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

Cell for highly expressing adenoviruses and method for preparing adenoviruses

A high-expression, adenovirus technology, applied in the field of biomedicine, can solve the problem of low recombination efficiency

Active Publication Date: 2015-07-22
汉恒生物科技(上海)有限公司
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantage is that the recombination efficiency is relatively low

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
  • Cell for highly expressing adenoviruses and method for preparing adenoviruses
  • Cell for highly expressing adenoviruses and method for preparing adenoviruses
  • Cell for highly expressing adenoviruses and method for preparing adenoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Construction of the 293 cell line stably and highly expressing the ABCB4 gene

[0069] The 293 cell line with stable and high expression of ABCB4 gene was constructed by lentiviral vector.

[0070] 1 Experimental materials

[0071] Reagent name

Manufacturer

Escherichia coli strain DH5α

Tiangen

restriction endonuclease

Fermentas

T4 ligase

Fermentas

Plasmid DNA Small, Massive Extraction Kit

Kang Wei Century

Gel Recovery Kit

Axygen

Agarose

Biowest

DNA ladder

GeneRay

[0072] Carrier and target gene information

[0073] 1) The pHBLV-CMVIE-IRES-puro carrier map is as follows figure 1 As shown, EcoRI and BamHI are insertion sites, and the pHBLV-CMVIE-IRES-puro vector sequence is shown in SEQ ID NO.:4.

[0074] The pHBLV-CMVIE-IRES-puro vector has the CMVIE promoter to promote the expression of the target gene, and contains the Puromycin resistance gene as a marker. ...

Embodiment 2

[0090] Example 2 lentiviral packaging

[0091] 1. Experimental process

[0092] Prepare the lentiviral shuttle plasmid and its auxiliary packaging original carrier plasmid. The three plasmid vectors were extracted with high purity and no endotoxin respectively, and co-transfected into 293T cells. After 8 hours of transfection, the complete medium was replaced. After 48 hours of culture, the collected The cell supernatant of the lentiviral particles was filtered by a 0.45um filter (Millpore Company), and the virus supernatant was concentrated by ultracentrifugation. After it is concentrated, a high-titer lentivirus concentrate is obtained.

[0093] 2. Experimental materials

[0094] 2.1 Lentiviral vectors, packaging cells and strains

[0095] The viral packaging system is a three-plasmid system consisting of pSPAX2 (the plasmid sequence is shown in SEQ ID NO.: 5), pMD2G (the plasmid sequence is shown in SEQ ID NO.: 6), pHBLV-CMVIE-IRES-puro-ABCB4 . Among them, pHBLV-CMVIE-...

Embodiment 3

[0121] Example 3 293A cells highly expressing ABCB4 are used for the packaging of adenovirus, comparing the virus export efficiency of normal 293A cells and 293A-ABCB4 cells

[0122] 1. Experimental materials

[0123] The map of recombinant adenovirus vector plasmid pHBAd-U6-GFP is as follows Figure 5 Shown (the plasmid sequence is shown in SEQ ID NO.:7).

[0124] Human embryonic kidney cell line 293A cells, 293A-ABCB4 (293A cells highly expressing ABCB4); DMEM was purchased from Hyclone Company; eukaryotic transfection reagent Lipofiter TM It is a product of Hanbio (Hanbio); fetal bovine serum and trypsin were purchased from Hyclone Company; 60mm culture dish, 10cm culture dish, 96-well plate, pipette (5ml, 10ml), 15ml and 50ml centrifuge tube All were purchased from NEST; double antibodies were purchased from Invitrogen; membrane filters were purchased from Millipore.

[0125] 2. Experimental method

[0126] 2.1 Mass preparation of recombinant plasmids

[0127] 1) Add ...

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 cell for highly expressing adenoviruses and a method for preparing the adenoviruses. Through the in-depth research of the inventor, the situation that the toxin production efficiency of the adenoviruses is greatly improved by adjusting and controlling the expression level of an ABCB4 gene in a 293A cell is accidentally found. The technical scheme adopted by the cell disclosed by the invention is used for packaging the adenoviruses, the total number of virus particles is increased by about 60%, the infectious titer is increased by about 10 times, and therefore the technical scheme adopted by the cell disclosed by the invention is especially suitable for efficiently preparing the adenoviruses.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to cells expressing adenovirus with high efficiency and a method for preparing adenovirus. Background technique [0002] Adenovirus is a double-stranded DNA virus without a shell. The genome is about 36kb long, and the capsid has a regular 20-hedron structure. Members of the adenoviridae family infect a fairly wide variety of post-mitotic cells, even cells from highly differentiated tissues such as skeletal muscle cells, lung cells, brain cells and heart cells. Because adenovirus can deliver its own genome into the nucleus, it has a wide range of hosts, can infect dividing and non-dividing cells, and has a large loading capacity of foreign genes, etc., so adenovirus has become the main candidate for expression and delivery of therapeutic genes. It is widely used in basic research and clinical trials. [0003] Viral vectors, as a new generation of transgenic vectors, have broad applicati...

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 Applications(China)
IPC IPC(8): C12N5/10C12N7/00C12N15/867C12N15/12C12R1/93
Inventor 蔡晓龙陈意雄邹杰王丽莎朱鹏飞李明明杨龙飞许曼蒂万颖曹青卞正雅韦唯
Owner 汉恒生物科技(上海)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