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

Method for constructing multi-copy kluyveromyces lactis expression vector efficiently in in-vitro manner

A Kluyveromyces cerevisiae and expression vector technology is applied in the field of efficient construction of multi-copy Kluyveromyces lactis expression vector in vitro, which can solve the problems such as the inability to meet the yield requirement, the expression amount and the stability to be improved, and achieve the improvement of protein expression. Expression level, the effect of increasing copy number

Inactive Publication Date: 2013-04-24
HUBEI UNIV +1
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The expression system commonly used by Kluyveromyces lactis is still unable to meet the increasing production requirements, and its expression and stability need to be improved

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
  • Method for constructing multi-copy kluyveromyces lactis expression vector efficiently in in-vitro manner
  • Method for constructing multi-copy kluyveromyces lactis expression vector efficiently in in-vitro manner
  • Method for constructing multi-copy kluyveromyces lactis expression vector efficiently in in-vitro manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Build in the above way Expression vector PPML 2 .

Embodiment 2

[0026] Example 2 Construction of the recombinant vector GG799-Bp-Bman containing the mannanase gene.

[0027] ,choose Bacillus subtilis ( Bacillus subtilis ) derived mannanase gene is the target gene;

[0028] 2、 Synthetic primers. The primers in Table 2 were designed with GeneTool software and synthesized for later use.

[0029] Primer name sequence Bman-f 5'atagcggccgcAAACCAATTGAAGCGCATACTGTGT 3 Bman-r 5'atacggtccgTTACTCAACGATTGGCGTTAAAAGAATC 3

[0030] sheet

[0031] 3. Construction of expression vector. Bacillus subtilis ( Bacillus subtilis) derived mannanase gene with primers Bman-f and Bman-r for PCR (98°C, 2min, 1 cycle; 98°C, 7s, 55°C, 30s, 72°C, 1min, 25cycle; 72°C, 8min, 1 cycle; stored at 4°C), and the product was recovered by agarose gel electrophoresis and sequentially treated with restriction endonuclease not I and Cpo Ⅰ Enzyme digestion, after recovery, connect to not I and Cpo Ⅰ Expression vector BpKLAC after ...

Embodiment 3

[0035] 1. Using restriction endonuclease Bam H I and Speech Ⅰ Double digestion instance 2 vectorBpKLAC 2 -manB, resulting in a biobrick body containing cohesive ends, named Fragment 1. restriction endonuclease BIn I and Sal Ⅰ Double digestion vector BpKLAC 2 -manB, to obtain the Biobrick body containing cohesive ends, named Fragment 2, with restriction endonucleases Bam H I and Sal Ⅰ Double digestion vector BpKLAC 2 -manB, resulting in biobrick vector fragment 3 containing cohesive ends.

[0036] Mix Fragment 1, Fragment 2 and Fragment 3 at a ratio of 3:3:1 (1.5 μl for Fragment 1 and 2, and 0.5 μl for Fragment 3), use TAKARA’s ligation kit Solution I ligase for 2 hours, and routinely transform into Escherichia coli competent cells DH10β were coated on LB plates containing ampicillin sodium and cultured at 37°C for 12 hours. Randomly pick colonies, extract plasmids, and use restriction endonucleases Bam H I and Sal Ⅰ Double enzyme digestion to verify whethe...

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 a method for constructing a multi-copy kluyveromyces lactis expression vector efficiently in an in-vitro manner. The method comprises the following steps: 1) 10 primers for PCR are designed and synthesized according to the gene sequence of a carrier pKLAC2 of kluyveromyces lactis GG799; 2), synthesizing the gene segments of yeast expression nuclear structures of four restriction enzyme loci; 3) constructing the expression vector BpKLAC2; 4) using the restriction enzyme loci to verify the accuracy of the expression vector through digestion; 5) inserting a target gene in the expression vector BpKLAC2 to become the single-copy expression vector containing the target gene; and 6) applying the biological bricking method to achieve the in-vitro multi-copy of the expression vector. Through the application of the method, the expression quantity and the stability of the target protein gene of the kluyveromyces lactis can be improved.

Description

technical field [0001] The present invention relates to an expression vector of Kluyveromyces lactis, in particular a method for efficiently constructing multi-copy Kluyveromyces lactis expression vector in vitro, which can artificially control the insertion copy number of exogenous genes and can be used for gene dosage effect research , is an advantageous modification of the yeast expression vector. Background technique [0002] In recent years, the yeast expression system has attracted extensive attention and has been gradually used in the production of pharmaceutical proteins. It not only has the advantages of fast growth like prokaryotic cell system, is convenient for gene manipulation, and can be cultivated on a large scale, but also has the post-translational processing and glycosylation modification capabilities of eukaryotic cells, and can produce recombinant proteins with biological activity, etc. features. As one of the simplest eukaryotes, Kluyveromyces lactis i...

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): C12N15/81C12N15/66C12R1/645
Inventor 马立新赵西选刘晓倩李晔星王亚平姚永兰
Owner HUBEI UNIV
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