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Plasmid vector and construction method thereof

A technology of plasmid vector and construction method, which is applied in the field of genetic engineering, can solve problems such as inability to orientate the cloning process, false positives outside the clone, deletion, etc., and achieve the effect of convenient protein purification

Active Publication Date: 2017-10-24
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

In addition, when the second method prepares T vectors, there will be problems such as low tailing efficiency and the sequences at both ends of the linear plasmid are sometimes partially deleted during the tailing process (Shuman S. Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J BiolChem. 1994 Dec 23;269(51):32678-32684.)
At present, the main problem of TA cloning is that fragments can be randomly inserted into the T vector in both positive and negative directions during the cloning process, which causes at least half of the false positives outside the clone, thus increasing the pressure of screening.
As a simple cloning method, TA cloning has the problem that the cloning process cannot be oriented (that is, only half of the cloned inserts and vectors are connected in the same way as expected)
In addition, commonly used T vectors cannot have both cloning and expression functions

Method used

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  • Plasmid vector and construction method thereof
  • Plasmid vector and construction method thereof
  • Plasmid vector and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1: the acquisition of plasmid vector pANY2

[0046] 1) Using plasmid pET3a as a template, PHis-for1 and PHis-rev1 as upstream and downstream primers for PCR amplification, the amplified product is named Fragment 1, to be used; PHis-for1: 5'- AAAAC TGCAG CACCA CCACC ACCAC CACTA AGGCTGCTAA CAAAG CCCGA AAGGA AGCTG-3' and PHis-rev1: 5'-GTAGT TTATC ACAGT TAAATTGCTA ACGCA GTCAG GGATA TCCGG ATATA GTTCC TCCTT TC-3'. The reaction system for PCR amplification is: 2 µl of 10 x Pfu buffer in a 20 µl reaction system, 10 µmol / L of primers, 2 U of Pfu enzyme, 0.2 mmol / LdNTPs, about 2 ng of template, supplemented with sterilized deionized water Qi; PCR amplification cycle program: 94°C pre-denaturation for 3 min, (94°C, 30 s; 63°C, 30 s; 72°C, 2 min) × 25, 72°C for 10 min final extension.

[0047] 2) Use plasmid pET9a as a template, PET9-for1 and PET9-rev1 as upstream and downstream primers for PCR amplification, and the amplified product is named fragment 2, to be used; PE...

Embodiment 2

[0059] Embodiment 2: the above-mentioned example obtains pANY2 and is used for the functional verification of directional TA clone

[0060] The process and principle of directional TA cloning using pANY2 are as follows: Figure 4 shown. The multiple cloning site region of plasmid pANY2 contains two AhdIs. After AhdI digestion, half of the recognition sequences of AvrII and NcoI are also generated at the end of the vector while introducing 3'-T; and the primer sequences for PCR amplification of insert fragments contains the other half of the recognition sequences of AvrII and NcoI. If the ligation direction is opposite to the expected one, new AvrII and NcoI restriction sites will be generated; and if the ligation direction is as expected, no new AvrII and NcoI restriction sites will be generated. In this way, reverse-linked clones can be eliminated by AvrII or NcoI digestion, thereby achieving the goal of directional TA cloning.

[0061] The specific cloning steps are as fo...

Embodiment 3

[0068] Embodiment 3: Obtaining pANY2 for the functional verification of background-free sticky-end cloning to the above-mentioned examples

[0069] The process and principle of background-free cohesive end cloning using pANY2 are as follows: Figure 7 -a shown. The multiple cloning site region of the plasmid pANY2 contains two BsaIs. After digestion with BsaIs, arbitrarily designed cohesive ends are introduced on both sides of the linear vector; the primer sequence for PCR amplification of the insert also contains the BsaI recognition site Point, can be arbitrarily designed cohesive ends can be generated by BsaI. Since the cohesive ends of the insert and the vector are perfectly complementary, the insert can be ligated into the vector by ligation. Adding BsaI enzyme for digestion during the ligation process can avoid contamination of the original plasmid. like Figure 7 As shown in -b, since the arbitrarily designed sticky end is not a palindromic structure, it can effecti...

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Abstract

The invention relates to specifically relates to a plasmid vector capable of realizing directional TA cloning, background sticky end-free cloning and expression, and a construction method thereof, belonging to the field of gene engineering. The plasmid vector is a closed circular double-stranded DNA and contains two multiple cloning sites, wherein a ccdB expression cassette exist between the two multiple cloning sites, and the ccdB expression cassette can realize constitutive expression of toxic protein ccdB, which is used as a negative selection marker, in most Escherichia coli strains. The multiple cloning sites are designed on the basis of the plasmid vector; and the plasmid vector is applicable to directional TA cloning and improved background sticky end-free cloning. Moreover, the plasmid vector is directly applicable to expression and purification of proteins. Each function of the plasmid vector is verified. The plasmid vector provided by the invention is expected to play an important role in the field of gene engineering due to its convenience and high efficiency.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a plasmid vector with the functions of directional TA cloning, background-free sticky end cloning and expression and a construction method thereof. Background technique [0002] Molecular cloning is the basis for studying gene structure and function, and is the core technology of molecular biology. Molecular cloning methods can be divided into two categories: ligase-dependent cloning and ligase-independent cloning. Among them, there are mainly three cloning methods relying on ligase, including: blunt end ligation, sticky end ligation and TA cloning. In recent years, various ligase-independent cloning strategies have also been developed rapidly, such as: CPEC, FastCloning, Gateway, LIC, OEC, PIPE, SLIC, SLiCE, USER and recombinase cloning technologies (Yao S, Hart DJ, An Y. Recent advances in universal TAcloning methods for use in function studies. Protein Eng Des Sel. 2016; 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/64
CPCC12N15/64C12N15/70
Inventor 安迎锋高和瑞高嵩张艺锋许淑敏刘霞
Owner SHENYANG AGRI UNIV
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