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Preparation method of SIV (simian immunodeficiency virus) vector

A carrier, calcium phosphate technology, applied in the field of medicine, can solve problems such as low production efficiency, inability to meet large-scale industrial production, complex preparation method and process, and achieve the effects of low production cost, large-scale industrial production, and increased DNA concentration.

Inactive Publication Date: 2013-04-24
SICHUAN BAILI PHARM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation method is complex in process and low in production efficiency, and cannot meet the requirements of large-scale industrial production

Method used

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  • Preparation method of SIV (simian immunodeficiency virus) vector
  • Preparation method of SIV (simian immunodeficiency virus) vector
  • Preparation method of SIV (simian immunodeficiency virus) vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: select 3 ordinary culture dishes (dish) of diameter 10cm and 3 same size culture dishes that Collagen-Type I has processed, adopt the same method to carry out the production of SIV-GFP, compare using two kinds of culture dishes in SIV carrier difference in productivity.

[0016] The specific method is: (DNA 32μg +1.5ml 2xBES Buffer) and 1.5ml 167mM CaCl 2 After mixing for 20 minutes, mix with 12ml DMEM medium, add 5ml / dish to three culture dishes with 293T / 17 cells (wash the cells with DMEM before adding), add 20% FBS at 5ml / dish after 3 hours The DMEM medium was cultured overnight, and the culture medium was removed on the second day. After washing with DMEM, DMEM containing 10mM NaB was added at 10ml / dish overnight. After 48 hours of transfection, the supernatant was collected and the titer of SIV-GFP was detected.

[0017] In the prior art, ordinary cell culture flasks are used for the production of SIV vectors. In the present invention, Collagen is us...

Embodiment 2

[0018] Example 2: Select common petri dishes (3 pieces / group) with a diameter of 10 cm, use the same conditions to prepare a DNA mixture, and mix the DNA mixture with DMEM medium, IMDM medium, and Opti-MEM medium respectively. Afterwards, it was added to a culture dish to produce SIV-GFP, and the difference in the productivity of SIV vectors using different media during transfection was compared.

[0019] In the prior art, DMEM medium is used for transfection, and the specific method is the same as in Example 1. The calcium phosphate co-precipitation transfection method is greatly affected by the change of pH, and the use of a medium with a stable pH can improve the transfection efficiency, which is conducive to increasing the production of SIV vectors. Opti-MEM is a medium developed by Invitrogen, with a relatively stable pH and can be widely used for transfection of various transfection reagents. The experimental results found that using Opti-MEM for the production of SIV v...

Embodiment 3

[0020] Example 3: select common petri dishes (3 pieces / condition) with a diameter of 10 cm, use different conditions (1 times DNA concentration, 1.5 times DNA concentration, 2.0 times DNA concentration) to prepare DNA mixture respectively, and mix DNA mixture and DMEM medium After adding into the culture dish to carry out the production of SIV-GFP, the specific method is the same as that of Example 1. The differences in the productivity of SIV vectors using different DNA concentrations during transfection were compared.

[0021] The SIV vector is produced by calcium phosphate co-precipitation transfection method. The DNA concentration during transfection has a certain influence on the transfection efficiency. Although the use of excess DNA will cause toxicity to the cells, appropriately increasing the DNA concentration within the allowable range will Improve transfection efficiency, which is beneficial to increase the production of SIV vector. The experimental results found t...

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Abstract

The invention discloses a preparation method of an SIV (simian immunodeficiency virus) vector. A culture dish is treated with collagen by a calcium phosphate coprecipitation transfection process; and in the transfection process, an Opti-MEM culture medium is used, and the DNA (deoxyribonucleic acid) concentration is increased. The invention has the advantages of ingenious concept and low production cost; and the prepared tolvaptan tablet has the advantages of high in-vitro dissolution, high drug bioavailability and favorable clinical curative effect.

Description

technical field [0001] The invention belongs to the technical field of medicine and relates to a preparation method of an SIV vector. Background technique [0002] SIV (Simian Immunodeficiency Virus) is Simian Immunodeficiency Virus, and SIV vector is a vector based on Simian Immunodeficiency Virus (SIV). [0003] At present, the common preparation method of SIV vector is the calcium phosphate co-precipitation transfection method, which is greatly affected by the change of pH value. The preparation method has complicated process and low production efficiency, and cannot meet the requirements of large-scale industrial production. Contents of the invention [0004] The object of the present invention is to provide a preparation method of SIV vector aiming at the deficiencies in the prior art. The preparation method has ingenious conception, simple process, low production cost, greatly improved production efficiency, and meets the requirements of large-scale industrial prod...

Claims

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Application Information

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IPC IPC(8): C12N15/867
Inventor 朱义游军朱亚峰长谷川户
Owner SICHUAN BAILI PHARM CO LTD
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