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Method for maintaining foreign gene in cell stably

A gene and host cell technology, applied in the field of stably maintaining foreign genes, can solve problems such as cell proliferation

Inactive Publication Date: 2010-09-29
JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, through trophic symbiosis, it is still possible for cells to proliferate after the disappearance of the recombinant vector

Method used

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  • Method for maintaining foreign gene in cell stably

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Effect test

preparation example Construction

[0105] As a procedure for preparing mutant host cells, in addition to the method of systematically deleting or inactivating a gene of interest existing on the host chromosome, it is also possible to give random gene deletion or inactivation mutations, followed by appropriate methods. A method for gene analysis or protein productivity evaluation.

[0106] To delete or inactivate a gene of interest, for example, homologous recombination can be used. That is, the DNA fragment containing a part of the target gene is cloned into an appropriate plasmid vector, the resulting circular recombinant plasmid or the DNA fragment on the linear chain is integrated into the host cell, and the parent is truncated by homologous recombination of a part of the target gene The target gene on the microbial genome is inactivated. Alternatively, a method such as PCR is used to construct a target gene introduced with an inactivating mutation caused by base substitution or base insertion, or a linear ...

Embodiment 1

[0136] [Example 1] Introducing a dye with tryptophanyl tRNA synthetase gene into a Bacillus subtilis strain in vitro gene

[0137] The extrachromosomal gene carrying the tryptophanyl tRNA synthetase gene, plasmid pDATS14, was constructed as follows. First, PCR amplification was performed using primers A and B using a general-purpose Bacillus subtilis-Escherichia coli shuttle plasmid, pHY300PLK (manufactured by Yakult Co., Ltd.), as a template. Restriction enzyme wxya After digestion, a circular plasmid is formed by ligating the two ends. This plasmid was used to transform Escherichia coli HB101 strain. A plasmid was prepared from the transformant and designated as pDA2. On the other hand, using the chromosome of Bacillus subtilis ISW1214 strain as a template, primers C and D were used to obtain a DNA fragment containing tryptophanyl tRNA synthetase gene and its adjacent region. restriction enzyme wxya The DNA fragment was digested and pre-treated with restriction en...

Embodiment 2

[0138] [Example 2] Used to destroy the tryptophanyl tRNA synthetase gene on the chromosome of Bacillus subtilis strain Construction of DNA Fragments

[0139] Using the chromosome of Bacillus subtilis ISW1214 strain as a template, using primers E and F, a DNA fragment of about 2.5 kb containing tryptophanyl tRNA synthetase gene and its adjacent regions was obtained. restriction enzyme EcoRI and SalI Digestion. post and pre pass EcoRI and SalI The digested universal plasmid pUC18 was ligated to obtain plasmid pTSAF. On the other hand, using the universal plasmid pC194 as a template and using primers G and H, a DNA fragment of about 1.6 kb containing the chloramphenicol resistance gene and its adjacent regions from Staphylococcus was obtained. Using pTSAF as a template, PCR reaction was carried out using primers I and J to obtain DNA fragments. The DNA fragment and a DNA fragment of about 1.6 kb containing the chloramphenicol resistance gene and its adjacent region ...

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Abstract

Disclosed is a method for preparing a protein or peptide encoded by a foreign gene by expressing the foreign gene, which comprises the steps of: inserting the foreign gene in such a manner that the foreign gene can be expressed, so as to prepare a recombinant vector carrying a gene encoding an aminoacyl tRNA synthetase in such a manner that the gene can be expressed; providing a mutant host cell or the like having the deletion of a chromosomal gene encoding the aminoacyl tRNA synthetase; transforming the mutant host cell with the recombinant vector to produce a transformant; and culturing the transformant to prepare a protein or peptide encoded by the foreign gene. It becomes possible to provide: a novel means for maintaining a recombinant DNA cloning vector in a host cell without the need of using any antibiotic or without any limitation in the composition of a culture medium; and a method for preparing a protein or peptide encoded by a foreign gene by utilizing the means.

Description

technical field [0001] The present invention relates to methods for stably maintaining foreign genes in cells. More specifically, the present invention relates to a recombinant vector comprising a gene encoding aminoacyl tRNA synthetase, a mutant host cell for expressing any foreign gene other than a gene encoding aminoacyl tRNA synthetase, and expression A method of producing a protein or peptide encoded by a foreign gene other than the gene encoding aminoacyl-tRNA synthetase. In addition, this application claims the priority of Japanese Patent Application No. 2007-1839831 filed on July 13, 2007 as a cross-reference to related applications, the entire contents of which are specifically incorporated herein as disclosure. Background technique [0002] When applying recombinant DNA technology in practice and trying to use the resulting transformed cells to produce target molecules, usually the presence of extrachromosomal genes in the transformed cells (that is, the target fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/15C12N1/19C12N1/21C12N5/00C12C5/10C12N9/42
CPCC12N9/93C12N15/64C12N15/821
Inventor 秦田勇二大田优佳莉日高祐子中村信之
Owner JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY
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