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High-yield G418 engineering strain, and construction method and application thereof

A construction method and technology of engineering bacteria, applied in the field of antibiotic pharmaceuticals, can solve problems such as interference, high cost of G418, pollution, etc., and achieve the effects of increasing production, simplifying production process, and reducing production cost

Inactive Publication Date: 2014-03-26
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, due to the complex metabolites of Micromonospora red orange, and the metabolites have similar chemical structures and similar physical and chemical properties, the production cost of G418 is high, the chromatographic separation cycle is long, the yield is low, the material consumption is large, and the pollution is serious. It is complex, and the product quality is unstable, and the by-products with similar structure to G418 may interfere at any time, causing great uncertainty to the quality control of G418 products, seriously affecting the stability, safety and effectiveness of the drug, which is an urgent need scientific puzzles solved

Method used

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  • High-yield G418 engineering strain, and construction method and application thereof
  • High-yield G418 engineering strain, and construction method and application thereof
  • High-yield G418 engineering strain, and construction method and application thereof

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Embodiment 1

[0040] (1) Construction of in-frame knockout plasmid for gene genQ

[0041]According to the corresponding sequence of the published gentamicin-producing bacterium Micromonospora echinospora echinospora biosynthetic gene cluster (GenBank accession number: AJ628149), two pairs of primers were designed on the upstream and downstream of the genQ gene:

[0042] Left arm upstream primer L1: 5'-CTG CATATG CTCACCGCCCGGC-3' (with NdeI restriction site);

[0043] Left arm downstream primer L2: 5'-CCC GAATTC TCCCTGTGACCTGTCG-3' (including EcoRI restriction site); amplify the left arm (genQ-L) 1937bp;

[0044] Right arm upstream primer R1: 5'-ACC GAATTC GCCGCTACCGATCACAC-3' (contains EcoRI restriction site);

[0045] Right arm downstream primer R2: 5'-GGC AAGCTT GATCGGAACCATCCGG-3' (including HindIII restriction site); amplified right arm (genQ-R) 1988bp.

[0046] Using the total DNA of wild-type Micromonospora aculeatus ATCC15835 as a template, high-fidelity DNA polymerase was u...

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Abstract

The invention discloses a high-yield G418 engineering strain, and a construction method and application thereof, and belongs to the field of antibiotic pharmacy. The high-yield G418 engineering strain is micromonospora echinospora deficient of a function of a gene genQ for encoding C-6' hydroxyl oxido-reductase, and the preservation number of the high-yield G418 engineering strain is CCTCCNO: M2013729. The construction method comprises the step of deleting the gene genQ from the micromonospora echinospora by using an in-frame deletion method, and comprises construction of genQ gene in-frame deletion plasmids, transformation of the micromonospora echinospora with the in-frame deletion plasmids and screening of a double-crossover mutant strain. According to the high-yield G418 engineering strain, and the construction method and application thereof, an in-frame deletion technology is used for deleting the gene genQ from the gentamicin producing strain micromonospora echinospora to block the synthesis of a component C of gentamicin and a related intermediate product and mainly generate G418; the G418 DeltagenQ is high in yield and purity, a production process for the G418 is simplified, and the production cost is lowered.

Description

technical field [0001] The invention belongs to the field of antibiotic pharmacy, and relates to a high-yield G418 engineering bacterium and its construction method and application. Background technique [0002] G418 (Geneticin) is an aminoglycoside antibiotic produced by Micromonospora rhodorangea with a structure similar to neomycin, gentamicin and kanamycin. Its chemical structure is shown in the following formula. In molecular genetics, G418 is the most commonly used resistance screening reagent for stable transfection. In recent years, G418 has also been widely used to remove contaminating fibroblasts in cell culture. The latest research reports that G418 and its derivatives can be used to treat human genetic diseases caused by nonsense mutations and have the effect of inhibiting viruses. [0003] [0004] However, due to the complex metabolites of Micromonospora red orange, and the metabolites have similar chemical structures and similar physical and chemical prop...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/74C12P19/50C12R1/01
Inventor 孙宇辉郭俊红邓子新
Owner WUHAN UNIV
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