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

Novel high-efficiency recombinant plasmid vector and application thereof

A carrier and high-efficiency technology, applied in the direction of recombinant DNA technology, introduction of foreign genetic material using carriers, biochemical equipment and methods, etc., can solve the problems of short service life, high cost, low possibility, low human allergic reaction, purification The effect of process difficulty and cost reduction

Inactive Publication Date: 2011-03-30
BEIJING NORTHLAND BIOTECH
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the GST affinity chromatography medium used in purification is relatively expensive and has the disadvantage of short service life.

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
  • Novel high-efficiency recombinant plasmid vector and application thereof
  • Novel high-efficiency recombinant plasmid vector and application thereof
  • Novel high-efficiency recombinant plasmid vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0027] Construction of Example 1pNL-06 Vector

[0028] Firstly, according to the idea of ​​invention and the construction principle of the prokaryotic expression vector, select the various elements that make up the plasmid vector: promoter, protein tag, resistance gene, self-replicating sequence in the prokaryotic, etc., and arrange their respective positions. Referring to the gene sequence of each constituent element in "GENE BANK" and related sequences in practical application, the base sequence of the recombinant plasmid pNL-06 was designed. The transformed and successfully screened recombinant was named pNL-06 (see figure 1 ), the constructed recombinant vector gene is 5231bp through plasmid whole gene sequencing. The plasmid has a nucleic acid sequence shown in SEQ ID NO: 1, and Table 1 is a description of each constituent element of the gene sequence in the sequence listing.

[0029] Table 1. Description of each paragraph of the gene sequence in the sequence listing

...

example 2

[0031] Example 2: Application of pNL-06 vector in recombinant human thymosin α1

[0032] 1. Construction of pNL-06 recombinant plasmid

[0033] Using the target gene as a template, PCR amplification obtains the target product.

[0034] Plasmid pNL-06 was digested with BamH I and EcoR I to recover a large fragment, and the thymosin α1 target gene fragment was ligated in a T4 ligase system at 18°C, and the recombinant plasmid obtained after screening. Then use CaCl 2 Transform Escherichia coli BL21 by the method, smear it on the LB plate containing 50 μg / ml kanamycin, select the positive colony and culture it to OD in the LB containing 30 μg / ml kanamycin 600 Add IPTG 0.1mM at 0.6-0.8 to induce 3-4 hours and centrifuge to collect the bacteria. After breaking the bacteria with 8M urea, a protein band of about 30kD appears in 15% SDS-PAGE electrophoresis. A positive reaction was detected by monoclonal antibody immunoblotting. At the same time, the thymosin α1 gene was recombine...

example 3

[0101] Example 3: Application of pNL-06 vector in recombinant human thymosin β4

[0102] 1. Construction of recombinant plasmids

[0103] Using the target gene as a template, PCR amplification obtains the target product.

[0104] Plasmid pNL-06 was digested with BamH I and EcoR I to recover large fragments, ligated the target gene fragments in T4 ligase system at 18°C, and obtained recombinant plasmids after screening. Then use CaCl 2 Coli BL21 was transformed by the method, spread on LB plates containing 50 μg / ml kanamycin, and the positive colonies were selected and cultured in LB containing 30 μg / ml kanamycin until OD 600 Add IPTG 0.1mM at 0.6-0.8 to induce 3-4 hours and centrifuge to collect the bacteria. After breaking the bacteria with 8M urea, a protein band of about 31kD appears in 15% SDS-PAGE electrophoresis. A positive reaction was detected by monoclonal antibody immunoblotting. At the same time, the thymosin α1 gene was recombined into the pGEX-4T-1 plasmid, an...

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 high-efficiency prokaryotic expression vector which comprises two tags of polyhistidine and GST (Glutathione S Transferase), is convenient to quickly purify an expression product according to an actual condition and assists interest protein to realize soluble expression in engineering mycetocyte. The high-efficiency prokaryotic expression vector also comprises kanaresistance genes at the same time, when bio-medicine interest protein is produced, the possibility of a final product for causing the anaphylactic reaction of a human body is reduced to the lowest, and moreover, the difficulty and the cost of a purification technology are greatly reduced. The invention has an important significance for increasing the purification efficiency and reducing the cost of a purpose product.

Description

(1) Technical field [0001] The invention relates to a construction method of a plasmid vector, and has achieved preliminary results in practical application, has broad application prospects, and belongs to the prokaryotic expression system in genetic engineering. (2) Background technology [0002] Plasmids are genetic components other than bacterial or cell chromatin, capable of autonomous replication, and symbiotic with bacteria or cells. Its characteristics are as follows: plasmid has self-replication function, bivalent closed circular DNA outside chromatin, one or more restriction endonuclease cleavage sites on its DNA molecule, and special genetic markers on its molecule Gene. The plasmid vectors used in molecular biology are not the plasmids that existed naturally in the original bacteria or cells, but have undergone many artificial modifications. Starting from different experimental purposes, people have designed various types of plasmid vectors, which have developed...

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): C12N15/70C12N15/74C12P21/02C12R1/19
Inventor 聂李亚马素永许松山汤晓闯刘国强赵娜娜
Owner BEIJING NORTHLAND BIOTECH
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