999-05-06 Life Technologies, Inc. Clonage recombinatoire au moyen d'acides nucleiques possedant des sites de recombinaison

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006189A1 (fr) * 1990-09-27 1992-04-16 Invitrogen Corporation Clonage direct d'acides nucleiques amplifies par reaction en chaine de la polymerase
WO1999021977A1 (fr) * 1997-10-24 1999-05-06 Life Technologies, Inc. Clonage recombinatoire au moyen d'acides nucleiques possedant des sites de recombinaison

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011027808A1 (fr) * 2009-09-04 2011-03-10 国立大学法人富山大学 Méthode spécifique pour la préparation de fragments d'adn assemblés comprenant des séquences dérivées de gènes cibles
CN102597257A (zh) * 2009-09-04 2012-07-18 国立大学法人富山大学 包含来自靶基因的序列的连接dna片段的特异性制作方法
US8999673B2 (en) 2009-09-04 2015-04-07 National University Corporation University Of Toyama Method for specifically producing a joined DNA fragment comprising a sequence derived from a target gene
CN102597257B (zh) * 2009-09-04 2015-09-09 国立大学法人富山大学 包含来自靶基因的序列的连接dna片段的特异性制作方法
JP5779502B2 (ja) * 2009-09-04 2015-09-16 国立大学法人富山大学 標的遺伝子由来配列を含む連結ｄｎａ断片の特異的作製方法

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