Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Expression vector based on URA3 gene and construction method thereof

A technology for expression vectors and construction methods, which is applied in the field of expression vectors in molecular biology, and can solve problems such as only applicable

Pending Publication Date: 2020-10-30
GUANGZHOU HUAZHEN PHARM CO LTD
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are a variety of commercial expression vectors available on the market, such as pET series vectors from Novagen Corporation for protein expression in Escherichia coli, pPICZ series vectors from Invitrogen Corporation for protein expression in Pichia pastoris, CLONTECH Laboratories, Inc. The pDsRed1, pEYFP series of vectors used in mammalian cells, etc., the main problem of these expression vectors is that they are only applicable to cells of a few common species, and must be used when encountering uncommon species without corresponding commercial expression vectors Traditional vector construction methods specifically construct vectors

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
  • Expression vector based on URA3 gene and construction method thereof
  • Expression vector based on URA3 gene and construction method thereof
  • Expression vector based on URA3 gene and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 experimental material and basic method

[0038] Strain plasmids and reagents

[0039] Escherichia coli JM109 was preserved by our laboratory; Kluyveromyces lactis GG799 was purchased from NEB Company; the cloning vector pMD-19T was purchased from Shanghai Bao Biological Company; pLVX-IRES-mCherry plasmid was preserved by our laboratory; restriction enzymes AflII, BamHI was purchased from Takara; DNA Marker and T 4 DNA ligase was purchased from Takara Company; plasmid extraction kit and gel cutting recovery kit were purchased from Omega Company; pEASY-Uni Seamless Cloning and Assembly Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; mCherry gene was from pLVX-IRES- For the mCherry plasmid, use the jcat online tool to optimize the yeast codon, and add a 6X his-tag protein tag to the C-terminus of mCherry, and the sequence is handed over to Shenzhen Huada Gene Technology Co., Ltd. to synthesize DNA; the primers used in this experiment shown ...

Embodiment 2

[0049] Embodiment 2 expression vector construction process

[0050] Extraction and inspection of yeast GG799 genomic DNA:

[0051] The frozen yeast GG799 was inoculated on a YPDA plate, and after culturing at a constant temperature of 30 for 3 days, a single colony was picked and inoculated in YPDA liquid medium, and cultured at 30 and 250 r / min for 3 days. The culture was placed in a 50 mL centrifuge tube and centrifuged at 10000 g for 5 min at 4 °C. Remove the supernatant, wash with 15ml sterile water, and centrifuge at 4°C, 10000g for 5min. Remove the supernatant, put it in a mortar and add liquid nitrogen, grind the bacteria into a fine powder and transfer it to a 2.0mL centrifuge tube. The fungal genome rapid extraction kit of OMEGA was used to extract, the operation process was carried out according to the instructions, and the obtained gDNA was detected by 1% agarose electrophoresis. For the GG799 genomic DNA extracted by OMEGA’s Fungal Genome Rapid Extraction Kit, s...

Embodiment 3

[0067] Embodiment 3 uses the expression vector of the present application Lactococcus lactis and Trichoderma reesei

[0068] The expression vector of this application was further used in Lactococcus lactis (prokaryote) and Trichoderma reesei (filamentous fungus) and all obtained satisfactory results. The Western Blotting results of Lactococcus lactis vector structure and protein are shown in Figure 8 and 9 , Western Blotting effect of Trichoderma reesei vector structure and protein see Figure 10 and 11 , it can be seen that the carrier of the present application is fully applicable to other species.

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 provides an expression vector based on a URA3 gene and a construction method of the expression vector. The vector sequentially comprises a URA3 homologous arm, a strong promoter, multiple cloning sites, a terminator, a URA3 homologous arm or a URA3 promoter or a URA3 terminator. The construction method comprises the following steps of: extracting DNA of a target species; designing aprimer for amplification to obtain a URA3 homologous arm, a strong promoter, a terminator, a URA3 homologous arm or a URA3 promoter element or a URA3 terminator element; obtaining other elements; andconnecting the recombinase with a plurality of elements to obtain the complete vector in one step.

Description

technical field [0001] The present invention belongs to the field of expression vectors in molecular biology. Specifically, the application provides a rapid construction expression vector based on URA3 gene and a construction method thereof. Background technique [0002] Expression vectors are vectors that add expression elements such as promoters, terminators, etc. on the basis of the basic skeleton of the cloning vector, so that the target gene can be expressed in the host. Expression vectors are an indispensable tool in genetic engineering. There are a variety of commercial expression vectors available on the market, such as pET series vectors from Novagen Corporation for protein expression in Escherichia coli, pPICZ series vectors from Invitrogen Corporation for protein expression in Pichia pastoris, CLONTECH Laboratories, Inc. The pDsRed1, pEYFP series of vectors used in mammalian cells, etc., the main problem of these expression vectors is that they are only applicable...

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): C12N15/63C12N15/66
CPCC12N15/63C12N15/66C12N9/88C12Y401/01023C12N2800/30
Inventor 李芳红梁志成赵子建梁秀怡邓木兰
Owner GUANGZHOU HUAZHEN PHARM CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com