Preparation method and application of low-temperature bacterium DNA ligase

A DNA ligase, low-temperature technology, applied in the field of genetic engineering, can solve the problems of low DNA ligation efficiency and limit the speed of gene cloning, and achieve the effect of high gene cloning efficiency and shortening time.

Inactive Publication Date: 2017-05-31
SUZHOU KUANGSHI JUNCHI BIOLOGICAL SCI & TECH
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Problems solved by technology

The optimal temperature of T4 phage DNA ligase is 30-40 degrees, which does not match the DNA ligation reaction temperature of gene cloning, resulting in low efficiency of DNA ligation, requiring long-term reaction, and the ligation time is as long as 12 hours, which greatly limits the speed of cloning

Method used

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  • Preparation method and application of low-temperature bacterium DNA ligase
  • Preparation method and application of low-temperature bacterium DNA ligase
  • Preparation method and application of low-temperature bacterium DNA ligase

Examples

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Effect test

Embodiment 1

[0029] Example 1 Preparation of DNA Ligase from Psychrophilic Bacteria Colwellia psychrerythraea

[0030] The first step is to design and synthesize the DNA ligase gene of psychrophilic bacteria Colwellia psychrerythraea, and insert it into the pET28 expression vector to construct the recombinant expression plasmid of DNA ligase. The N-terminal of recombinant psychrophilic bacteria Colwelliapsychrerythraea DNA ligase has 6 consecutive histidine affinity purification tags derived from pET28 vector, which is used for immobilized nickel ion affinity chromatography purification.

[0031] In the second step, DNA ligase of psychrophilic bacterium Colwellia psychrerythraea was recombinantly expressed. The psychrophilic bacterium Colwellia psychrerythraea DNA ligase recombinant expression plasmid was transformed into Escherichia coli expression host BL21 (DE3), and the psychrophilic bacterium Colwellia psychrerythraea DNA ligase recombinant expression strain was obtained. The express...

Embodiment 2

[0036] Example 2 Preparation of psychrophilic bacteria Photobacterium profundum DNA ligase

[0037]The first step is to design and synthesize the DNA ligase gene of the psychrophilic bacterium Photobacterium profundum, and insert it into the pET28 expression vector to construct the recombinant expression plasmid of the DNA ligase. The N-terminus of recombinant psychrophilic bacterium Photobacterium profundum DNA ligase has 6 consecutive histidine affinity purification tags derived from pET28 vector, which is used for immobilized nickel ion affinity chromatography purification.

[0038] In the second step, DNA ligase of Photobacterium profundum was recombinantly expressed. The psychrophilic bacterium Photobacterium profundum DNA ligase recombinant expression plasmid was transformed into Escherichia coli expression host BL21 (DE3), and the psychrophilic bacterium Photobacterium profundum DNA ligase recombinant expression strain was obtained. The expression strain was cultivated...

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Abstract

The invention discloses a preparation method and application of low-temperature bacterium DNA ligase. The prepared low-temperature bacterium DNA ligase has the advantages that the optimal temperature range of the enzyme activity of the low-temperature bacterium DNA ligase is 4-20 DEG C, ligation products of the DNA ligase which has reaction for 5 minutes in the temperature range are larger than 90%, and the DNA ligase is quite suitable for the low-temperature DNA ligation reaction in gene cloning; due to the fact that the DNA ligation reaction of the gene cloning is low in temperature, the optimal temperature of traditional T4 phage DNA ligase is 30-40 DEG C and the optimal temperature of the traditional T4 phage DNA ligase is not matched with the temperature of the DNA ligation reaction of the gene cloning, DNA ligation efficiency is low, long-time reaction is needed, ligation time reaches up to 12 hours, and gene cloning speed is limited greatly; the DNA ligase has the highest catalytic activity under low temperature, the DNA ligation reaction time is shortened greatly, and gene cloning speed is increased.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method and application of a low-temperature bacterial DNA ligase. Background technique [0002] The emergence of restriction endonuclease and DNA ligase has promoted the in vitro recombination technology of DNA, that is, gene cloning technology. Gene cloning technology is the basis of modern molecular biology and genetic engineering, which greatly promotes the application of protein recombinant expression and engineering strain transformation. The DNA ligase currently used in gene cloning is T4 phage DNA ligase. The DNA ligation reaction temperature of gene cloning is carried out in the range of 4-16 degrees. The optimal temperature of T4 phage DNA ligase is 30-40 degrees, which does not match the DNA ligation reaction temperature of gene cloning, resulting in low efficiency of DNA ligation, requiring long-term reaction, and the ligation ti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/52C12N9/00
CPCC12N9/93C12N15/70C12N2800/101C12N2800/22C12Y605/01001
Inventor 刘喜朋
Owner SUZHOU KUANGSHI JUNCHI BIOLOGICAL SCI & TECH
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