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Non-viral transfection reagent, preparation method of complex of non-viral transfection vector reagent and plasmid DNA, and application thereof

A complex, non-viral technology, used in recombinant DNA technology, pharmaceutical formulations, genetic material components, etc., can solve the problem of low transfection efficiency, reduce costs, improve product yield and quality, and simple operation programs.

Inactive Publication Date: 2012-04-04
ABZYME BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the calcium phosphate co-precipitation method has lower transfection efficiency than viral vectors or liposomes, and has strong cell dependence

Method used

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  • Non-viral transfection reagent, preparation method of complex of non-viral transfection vector reagent and plasmid DNA, and application thereof
  • Non-viral transfection reagent, preparation method of complex of non-viral transfection vector reagent and plasmid DNA, and application thereof
  • Non-viral transfection reagent, preparation method of complex of non-viral transfection vector reagent and plasmid DNA, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0014] 1. Preparation of PEI for transfection

[0015] 1) Pour 450ml of ultrapure water prepared by Milli-Q into a 500ml beaker;

[0016] 2) Weigh 500 mg linear PEI (purchased from Poly Science Company) on the balance, add it to the beaker, and stir it continuously with a magnetic stirrer;

[0017] 3) Add pre-prepared 12M concentrated HCl dropwise to make the pH<2.0 (about 800 μl is needed). Stir the PEI solution for about 2-3 hours;

[0018] 4) Add pre-prepared 10M NaOH solution to adjust the pH to 7.0 (about 500 μl is required). Dilute the volume of PEI solution to 500ml with Milli-Q ultrapure water.

[0019] 5) Filter the PEI solution with a 0.22-μm filter membrane, aliquot into 1ml / tube, and store at -20°C.

[0020] 2. Calcium Chloride and 2x HEPES Preparation

[0021] 1) Prepare 2M calcium chloride solution

[0022] 2) Prepare 2×HEPES solution and adjust the pH to 7.0~7.2

[0023] 3) Filter the calcium chloride solution with a 0.22-μm filter membrane, store in 2×HE...

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Abstract

The present invention relates to a non-viral transfection vector, a preparation scheme for a complex of the non-viral transfection vector and plasmid DNA, and an application technology thereof. A purpose of the present invention is to provide a transfection reagent and a preparation method for a complex of the transfection reagent and plasmid DNA. With adopting the method of the present invention, the required transfection reagent and the transfected complex can be prepared with the simple operation scheme and the low price. The technology of the present invention is adopted in protein production or gene therapy and other researches, such that the cost can be significantly reduced, the product yield and the product quality can be improved.

Description

technical field [0001] The invention relates to a non-viral transfection vector and a preparation scheme and application technology of a complex with plasmid DNA. Background technique [0002] 1. Conventional transfection method [0003] Transfection is a technique for introducing exogenous genes into eukaryotic cells. It is widely used in protein structure and positioning, gene function and component research, gene expression regulation and gene therapy, and has become an important aspect in the field of biology. tool. Transfection can be roughly mediated by three types of methods: physical, chemical and biological. Common physical mediation methods include electroporation, microinjection and gene gun; there are many chemical mediation methods, such as lipofection, calcium phosphate co-precipitation method, and cationic substance mediation method; biological mediation method At present, the most common methods are various virus-mediated transfection methods. Using virus...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/85A61K48/00
Inventor 柴笑梅朱月珍杨宣武周慧芳卢守英
Owner ABZYME BIOTECH
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