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New type recombined plasmid of carrier framework for adenovirus and application

A technology of recombinant adenovirus and backbone plasmid, applied in the fields of application, biochemical equipment and methods, botany equipment and methods, etc., can solve the influence of virus genetic background and activity, the adenovirus genome is prone to mutation, and maintain the pressure of adenovirus survival And other issues

Inactive Publication Date: 2006-07-05
AGTC GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the AdMax system, it only takes 2 to 4 weeks to complete the whole process from plasmid construction to recombination, and the success rate is greater than 98% (95% of the clones contain the target gene), because recombination is in eukaryotic cells Completed, the survival pressure on the adenovirus is maintained, so it helps to maintain the integrity of the recombinant adenovirus genome; while the success rate of the AdEasy system is only 18-34% (Stratagene's new version of the AdEasy XL system has a success rate of 94% %), and the virus genome recombination is completed in prokaryotic cells (BJ5183), theoretically, the adenovirus genome that has lost the survival pressure is more likely to mutate, and the genetic background and activity of the virus may be affected

Method used

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  • New type recombined plasmid of carrier framework for adenovirus and application
  • New type recombined plasmid of carrier framework for adenovirus and application
  • New type recombined plasmid of carrier framework for adenovirus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: Construction process of pBHG-fiber5 / 35 plasmid

[0055] by figure 2 The indicated pBHGlox(delta)E1, 3Cre plasmid is the material, according to figure 1 The process shown in the construction of the backbone plasmid disclosed by the present invention, the steps are as follows:

[0056] (1) The backbone plasmid pBHGlox(delta)E1 and 3Cre in the AdMax system were double-digested with SpeI and XbaI, and the large fragment obtained after enzyme digestion was retained;

[0057] (2) The small fragment obtained by double enzyme digestion is figure 1 Fragment I in is cloned into pBluescript II KS to obtain intermediate plasmid I;

[0058] (3) double-digest the intermediate plasmid I with PacI and AflII, and retain the large fragment obtained by enzyme digestion;

[0059] (4) Using AdEasy5 / 35 as a template to amplify the fiber5 / 35 sequence with PacI and AflII restriction sites at both ends, which is figure 1 Fragment II shown in;

[0060] (5) connecting the fib...

Embodiment 2

[0062] Example 2: Using the recombinant adenovirus vector backbone plasmid pBHG-fiber5 / 35 of the present invention to package a recombinant adenovirus with foreign genes

[0063] The following operations are carried out under aseptic conditions, the principle of packaging poisoning is as follows: Figure 4 Shown:

[0064] 1. Resuscitate HEK-293 cells:

[0065] 1.1 Take a piece of HEK-293 cells frozen in liquid nitrogen, and quickly thaw it in warm water at 35-42°C;

[0066] 1.2 Centrifuge the cells at room temperature, 1000 rpm for 5 minutes;

[0067] 1.3 Discard the supernatant, add 1mL DMEM containing 10% FBS, and gently pipette to resuspend the cells;

[0068] 1.4 Add the resuspended cells to a 25 cm- 2 culture flask at 5% CO 2 , cultured overnight at 37°C;

[0069] 2. Transfection of HEK-293 cells

[0070] 2.1 When the cells are cultured until the confluence reaches 80%, 2 to 6 hours before transfection, change the medium of the cells and replace with fresh DMEM con...

Embodiment 3

[0073] Example 3: After co-transfecting cells with a shuttle plasmid with an exogenous gene sequence encoding GFP and a loxP site and the backbone plasmid of the present invention, the Ad5F35 recombinant adenovirus expressing the exogenous gene GFP is packaged

[0074] 1. Resuscitate HEK-293 cells:

[0075] 1.1 Take a piece of HEK-293 cells frozen in liquid nitrogen, and quickly thaw it in warm water at 35-42°C;

[0076] 1.2 Centrifuge the cells at room temperature, 1000 rpm for 5 minutes;

[0077] 1.3 Discard the supernatant, add 1mL DMEM containing 10% FBS, and gently pipette to resuspend the cells;

[0078] 1.4 Add the resuspended cells to a 25 cm- 2 culture flask at 5% CO 2 , cultured overnight at 37°C;

[0079] 2. Transfection of HEK-293 cells

[0080] 2.1 When the cells are cultured until the confluence reaches 80%, 2 to 6 hours before transfection, change the medium of the cells and replace with fresh DMEM containing 10% FBS;

[0081] 2.2 Co-transfect the cells wi...

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Abstract

This invention publishes a new recombined framework plasmid of adenovirus vector and its application. The framework plasmid constructed by this vector can be used to obtain Ad5F35 recombined adenovirus containing foreign genes quickly, efficiently and easily, with a significantly promoted yield. The technological realization of this invention is to replace the spiked gene sequence coded with Ad5 in pBHGlox (delta) E1, 3Cre framework plasmid with spiked gene sequence coded with Ad35. It has wide application in packaging Ad5F35 recombined adenovirus vector containing foreign genes quickly, steadily and proliferously, and infecting target cells such as malignant tumor cells, human peripheral blood cells and hematopoietic stem cells which are usually difficult to infect with conventional virus vectors, and is further applicable in the field of genetic therapy, research on genetic function, antisense and bacterin development.

Description

technical field [0001] The invention relates to a novel recombinant adenovirus vector backbone plasmid and its application. Background technique [0002] Adenoviral vector is currently one of the most widely used viral vectors, and it is widely used in gene therapy, gene functional research, antisense therapy, vaccine development and other fields. There are two main adenovirus packaging systems commonly used at present: AdEasy system and AdMax system. Among them, the AdEasy system consists of a shuttle plasmid, a backbone plasmid, and a prokaryotic cell (BJ5183). After the foreign gene sequence is constructed into the shuttle plasmid, the recombinant adenovirus is produced by recombining the viral genome with the backbone plasmid in the prokaryotic cell. The AdMax system consists of a shuttle plasmid containing a loxP site, a backbone plasmid, and HEK-293 cell lines. After the foreign gene sequence is constructed into the shuttle plasmid, recombinant adenovirus is produced ...

Claims

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Application Information

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IPC IPC(8): C12N15/861
Inventor 吴小兵袁振华刘宁赵革新高金华董小岩
Owner AGTC GENE TECH CO LTD
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