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

A chs4 element-based mammalian cell attachment expression vector, expression system, preparation method and application

An expression vector, mammalian technology, applied in the field of genetic engineering and gene therapy, can solve the problems of transgene silencing, low copy number, poor vector stability, etc., achieve long-lasting expression, good safety, and overcome the effects of transgene silencing

Active Publication Date: 2019-04-09
XINXIANG MEDICAL UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the first generation of MAR-mediated episomal expression vector pEPI was confirmed, a number of improved studies have been carried out, such as inserting regulatory elements or reducing bacterial sequences to make up for the lack of vectors, but transgene silencing often occurs in the research
Although the vector can remain attached to the host cells after transfection, the stability of the vector is poor and the copy number is low. Studies have shown that about 90% of the vector molecules are gradually lost during passage.

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
  • A chs4 element-based mammalian cell attachment expression vector, expression system, preparation method and application
  • A chs4 element-based mammalian cell attachment expression vector, expression system, preparation method and application
  • A chs4 element-based mammalian cell attachment expression vector, expression system, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction of the episomal expression vector pES4, the steps are as follows:

[0038] 1) Synthesis of cHS4 sequence

[0039] The cHS4 sequence was synthesized according to the literature (Saunders F, Sweeney B, Antoniou, et al. Chromatin function modifying elements in an industrial antibody production platform-comparison of UCOE, MAR, STAR and cHS4 elements.2015, PLoS One 10:e0120096.) for directional cloning and the identification of subsequent vectors, the restriction sites of BspE I and BsrG I were respectively introduced at the 5' end of the sequence. Since the pES4 vector was constructed using seamless cloning technology, the company added 15 bp homologous sequences to both ends of the sequence when synthesizing the cHS4 sequence. The synthetic cHS4 sequence is as follows:

[0040]

[0041] Wherein, the italic part represents the homologous sequence, and the underlined part is the enzyme cutting site.

[0042] 2) Construction of pES4 plasmid vector

[...

Embodiment 2

[0051] The construction of mammalian cell expression system, the steps are as follows:

[0052] 1) Synthesis of IFN-β sequence

[0053] According to the IFN-β sequence published by NCBI (GenBank: BC096151.2, bases 1 to 564), the company synthesized IFN-β (as shown in SEQ ID NO: 3, with 15 bp vectors added to both ends of the sequence) Homologous sequence, and IFN-β is inserted downstream of eGFP), which was completely consistent with the sequence registered in GenBank by sequencing analysis.

[0054] 2) Construction of the episomal expression vector pES4-IFN-β containing the IFN-β sequence

[0055] Digest the pES4 plasmid with BsrG I. The enzyme digestion system is: 1 μg / μL pES4 plasmid 10 μL, 10×NE buffer 2 μL, BsrG I enzyme 0.5 μL, add triple distilled water to 20 μL; mix well, and digest in a 37°C water bath 8h. After the enzyme digestion, 1.5% agarose gel electrophoresis was performed to recover the digested pES4 linear plasmid DNA.

[0056] 10 μL In-Fusion Cloning lig...

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

PropertyMeasurementUnit
fluorescenceaaaaaaaaaa
fluorescenceaaaaaaaaaa
Login to View More

Abstract

The invention discloses a mammalian cell attachment body expression vector, an expression system, a preparation method and an application based on a cHS4 element, belonging to the technical field of genetic engineering and gene therapy. Chicken hypersensitive site 4 (cHS4) is a classical insulator element with functions of enhancing hadron blocking and heterochromatin barrier, which can minimize the main cause-position effect of transgene silencing and reduce exogenous priming The probability of abnormal expression of adjacent genes caused by promoters and enhancers. In the present invention, the cHS4 sequence is introduced into the multi-cloning site of the vector, that is, the downstream of eGFP. On the one hand, it can overcome transgene silencing, and on the other hand, it can mediate the stability, high efficiency and high efficiency of exogenous genes in mammalian cells without selection pressure. Long-lasting expression and better security.

Description

technical field [0001] The invention relates to a cHS4 element-based mammalian cell attachment expression vector, and also relates to an expression system, preparation method and application comprising the attachment expression vector, and belongs to the technical field of genetic engineering and gene therapy. Background technique [0002] With the continuous development of molecular biology and biotechnology, people have a deeper understanding of the pathogenesis of genetic diseases, and on this basis, a large number of disease-causing genes have been discovered. There is even more scope for treating diseases by replacing faulty genes in damaged cells. Gene therapy refers to the direct introduction of exogenous normal genes into the target cells of the diseased part of the patient through gene transfer technology, and by controlling the expression of the target gene, inhibiting, correcting, replacing or compensating for defective or abnormal genes, a method of treating gene...

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): C12N15/85C12N15/66
CPCC12N15/66C12N15/85C12N2800/107C12N2830/40
Inventor 赵春澎王天云王小引张玺徐光华付笑笑高向征白可可
Owner XINXIANG MEDICAL 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