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

Human and mammal cell expression vector and application thereof

An expression vector and mammalian technology, applied in the field of genetic engineering, can solve problems such as unsatisfactory expression levels of transgenes, and achieve the effect of increasing expression and increasing expression levels

Active Publication Date: 2014-03-19
河南普诺易生物制品研究院有限公司 +1
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, MAR is still suboptimal in increasing transgene expression levels

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
  • Human and mammal cell expression vector and application thereof
  • Human and mammal cell expression vector and application thereof
  • Human and mammal cell expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Construction of expression vector pCAD

[0023] 1.1 Construction of expression vector pCATG containing neomycin eukaryotic resistance gene selection complex

[0024] 1.1.1 PCR amplification of neomycin eukaryotic resistance gene selection complex

[0025] According to the sequence of neomycin eukaryotic resistance gene selection complex (GenBank accession number is EF437957.1), primers P1 and P2 were designed, and the 5′ ends of the primers were respectively introduced with Sal I restriction sites. The primer sequences are as follows (the underline is the restriction site dot): P1:5′-GTC GTC GAC CTGTGGAATGTGTGTCAGTTAGGGTGTGGA-3'; P2: 5'-AGC GTC GAC CAGACATGATAAGATACATTGATGAGA-3'. Using the extracted pCDNA3.1 plasmid as a template, primers P1 and P2 were used to amplify the sequence of the neomycin eukaryotic resistance gene selection complex, and the conventional PCR method was used for amplification. The PCR system is shown in Table 1.

[0026] Table 1...

Embodiment 2

[0036] Example 2 Construction of expression vectors pCAC1 and pCAC2

[0037] 1.2.1 PCR amplification of DNA fragments of different sizes

[0038] Design the following primers with reference to the Enhanced green fluorescent protein (EGFP) gene fragment sequence of the pEGFP-C1 vector (GenBank accession number U55763.1): P5:5'-CCGGCTAGCGCTACCGGTCGCCA-3';P6:5'-CGCAGATCTCTGAGTGCGGACTTG-3';P7 : 5'-AATAGATCTCGGCGCGGGTCTTGTA-3'. P5 and P6 are used to amplify 750bp fragments, P5 and P7 are used to amplify 350bp fragments, and at the same time, BglII / NheI restriction sites are introduced, and the extracted pEGFP-C1 plasmid is used as a template. The PCR reaction system is the same as before. PCR The reaction conditions were as follows: 95°C for 3min; 94°C for 40s, 60-56°C for 30s, 72°C for 40s, 4 cycles for each annealing temperature, and finally 55°C for 30 cycles; 72°C for 3min. The amplified product was recovered by agarose gel electrophoresis and sent to a biological company for...

Embodiment 3

[0041] Example 3 Using the expression vector of the present invention to express VEGF gene in Chinese hamster ovary (CHO) cells

[0042] (1) Construction of pCAD-VEGF vector containing vascular endothelial growth factor (VEGF)

[0043] According to the human VEGF gene cDNA sequence (GenBank accession number is AF022375.1), PCR primers P8 and P9 were designed. In order to realize directional cloning, NcoI / XbaI restriction sites were respectively introduced at the 5′ ends of the primers. The primer sequences are as follows: P8: 5′-GGC CCATGG ATGAACTTTCTGCTGTCTTGGGTG-3';P9:5'-GCG AAGCTT TCACCGCCTCGGCTTGTCACATCTG-3'. Fetal myocardial tissue was taken, total RNA was extracted by Trizol method, and its integrity was identified by 1% formaldehyde-denatured agarose gel electrophoresis. In the 25 μl reaction system, add 1 μg of RNA, 1 μl of dNTP (10 mmol / L), 1 μl of P8 and P9 primers with a concentration of 20 μmol / L, 1 μl of AMV-Taq DNA polymerase (5 U / μl) and AMV reverse transcr...

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 human and mammal cell expression vector and application thereof. A nucleotide sequence of the expression vector is shown in Seq ID. No.1 or Seq ID No.2; the expression vector is the expression vector built by taking pCAT3-control as a starting vector. By adopting the expression vector disclosed by the invention, the expression level of the carried target gene can be significantly improved, and compared with the expression vector which contains MAR (matrix attachment region) and does not contain EGFP (enhanced green fluorescence protein), expression of the target gene can be significantly improved under the same conditions.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a human and mammalian cell expression vector and its application. Background technique [0002] Gene engineering, also known as recombinant DNA technology, is based on scientific research or production needs, at the molecular level, using artificial methods to extract or synthesize the genetic material (DNA fragments) of different organisms, cutting and splicing in vitro to form recombinant DNA, Then the recombinant DNA is recombined with the vector, and then introduced into the recipient cells without the DNA, replicated and expressed, to produce products that meet human needs or create new biological traits, and make them stably inherited to the next generation. According to the cloning and expression system of the target gene, it can be divided into prokaryotic genetic engineering, plant genetic engineering, animal genetic engineering and yeast genetic...

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): C12N15/85C12N15/66
Inventor 张俊河王天云董卫华王芳赵春澎柴树洁王小引王俐杨瑞李琴
Owner 河南普诺易生物制品研究院有限公司
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