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Fusion proteins for regulating and controlling CCR5 and CXCR4 genes and regulation and control method

A technology of gene activity and species, applied in the field of fusion proteins, can solve problems such as hindering application

Active Publication Date: 2014-04-16
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although TALE still has many problems to be solved by in-depth research, this does not hinder its application.

Method used

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  • Fusion proteins for regulating and controlling CCR5 and CXCR4 genes and regulation and control method
  • Fusion proteins for regulating and controlling CCR5 and CXCR4 genes and regulation and control method
  • Fusion proteins for regulating and controlling CCR5 and CXCR4 genes and regulation and control method

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0071] Example 1 TALEN plasmid amplification;

[0072] First, take 50 μl of competent cells DH5α (TIANGEN) from the -80°C refrigerator, and thaw on ice for about 10 minutes. Then add TALEN plasmid DNA to the competent cell suspension (50ul competent cells can be saturated by 500nG supercoiled DNA), gently rotate the centrifuge tube to mix the contents, and let it stand in an ice bath for 30min. Immediately after the heat shock in a water bath at 42°C for 90 seconds, place it on ice to cool for 3-5 minutes quickly after the heat shock, then add 500ul LB liquid medium (without antibiotics) to the tube, shake well and take 100μl to spread on On the screening plate containing Amp, place it face up for half an hour. After the bacterial solution is completely absorbed by the medium, invert the culture dish and incubate at 37°C for 16 hours.

[0073] Pick a single clone on the culture plate and put it in 3ml Amp LB medium, and culture it in a shaker at 37°C; after 16 hours, inoculat...

example 2

[0074] Example 2TALEN plasmid extraction (using Plasmid Plus Maxi)

[0075] Do the following preparations first. Add RNase A enzyme to P1 buffer, mix, and store at 2-8°C; then add RNase A in a ratio of 1:1000 Check whether solution P2 contains SDS precipitate. If so, place it at 37°C for a short time until it is completely dissolved; before use, add ethanol (96-100%) to solution PE (see bottle label).

[0076] Next, a large amount of plasmid extraction is performed. Collect 100 mL of Escherichia coli liquid obtained by overnight shaking with a centrifuge at 4°C and 6000×G for 15 minutes. Then resuspend the bacteria with 5ml of solution P1 and mix them repeatedly with a shaker. Then add 5ml of solution P2, and gently invert the bottle up and down 4-6 times until the bacteria are completely lysed and appear sticky. The lysed solution was then placed at room temperature for 3 min. If LyseBlue reagent has been added, the cell suspension will turn blue. Put the new QIAfilt...

example 3

[0077] Example 3Ghost cell cell culture

[0078] Before the cells were passaged, the cells were observed under the microscope to cover the bottom of the culture dish. Then put the cell culture medium (DMEM), PBS, and trypsin at room temperature for half an hour, and at the same time irradiate the cells with ultraviolet rays for 30 minutes before experimenting on the ultra-clean bench.

[0079] First use a pipette to suck away the original culture solution in the overgrown single-layer Ghost cell culture bottle, add 1ml of 0.25% trypsin solution to digest the cells (the volume added depends on the size of the specific culture dish. For example, a 10cm culture dish The cultured cells can be digested with 5ml 0.25% trypsin solution), and placed in a 37°C incubator to digest the cells for about 5 minutes; observe the changes of cell adhesion and shape under an inverted microscope, when the original adherent cells gradually tend to be round , floated up and began to disperse into ...

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PUM

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Abstract

The invention relates to transcription activator like effectors (TALE). The TALE can identify and bind with DNA fragments in human CCR5 gene active regions or CXCR4 gene active regions. The human CCR5 gene active regions comprise three regions and the CXCR4 gene active regions comprise two regions. The TALE binds with endonuclease so that a fusion protein TALEN is formed. Through TALEN specific loci aiming at the CCR5 or CXCR4 gene, a shearing process is carried out so that after gene regulation and control, the cell has HIV resistance.

Description

technical field [0001] The present invention relates to the method for the human CCR5 and CXCR4 gene change, and the fusion protein used in this method, particularly relate to including class transcription activator (Transcription Activator Like Effectors, TALE) and endonuclease / partial endonuclease A fusion protein, and a method for altering human CCR5 and CXCR4 genes using the fusion protein. Background technique [0002] According to the joint assessment of the AIDS epidemic in China by the Ministry of Health, UNAIDS and WHO in 2011, by the end of 2011, it was estimated that there were 780,000 (620,000-940,000) living HIV-infected and AIDS patients in China, including newly-infected HIV-infected AIDS has become a serious public health problem in my country. [0003] During the development of AIDS, the contest between HIV and the human immune system has not stopped for a moment, but so far no infected person has been able to spontaneously eliminate HIV. This is because th...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N9/22C12N15/12C12N15/55C12N15/861C12N5/10
CPCC07K14/195C07K14/7158C07K2319/80C12N9/22C12N15/85
Inventor 张林琦庄峰锋史宣玲石冰洁李娟李绍路
Owner TSINGHUA UNIV
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