DNA molecule for expressing acyltransferase in pichia pastoris and application thereof

A technology of DNA molecules and Pichia pastoris, applied in the direction of transferase, recombinant DNA technology, application, etc., to achieve good biological safety, simple operation, and avoid the effect of endotoxin residues

Inactive Publication Date: 2015-04-29
TIANJIN UNIV OF COMMERCE
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been breakthroughs in lactonases capable of degrading N-acyl homoserine lactones (AHLs), such as the technical solutions disclosed in patent documents with application numbers 201010541821.8, 200910088317.4, 2010...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • DNA molecule for expressing acyltransferase in pichia pastoris and application thereof
  • DNA molecule for expressing acyltransferase in pichia pastoris and application thereof
  • DNA molecule for expressing acyltransferase in pichia pastoris and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Cloning of acyl homoserine acyltransferase ahlM gene and construction of plasmid pPlCZαA-ahlM.

[0047] 1. Cloning of the acyl homoserine acyltransferase gene ahlM

[0048] The acyl homoserine acyltransferase ahlM gene was found from the Streptomyces sp.M664 strain genome database. The sequence number of the ahlM gene in Genebank is AAT68473.1, and the nucleotide sequence is shown in SEQ ID NO:1 , the amino acid sequence is shown in SEQ ID NO:2. Entrusted Shanghai Yihong Biotechnology Co., Ltd. to synthesize the full nucleotide sequence of the acyl homoserine acyltransferase ahlM gene onto the pUC57 plasmid to obtain the pUC57-ahlM plasmid.

[0049] 2. Construction of pPlCZαA-ahlM recombinant plasmid containing acyl homoserine acyltransferase ahlM gene expressed in Pichia pastoris

[0050] The P1 upstream primer shown in SEQ ID NO:3 and the P2 downstream primer shown in SEQ ID NO:4 were designed according to the nucleotide sequence of the gene encoding acyl...

Embodiment 2

[0058] Embodiment 2, construction and identification of recombinant yeast strain

[0059] 1. For the linearization of the pPICZαA-ahlM recombinant plasmid, the pPICZαA-ahlM recombinant plasmid was digested with SacⅠ restriction endonuclease, and the enzyme digestion system was as follows: the concentration was 100ng / μL pPICZαA-ahlM recombinant plasmid 100μL, SacⅠenzyme 10μL, 30 μL of 10× buffer, 160 μL of water. Enzyme digestion in a water bath at 37°C for 12 hours. After the reaction, add the enzyme digestion system to an equal volume of extract solution consisting of phenol: chloroform: isoamyl alcohol for extraction, wherein the volume ratio of phenol: chloroform: isoamyl alcohol is 25: 24:1. Then centrifuge at 15,000×g for 10min and take the upper aqueous phase, add 2 times the volume of -20°C cold absolute ethanol, mix well, put it into a refrigerator at -20°C for alcohol precipitation for 2h, and then centrifuge again (15,000× g, 10 min), carefully suck off the ethanol...

Embodiment 3

[0071] Example 3, Induced Expression of Recombinant Yeast Strains

[0072] 1. Select 3 clones from the recombinant yeast with the deposit number CGMCC No.10362, inoculate them in 5 mL of YPDS liquid medium containing 100 μg / mL bleomycin, and culture them at 30° C. and 200 rpm / min for 12 hours.

[0073] 2. The culture is inoculated in 20mL BMGY medium according to the inoculum size of 1%, and cultured at 30°C and 200rpm / min for 16-20 hours until OD 600nm Between 2.0-6.0. Centrifuge at 6000rpm for 5min at room temperature, collect the recombinant yeast cells, and then resuspend the cells with BMMY medium to OD 600nm is about 1.0. Continue to culture at 28° C. with shaking at 200 rpm / min for 72 hours, wherein anhydrous methanol is added every 24 hours until the final concentration of methanol is 1%.

[0074] 3. Take 50 mL of the recombinant yeast liquid induced in step 2 for 72 hours and centrifuge at 5000 rpm / min for 10 min, collect the supernatant after centrifugation, lyoph...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a DNA molecule for expressing acyltransferase in pichia pastoris and an application thereof and provides a DNA sequence of acyl-homoserine acyltransferase genes ahlM, a recombinant expression vector containing the DNA molecule, an expression box, a transgenic cell line or recombinant bacteria and an application of the DNA in the aspect of degrading signal molecules N-acyl-homoserine lactone. The nucleotide sequence of the DNA molecule is shown as SEQ ID NO:1. The acyl-homoserine acyltransferase specifically catalyzes hydrolysis of acyl-homoserine amido bonds, the requirement on the pH value of the reaction is low, a hydrolysis reaction can be carried in a wide pH range, and wide application of the acyl-homoserine acyltransferase is promoted. In addition, the acyltransferase has different wide properties from the substrate of lactonase, and the acyl-homoserine acyltransferase has high substrate specificity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA molecule of acyltransferase, and its expression and application in Pichia pastoris. Background technique [0002] At present, antibiotics widely used clinically target the important life metabolic processes of bacteria such as protein synthesis, nucleic acid synthesis, cell wall synthesis and folic acid synthesis, directly kill microorganisms or inhibit the growth of microorganisms. Under the selection of this survival pressure, pathogenic microorganisms gradually develop drug resistance [Rasmussen T B, Bjarnsholt T, Skindersoe M E, et al. Screening for quorum-sens inginhibitors (QSI) by use of a novel genetic system, the QSI selector . J Bacteriol, 2005, 187:1799 1814]. Existing drugs cannot solve the problems caused by drug resistance and emerging pathogens, so it is necessary to broaden the thinking to find new targets to develop new antibacterial drugs. [0003] In recent...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/54C12N15/81C12N1/19C12N9/10A61K38/45A61P31/04C12R1/84
Inventor 阮海华杜康龙张振奇
Owner TIANJIN UNIV OF COMMERCE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap