Transfection agent for in-vivo gene expression protein system, and in-vitro transfection method of transfection agent

A technology for gene expression and expression system, which is applied to the transfection agent of gene expression protein system in vivo and the field of in vitro transfection, can solve the problems of few positive charges, poor transfection efficiency, low transfection efficiency, etc., and achieves toxicity. Small, reduced work links, high efficiency

Pending Publication Date: 2020-08-11
苏州世纪坐标生物科技有限公司
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

At the same time, chitosan is still a kind of cationic polymer because of its free amino group and has good biocompatibility. It has been developed as a DNA transfection carrier, but because of its small number of positive charges, the transfection efficiency is low.
[0003] Poly-L-Lysine (Poly-L-Lysine, PLL) is a degradable cationic polypeptide with strong cationic properties and can bind to DNA well, but the transfection efficiency is poor

Method used

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  • Transfection agent for in-vivo gene expression protein system, and in-vitro transfection method of transfection agent
  • Transfection agent for in-vivo gene expression protein system, and in-vitro transfection method of transfection agent
  • Transfection agent for in-vivo gene expression protein system, and in-vitro transfection method of transfection agent

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preparation example Construction

[0041] Wherein the preparation method of the plasmid having the protein gene sequence to be expressed comprises the following steps:

[0042] Step 1, construct gene carrier, such as figure 1 The vector shown is based on a circular plasmid vector. The sequence of the circular plasmid vector is shown in Sequence Table 1. The CMV (CMV enhancer+CMV promoter) in the circular plasmid is used as the promoter, containing Poly(A), circular The sequence of the plasmid vector is shown in Sequence Table 1, and the total length is 1293bp;

[0043] Step 2, insert the protein gene sequence to be expressed between the CMV promoter and the Poly(A) on the gene vector constructed in the step 1, taking the insertion of a fluorescent protein gene fragment as an example, its structure is as follows figure 2 As shown, the gene sequence is shown in sequence 2.

[0044] The preparation method of the chitosan polylysine polymer for the transfection agent of gene expression protein system in vivo as ...

Embodiment 1

[0050] 1. In vitro transfection, taking 293T cells as an example:

[0051] In the first step, transfection was carried out in a 6-well cell culture plate, and the cells were divided into 5X10 5 Inoculate the cells / well, add 2-5ml complete medium (medium + 10% serum), and culture overnight;

[0052] In the second step, take 5 μg of fluorescent plasmid / well, mix it with chitosan polylysine polymer at a volume ratio of 1:3, and mix well;

[0053] In the third step, add 100 μl of OMEM medium and mix thoroughly;

[0054] In the fourth step, add 20 μl of 50% PEG3350 and mix well;

[0055] In the fifth step, the mixture is added to the cell culture system of the six-well plate;

[0056] The sixth step, replace the culture medium after 24 hours;

[0057] The seventh step is to continue to observe for 48 hours.

[0058] like Figure 4 As shown, after 293T cells were transfected, observed after 24 hours, the cell expression fluorescence efficiency was about 30%, as shown in Figu...

Embodiment 2

[0066] 1. In vitro transfection, taking 293T cells as an example:

[0067] In the first step, transfection was carried out in a 6-well cell culture plate, and the cells were divided into 5X10 5 Inoculate the cells / well, add 2-5ml complete medium (medium + 10% serum), and culture overnight;

[0068] In the second step, take 5 μg of fluorescent plasmid / well, mix it with chitosan polylysine polymer at a volume ratio of 1:3, and mix well;

[0069] In the third step, add 100 μl of OMEM medium and mix thoroughly;

[0070] In the fourth step, add 20 μl of DMSO and mix well;

[0071] In the fifth step, the mixture is added to the cell culture system of the six-well plate;

[0072] The sixth step, replace the culture medium after 24 hours;

[0073] The seventh step is to continue to observe for 48 hours.

[0074] like Figure 7 As shown, after 293T cells were transfected, observed after 24 hours, the cell expression fluorescence efficiency was about 30%, as shown in Figure 8 As...

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Abstract

The invention provides a method for in-vitro transfection by using a transfection agent of an in-vivo gene expression protein system. The method comprises the following steps of 1, inoculating cells,adding a complete culture medium, and culturing overnight; 2, mixing protein genetic sequence plasmid to be expressed and chitosan poly-l-lysine polymer according to a volume ratio of 1 to 3, and fully and uniformly mixing; 3, adding a culture medium into the mixed solution obtained in the step 2, and fully and uniformly mixing; 4, adding the mixed solution obtained in the step 3 into a cell culture system obtained in the step 1, and culturing for 24 hours; and 5, replacing the culture medium, and continuously observing for 48 hours until the culture is finished. The beneficial effects of theinvention is as follows: the chitosan poly-l-lysine copolymer is designed by using the characteristics of chitosan and poly-l-lysine, the cell transfection efficiency of DNA can be maintained at about80%, the cell keeps a certain viability (98%), the transfection reagent efficiency is high, and the toxicity is low.

Description

technical field [0001] The present invention relates to the field of gene expression protein systems in vivo, more specifically to a transfection agent for in vivo gene expression protein systems and an in vitro transfection method thereof. Background technique [0002] The non-viral transfection is effectively absorbed by the cells to the inside of the cells to achieve the effect of gene transfection. This method is simple and practical, and has relatively low damage to cells and high transfection efficiency. Different positive ions are used to introduce foreign genes into eukaryotic cells, so that cells can express specific genes, which is safe and proven. Modified properties. Transfection methods can be divided into two types: physically mediated and chemically mediated. Physical methods mainly rely on external help to pierce the cell membrane to introduce genes into cells, including electroporation, microinjection, etc.; chemically mediated methods, such as calcium pho...

Claims

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Application Information

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IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 赵钢史铭哲张达
Owner 苏州世纪坐标生物科技有限公司
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