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A cell strain expressing adenovirus and a method for efficiently preparing adenovirus

An adenovirus and cell technology, applied in the field of biomedicine, can solve problems such as low recombination efficiency

Active Publication Date: 2019-06-11
汉恒生物科技(上海)有限公司
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  • 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

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  • A cell strain expressing adenovirus and a method for efficiently preparing adenovirus
  • A cell strain expressing adenovirus and a method for efficiently preparing adenovirus
  • A cell strain expressing adenovirus and a method for efficiently preparing adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Construction of 293 cell line stably and highly expressing PVT1 gene

[0068] The 293 cell line with stable and high expression of PVT1 gene was constructed by lentiviral vector.

[0069] 1. Experimental reagents

[0070] 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

[0071] Carrier and target gene information

[0072] 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.

[0073] 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.

[0074] 2) ...

Embodiment 2

[0091] Example 2 lentiviral packaging

[0092] 1. Experimental process

[0093] 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.

[0094] 2. Experimental materials

[0095] 2.1 Lentiviral vectors, packaging cells and strains

[0096] 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-PVT1 . Among them, pHBLV-CMVIE-I...

Embodiment 3

[0119] Example 3 293A cells highly expressing PVT1 are used for the packaging of adenovirus, comparing the toxicity efficiency of normal 293A cells and 293A-PVT1 cells

[0120] 1. Experimental materials

[0121] The recombinant adenovirus vector plasmid map is as follows Figure 5 shown.

[0122] Human embryonic kidney cell line 293A cells and 293A-PVT1 cells; 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.

[0123] 2. Experimental method

[0124] 2.1 Mass preparation of recombinant plasmids

[0125] 1) Add 2ml of the bacterial solution in the logarithmic growth phase to 100mL LB medium containing 100ug / m...

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Abstract

The invention discloses a cell for efficiently expressing adenoviruses and a method for preparing the adenoviruses. Deep researches accidentally show that the virus production efficiency of the adenoviruses can be greatly improved by regulating and controlling the expression level of PVT1 genes in 293A cells. According to the technical scheme, when the adenoviruses are packaged, total virus particles are increased by about 60 percent, and the infectivity titer is increased by about 10 times, so that the technical scheme is particularly applied to the efficient preparation of the adenoviruses.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a cell strain expressing adenovirus and a method for efficiently 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 applicatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N7/00C12R1/93
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
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