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Novel DNA fragment, recombinant vector containing the same, transformant transformed therewith and utilization of the same

一种转化体、片段的技术，应用在转化体领域，能够解决重组蛋白质工序变复杂等问题

Inactive Publication Date: 2010-09-08
JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the problem of complicating the steps for collecting and purifying the recombinant protein also arises

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0076] (D) Preparation method of recombinant protein of the present invention

[0077] The present invention includes a method of inoculating and culturing the transformant of the present invention in a suitable medium according to a known method, and collecting the recombinant protein from the culture.

[0078] The nutrient medium for cultivating the transformant of the present invention may be any of a natural medium and a synthetic medium as long as it appropriately contains carbon sources, nitrogen sources, inorganic substances, and micronutrients necessary for the strain used as needed. kind.

[0079] As the carbon source of the nutrient medium for cultivating the transformant of the present invention, as long as it can be assimilated by the transformant, for example, glucose, maltose, fructose, mannose, trehalose, sucrose, mannitol, and sorbitol can be used. , starch, dextran, molasses and other sugars or organic acids such as citric acid and succinic acid or fatty acid...

Embodiment 1

[0090] [Example 1] Construction of a plasmid for searching gene regulatory regions

[0091] The vector pPTCF for searching DNA fragments of gene regulatory regions was constructed by the following method.

[0092] Since Microcormum autogenus JAMB-A7 ( Microbulbifer sp.JAMB-A7) strain (deposit number; FERM BP-8320) chromosomal DNA as a template, using the promoter A described in SEQ ID NO: 4 and the promoter B described in SEQ ID NO: 5 in the sequence listing to carry out PCR , to obtain DNA fragment A. The resulting DNA fragment A was treated with a restriction enzyme Eco RI processing, with pre-used Eco RI-treated pHY300PLK (manufactured by Yakult Co., Ltd.), which is a Bacillus subtilis-Escherichia coli shuttle plasmid, was ligated to construct a circular plasmid B. The circular plasmid B was used to transform Escherichia coli HB101 strain to obtain transformant C. Circular plasmid B was prepared from transformant C, and the plasmid having a polylinker site derived ...

Embodiment 2

[0093] [Example 2] Obtaining of gene regulatory region (1)

[0094] Bacillus JAMB750 was treated with the restriction enzyme Sau3AI ( Bacillus sp.JAMB750) strain (Accession No.: FERM AP-20227) chromosomal DNA, which was combined with restriction enzyme Bam The HI-cleaved plasmid pPTCF constructed in Example 1 was mixed, and a ligation reaction was performed with T4 DNA ligase to obtain a ligation reaction solution D. The obtained ligation reaction solution D was used to transform Bacillus subtilis to obtain group E of transformants. As the regeneration medium for culturing the transformant group E, 8% sodium succinate, 1% agar, 0.5% casamino acid, 0.5% yeast extract, 0.15% potassium dihydrogen phosphate, 0.35% dipotassium hydrogen phosphate, DM3 medium composed of 0.5% glucose, 0.4% magnesium sulfate, 0.01% bovine serum albumin, 0.001% methionine, 0.001% leucine and 7.5 μg / ml tetracycline. The transformant group E was cultured on DM3 medium, and about 2000 clones with ag...

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PUM

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Abstract

A DNA fragment containing a gene which encodes a specific gene regulatory region alone or the gene regulatory region together with a signal peptide; a recombinant vector containing the DNA fragment; a transformant containing the recombinant vector; and a method of producing a recombinant protein by using the transformant.Thus, it becomes possible to produce a protein in a large amount at a high efficiency regardless of the kind of the recombinant protein.

Description

technical field [0001] The present invention relates to DNA fragments for the production of recombinant proteins, having base sequences for regulating transcription and base sequences encoding signal peptides, recombinant vectors containing the fragments, transformants obtained by using them, and their applications. In addition, this application claims the priority of Japanese Patent Application No. 2007-183934 filed on July 13, 2007 as a cross-reference of related applications, and the entire description thereof is specifically incorporated herein as an disclosure. Background technique [0002] With recent advances in genetic engineering, various organisms can be used as host cells to produce proteins. In particular, host-vector systems for introducing recombinant vectors containing foreign genes into host cells are well known. If a host-vector system is used, recombinant proteins can be obtained without inserting foreign genes into the chromosome of the host cell. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12N9/24C12N9/42C12P21/02
CPCC07K2319/02C12Y302/01004C12N9/2468C12P21/02C12N9/24C12N15/63C12Y302/01081C12N15/75C12N15/70C12N9/2437
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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