Gene pme16A for encoding metalloprotease and application thereof

A technology of metalloprotease and pme16a, which is applied in application, genetic engineering, plant genetic improvement, etc., to achieve good hydrolysis ability and make up for the shortage.

Inactive Publication Date: 2015-01-21
GUANGXI UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Metalloprotease-producing strains can be screened by pure culture technology, but due to the limitations of various conditions, about 99% of microorganisms in nature cannot be achieved by pure culture technology, and there are certain limitations in the discovery of new genes. Therefore, macro More and more scientists pay attention to genome technology in discovering new genes

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
  • Gene pme16A for encoding metalloprotease and application thereof
  • Gene pme16A for encoding metalloprotease and application thereof
  • Gene pme16A for encoding metalloprotease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A clone of gene pme16A encoding novel metalloprotease mainly includes the following steps 1, 2, 3 and 4.

[0028] Step 1: extracting and purifying metagenomic DNA from activated sludge of polluted water samples, the method steps are as follows.

[0029] (1) First prepare the DNA extraction solution: 100mM sodium phosphate buffer (pH8.0), 1% (w / v) CTAB, 100mM EDTA (pH8.0), 0.3M NaCl, 0.01mM Tris-HCl (pH8.0 );

[0030] (2) PVPP pickling treatment: Weigh 10g PVPP, add 3M HCl, soak for 12h, filter with filter paper, wash and stir PVPP with 20mM potassium phosphate buffer (pH7.4), repeat several times until the suspension reaches neutrality, and PVPP filter and dry at 50°C;

[0031] (3) Weigh 1 g of the sample into a 15 mL centrifuge tube, add 0.25 g of acidified PVPP, 4 mL of extraction buffer, mix well, freeze and thaw at 70°C and liquid nitrogen three times, each time for 30 min. After the last dissolution, add SDS to a final concentration of 1%, mix evenly by inversio...

Embodiment 2

[0051] Construction of pme16A gene expression recombinant plasmid.

[0052] The plasmid pET-32a(+) provided by Novagen, USA was used as the expression vector and E.coli BL21(DE3)pLysS was used as the host cell for heterologous high-efficiency expression of the target gene.

[0053] According to the pme16A gene sequence, primers were designed using relevant software. Forward amplification primer F1: 5'-GC GAATTC ATGCCCCTTTATCAGACCTTC-3', introduce EcoRI restriction site; reverse amplification primer R1: 5'-AA CTCGAG GGGCGATTCGAGCTC-3', add PstI restriction site. Using pGXM16 as a template, use Pfu DNA polymerase to carry out PCR reaction to amplify the pme16A gene, such as Image 6 shown. The expression vector pET-32(a)+ plasmid was extracted, digested with EcoRI and PstⅠ, purified and ligated with the pme16A amplified product that had also undergone double digestion and purification, and the expression recombinant plasmid was constructed, and transformed into E .coli DH...

Embodiment 2

[0054] For example two, please refer to Image 6 . Image 6In order to express the enzyme digestion results of the recombinant plasmid pGXPME16A, 1 is 1kb Ladder Maker, and the fragment sizes from top to bottom are: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb, 1.0kb, 750bp, 500bp, 250bp; 2 is the result after digesting pGXPME16A with EcoR Ⅰ and Pst Ⅰ.

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 relates to a gene pme16A for encoding metalloprotease. A nucleotide sequence of the gene pme16A is as shown in a sequence table No.1; a sequence of an amino acid residue of the gene pme16A is as shown in a sequence table No.2. DNA (Deoxyribose Nucleic Acid) in the sequence table 1 is started with an initiation codon ATG and is end with a termination codon TAA from a first nucleotide to a 1461th nucleotide at an end 5'; a complete ORF (nucleotides 1 to 1461) exists; initiation codons ATG of the gene pme16A are from the first to third nucleotides at the end 5'; the termination codons TAA are from the 1459th to 1461th nucleotides at the end 5'. The gene pme16A provides a novel material for industrial production application of the metalloprotease, can effectively make up the current situation of shortage of animal and plant source metalloprotease raw materials and has important research value and application prospect.

Description

technical field [0001] The invention relates to a gene pme16A encoding metalloprotease and its application. Background technique [0002] Metalloprotease (EC3.4.24.-) refers to a class of protease whose active center depends on metal ions. Most metalloproteases mainly rely on divalent cations, such as zinc ions, copper ions, cobalt ions, and manganese ions. where Zn 2+ most common. Metalloproteases are easily and strongly inhibited by metal chelators. Metalloproteases are widely distributed and specific in nature, and have important economic and application values. Due to the different sources of metalloproteases, most of them have their own distinctive characteristics. Generally, it has the characteristics of high temperature resistance or low temperature resistance, resistance to organic solvents, alkali resistance, and heat sensitivity. At present, metalloproteases are mainly used in food, detergent, cosmetics, antineoplastic drugs and disease mechanism research, an...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/10
Inventor 蒋承建黄捷古恒森曾蓉陈高申佩弘武波
Owner GUANGXI UNIV
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