Unlock instant, AI-driven research and patent intelligence for your innovation.

Target gene entraining system using histone as core sequence

A histone and sequence technology, applied in the fields of molecular biology and gene therapy, can solve problems such as being unsuitable for large-scale production and complex preparation process

Inactive Publication Date: 2004-01-28
NEWORGEN
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of this patent application is that the polycationic polypeptide needs to be chemically synthesized, and then linked to the ligand oligopeptide, endocytic body release oligopeptide, etc., so the preparation process is complicated and not suitable for large-scale production

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Target gene entraining system using histone as core sequence
  • Target gene entraining system using histone as core sequence
  • Target gene entraining system using histone as core sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Obtaining of the expression sequence of each fusion protein

[0092] to construct GE7-Histone H1 0 -HA20 and GE7-Histone H1 0 97-193 The expression sequence of -HA20 is an example:

[0093] 1. Linker-Histone H1 0 - Linkers and Linkers - Histone H1 0 97-193 - Obtaining of linker expression sequences:

[0094] To ensure the fusion protein GE7-histone H1 expressed in genetic engineering 0 -HA20 and GE7-Histone H1 0 97-193 - In HA20, GE7 and HA20 each play an independent biological function, in their interaction with histone H1 0 and histone H1 0 97-193 designed by (Gly) 4 The oligopeptide composed of Ser serves as a flexible structure (hereinafter referred to as linker) to maintain the structural stretchability of GE7 and HA20 functional peptides.

[0095] The primers shown in Table A were designed and synthesized.

[0096] Table A Primers Primer Sequence SEQ ID NO: Primer 1-Histone-L 5'- TGGTGGCGGTTC TACCGAGAATTCCACGTCC-3′ 19 Primer 2-Histone 97-...

Embodiment 2

[0104] Example 2 Histone H1 0 Expression, separation and purification of a series of fusion proteins with backbones:

[0105] 1. Induction, expression:

[0106] The expression plasmids were respectively transformed into competent cells expressing the host strain HMS174(DE3), and the preparation and transformation conditions of the competent cells were carried out according to "Molecular Cloning". The transformed competent bacteria were smeared on ampicillin LB agar plate and kept overnight at 37°C. Pick a single colony in 5ml LB containing ampicillin resistance, shake overnight at 37°C, dilute 1:50 in 1000ml LB and culture until the bacterial density OD600nm is about 0.6, add IPTG to a final concentration of 1mM, and induce at 30°C for 3 hours. The bacteria were collected by centrifugation, 40 ml of ultrasonic solution (50 mM Tris·Cl, pH 8.0, 10 mM EDTA, 0.5 mM PMSF) was added, and the ice bath was ultrasonicated for 1 hour. After ultrasonication, the bacterial liquid was c...

Embodiment 3

[0116] Example 3 Histone H1 0 The complex polypeptide and DNA as the backbone form a complex

[0117] The combination of DNA and each fusion protein or different fusion proteins is mixed according to the mass ratio of 1:3, and left at room temperature for one hour, so as to be used in in vitro and in vivo biological function tests. The complex containing 0.2 μg of plasmid DNA was used for 1% agarose electrophoresis identification to confirm that the plasmid DNA had been completely suppressed in the sample well. The final concentration of DNA in the complex was 0.2 μg / μl. See attached drawings 15-20.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a target gene introduction system using histone as core sequence, its preparation method and application. Said gene introduction system includes fusion protein using histone as core sequence, it contains (a). histone element which is selected from H1 to the power 0 full-length sequence or its fragment richyl containing lysine; (b) receptor identification oligopeptide element LOP; and (c) arbitrarily-selected endocytic corpusculum release oligopeptide EROP. Said system not only can high-effectively introduce exogenous DNA, but also can be conveniently prepared byusing gene engineering technique.

Description

technical field [0001] The present invention relates to the field of molecular biology and gene therapy, more specifically, relates to a gene introduction system with histone as the core sequence, its preparation method and application. Background technique [0002] Gene therapy is to introduce foreign DNA into specific cells of the human body to produce therapeutic effects and achieve the purpose of treating diseases. To this end, it is necessary to have a safe and effective gene transfer system. At present, there are two main types of methods that can effectively transfer foreign DNA into human cells, one is the method mediated by viral vectors, and the other is the method mediated by non-viral vectors. guide method. [0003] Fritz et al. have expressed histone H1 in Escherichia coli 0 The C-terminal region, but only with the participation of liposomes and chloroquine, can DNA be introduced into eukaryotic cells (Hum. Gene Ther. 1996, 7: 1395-1404). Furthermore, the sys...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): A61K48/00C07K14/47C12N15/87
CPCC12N2810/80C07K14/47A61K48/00C12N15/87C07K2319/01
Inventor 顾健人杨胜利龚毅朱景德陈燕戴菲寒任常青田培坤
Owner NEWORGEN