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

Recombinant retroviral vector and application thereof

A technology of retroviruses and vectors, applied in the field of genetic engineering, can solve problems that need to be further improved and the improvement range is not large, and achieve the effects of improved effects, stable expression levels, and good application prospects

Active Publication Date: 2017-03-22
四川丰讯科技发展有限公司
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For example, Xu Jian et al., "Establishment and application of a high-efficiency expression system based on retroviral vectors", Academy of Military Medical Sciences of the Chinese People's Liberation Army, Cell Biology, 2008, Master published the relative fluorescence of EGFP-positive cells obtained by retrovirus PQCXIN The intensity is about 2 times of the relative fluorescence intensity of EGFP-positive cells transfected with pcDNA3.1 / EGFP plasmid, indicating that it is easier to obtain high-efficiency expression of exogenous gene cell lines with retroviral vector PQCXIN than plasmids, but the increase is not large. To be further 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
  • Recombinant retroviral vector and application thereof
  • Recombinant retroviral vector and application thereof
  • Recombinant retroviral vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of retroviral vector of the present invention

[0029] Such as figure 1 Shown to construct the retroviral vector PQCEDHFR of the present invention:

[0030] Take the retroviral vector PQCXIN (its structure is as figure 1 As shown, purchased from clontech company), using restriction sites AgeI (available from NEB company) and XhoI (available from NEB company) to excise IRES and Neo;

[0031] Connect the two genes of IRES and DHFR (sequence shown in SEQ NO:1) by overlap extension PCR method to obtain the gene fragment IRESDHFR. Design restriction sites AgeI and XhoI at both ends, and use restriction sites AgeI for PQCXIN Link with XhoI to remove IRES and Neo to construct the vector PQCDHFR;

[0032] Using the XbaI (purchased from NEB company) and NotI (purchased from NEB company) restriction sites on the vector PQCDHFR, the promoter CMV was replaced with the PCEF promoter (sequence shown in SEQ NO: 2) to construct the reverse transcription of the present in...

Embodiment 2

[0034] Example 2 Transfection of cells with retroviral vector of the present invention carrying EGFP gene

[0035] 1. Experimental materials

[0036] The retroviral vector PQCEDHFR of the present invention (the nucleotide sequence is shown in SEQ NO: 3): prepared according to the method of Example 1.

[0037] Control plasmid: pcDNA3.1(+) (purchased from Invitrogen) was digested with XmaI (purchased from NEB) and BstBI (purchased from NEB) by conventional molecular biology methods to remove the screening gene Neo, and then connected to The DHFR gene was cut with the same restriction enzymes, and the vector pcDNA3.1 / DHFR was successfully constructed, see attached figure 1 And SEQ NO: 4.

[0038] 2. Experimental method

[0039] Connect the EGFP reporter gene at EcoRV of pcDNA3.1 / DHFR and name it pcDNA3.1 / DHFR / EGFP as attached figure 2 As a control vector, the vector PQCEDHFR was digested with NotI (purchased from NEB) and Pad (purchased from NEB), and then connected to the same digested ...

Embodiment 3

[0055] Example 3 Transfection of cells with the retroviral vector of the present invention carrying TNK-tpa gene

[0056] 1. Experimental materials

[0057] In order to further verify the feasibility of this method, the foreign gene TNK-tpa was used as the test gene construction vector pcDNA3.1 / DHFR / TNK-tpa and PQCEDHFR / TNK-tpa( figure 1 , 2 ).

[0058] 2. Experimental method

[0059] According to the transfection and infection methods and the method of screening pressurized amplification genes in Example 2, a mixed clone population of positive cells expressing the TNK-tpa gene was obtained.

[0060] Separately monoclonal cells into a 96-well plate by limiting dilution. When the monoclonal cells grow to 1 / 2-2 / 3 of the entire well plate in about 2 weeks, use the in vitro fibrin agar plate lysis method to detect TNK-tPA In vitro fibrinolytic activity, the expression of cell supernatant was determined, and 10 high-expressing cell lines were selected respectively. The results are shown in ...

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 discloses a recombinant retroviral vector which is a retroviral vector replacing a screening gene Neo with a DHFR gene and replacing a promoter CMV with a promoter PCEF in a retroviral vector PQCXIN. The nucleotide sequences of the DHFR gene and the promoter PCEF are as shown in SEQ NO:1 and SEQ NO:2 respectively. The invention further discloses application of the recombinant retroviral vector. The recombinant retroviral vector PQCEDHFR carries foreign genes to transfect mammalian cells, the transfection ratio is high, the expression level of the foreign genes is high, and the expression level of the foreign genes can be steady, stable and high-yield. By the adoption of recombinant retroviral vector and application thereof, the recombinant retroviral vector PQCEDHFR can easily screen the foreign gene high-expressed cell lines, and is good in application prospect.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a retrovirus vector and its use. Background technique [0002] Mammalian cells are the main expression system for the production of recombinant protein drugs. Currently, biotech drugs produced using mammalian cell expression systems have exceeded 70% of the global sales of biotech drugs. In the production of recombinant protein drugs using mammalian cells as the production system, obtaining a cell line that efficiently and stably expresses the foreign target protein plays an important role in the entire production process. [0003] The main factors affecting the establishment of a recombinant protein high-expressing cell line are: the integration position of the foreign gene in the chromatin; the copy number of the foreign gene and the expression efficiency of the expression vector. Therefore, to establish a high-expressing cell line, first integrate the foreign gene into...

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): C12N15/867C12N5/10
CPCC12N15/86C12N2740/10043C12N2830/00
Inventor 罗世超李复岁
Owner 四川丰讯科技发展有限公司
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