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Heat-resistant nucleic acid degrading enzyme expression vector, construction method and application thereof

A nucleic acid degradation and construction method technology, applied in the field of genetic engineering, can solve problems such as unfavorable preservation and application, limited application scope, heat resistance, etc., and achieve the effects of excellent thermal stability, improved removal efficiency, and improved adaptability

Active Publication Date: 2022-06-24
河南省儿童医院郑州儿童医院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the DNase I enzyme needs to be stored below -20°C, and it is not heat-resistant. It is easy to inactivate at room temperature, and it will be quickly inactivated by heating at 65°C for 10 minutes, which is not conducive to the preservation and application of the enzyme, which greatly limits its practical application. Application range

Method used

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  • Heat-resistant nucleic acid degrading enzyme expression vector, construction method and application thereof
  • Heat-resistant nucleic acid degrading enzyme expression vector, construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] The thermostable nucleic acid degrading enzyme expression vector of this embodiment is constructed by introducing a recombinant DNase I enzyme gene into a Saccharomyces cerevisiae vector, and the nucleotide sequence of the recombinant DNase I enzyme gene is shown in SEQ ID No.1.

Embodiment 2

[0033] The construction method of the thermostable nucleic acid degrading enzyme expression vector of the present embodiment describes the construction of the thermostable nucleic acid degrading enzyme expression vector in Example 1, including the following steps:

[0034] 1) Obtain the complete nucleotide sequence of the DNase I enzyme gene, and then remove the nucleotides at the 1-66bp site (the complete bovine pancreas DNase I enzyme gene sequence is shown in SEQ ID No.3, and the GenBank accession number is M60606. 1);

[0035] 2) Obtain the tag gene with a nucleotide region of 2127-2495bp in the Taq DNA polymerase (the nucleotide sequence of the tag gene is shown in SEQ ID No.2), and then use the tag gene tandem step 1) to remove the 1-66bp site The DNase I enzyme gene sequence obtained after the nucleotide sequence was combined with the codon preference of Saccharomyces cerevisiae, and the codon optimization was carried out using DNAWORKS tools to improve translation effi...

Embodiment 3

[0038] The application of the expression vector of the thermostable nucleic acid degrading enzyme in this embodiment, specifically, the application of the expression vector in the preparation of the thermostable nucleic acid degrading enzyme for the removal of nucleic acid aerosol pollutants.

[0039]During specific application: the method for obtaining the recombinant DNase I enzyme protein by using the heat-resistant nucleic acid degrading enzyme expression vector is: amplifying the recombinant DNase I enzyme gene expression vector with homologous recombination arm by PCR as a repair template, and recombining the recombinant DNase I enzyme gene The expression vector is inserted into the multi-copy Ty2 retrotransposon of Saccharomyces cerevisiae by means of homologous recombination to realize multi-copy gene expression. The repair template and pCas-ty2 plasmid were co-transformed into Saccharomyces cerevisiae, and URA-deficient plates were used for clone screening. Screen out...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a heat-resistant nucleic acid degrading enzyme expression vector as well as a construction method and application thereof. The expression vector is constructed by introducing a recombinant DNase I enzyme gene on a saccharomyces cerevisiae vector, and the nucleotide sequence of the recombinant DNase I enzyme gene is as shown in SEQ ID No. 1. The recombinant DNase I enzyme gene in the expression vector disclosed by the invention is the recombinant DNase I enzyme gene which is cloned for the first time and has a heat-resistant property, and the length of an open reading frame sequence is 1119bp. An enzyme heat resistance analysis result shows that an expression vector containing the recombinant gene can effectively improve the heat stability and environmental adaptability of DNase I enzyme, meanwhile, is beneficial to improving the activity of the recombinant DNase I enzyme and enhancing the removal efficiency of nucleic acid pollution, and effectively solves the problems of storage, transportation and use of enzyme for removing nucleic acid aerosol pollution in a PCR (Polymerase Chain Reaction) laboratory.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a heat-resistant nucleic acid degrading enzyme expression vector, a construction method and an application thereof. Background technique [0002] With the surge in market demand for novel coronavirus testing, PCR technology has been popularized and promoted on a large scale, and a large number of PCR testing laboratories have been successively established across the country. However, in the daily operation of the laboratory, the problem of nucleic acid aerosol contamination is often encountered. Nucleic acid aerosol pollution, that is, DNA / RNA aerosol pollution, is a kind of air pollution that easily occurs in the laboratory. Under normal circumstances, the surface friction between air and liquid, centrifuge centrifugation, violent shaking of reaction tubes, PCR cap opening, pipette repeated sampling, pollutant leakage, etc. will generate nucleic acid aero...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/64C12N9/22B01D53/84C12R1/865
CPCC12N15/81C12N15/64C12N9/22C12Y301/21001B01D53/84C12N2800/22C07K2319/35Y02A50/20
Inventor 岳凌清张德鹏于恩琪刘云龙
Owner 河南省儿童医院郑州儿童医院