DNA sequence regulating eukaryotic gene transcription, and its binding proteins

A gene and actin technology, applied in the field of DNA sequences regulating eukaryotic gene transcription and their binding proteins, can solve problems such as unclear molecular mechanisms of acting factors

Inactive Publication Date: 2012-08-01
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, introns are important regulatory sequences of the var gene family, but the corresponding acting factors (nucleoproteins) and their molecular mechanisms are still unclear

Method used

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  • DNA sequence regulating eukaryotic gene transcription, and its binding proteins
  • DNA sequence regulating eukaryotic gene transcription, and its binding proteins
  • DNA sequence regulating eukaryotic gene transcription, and its binding proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1, Amplification of Plasmodium falciparum var gene intron sequence and probe preparation

[0086] (1) Intron sequence amplification of var gene

[0087] In order to prepare EMSA probes, it is first necessary to amplify the var gene intron sequence from the genome of Plasmodium falciparum 3D7 strain (see Chinese patent application 200610118947.8), clone it into a vector and perform sequencing. Since the AT content in the non-coding region of Plasmodium falciparum can be as high as more than 90%, ordinary Taq enzymes cannot effectively amplify these sequences, and it is difficult to ensure the correctness of the sequences. In this example, in order to balance amplification efficiency and sequence fidelity, the inventors all used Toyobo's KOD-Plus high-fidelity enzyme. The specific reaction preparation and amplification procedures are as follows:

[0088] 10x KOD-Plus reaction buffer: 5 μl;

[0089] MgCl (2mM): 5μl;

[0090] dNTP (2mM): 5μl;

[0091] Upstream ...

Embodiment 2

[0108] Example 2, Preliminary Extraction of Total Nucleoprotein of Plasmodium falciparum Hainan Strain

[0109]1. Get 100ml of Plasmodium falciparum 3D7 strain culture cultured in vitro, the parasite rate is about 10% (the number of erythrocytes infected by Plasmodium per 100 erythrocytes), 2000rpm, 5min to remove the supernatant.

[0110] 2. Add 20ml of 0.15% (w / v) saponin / PBS solution, mix well, 4°C, 30min.

[0111] 3. 8000rpm, 4°C, 10min, remove the supernatant.

[0112] 4. Wash 3 times with 20ml of cold 1xPBS solution, 4°C, 8000rpm, 10 minutes each time.

[0113] 5. Add 1.5ml complete cell lysate (20mM Hepes pH7.9, 10mM KCl, 1mM EDTA, 1mM EGTA, 1mM DTT, 1× protease inhibitor mixture, 0.65% (v / v) NP-40), mix well, Shake for 10 seconds and place in an ice-water bath for 10 minutes.

[0114] 6. 8000rpm, 5min, carefully draw the supernatant, which is the Plasmodium cytoplasmic protein, and store it at -80°C after aliquoting.

[0115] 7. Repeat 2 times to fully wash the nuc...

Embodiment 3

[0120] Embodiment 3, EMSA experiment identification intron-nucleoprotein complex

[0121] In this example, the inventor mainly used Pierce's LightShift Chemiluminescent EMSA kit to identify nucleoproteins (see the Pierce 20418 product manual for specific methods). Its main steps are as follows:

[0122] A. Preliminary experiment, initially establish the EMSA system, and explore the optimal amount of probe;

[0123] B. EMSA analysis

[0124] (1) Prepare the corresponding non-denaturing PAGE gel according to the size of the probe.

[0125] (2) Use 0.5×TBE as electrophoresis buffer, 4°C, 100V, pre-electrophoresis for 1 hour.

[0126] (3) According to different experimental purposes, design the corresponding EMSA reaction system (taking 20 μl system as an example), and each system is shown in Table 1.

[0127] Table 1

[0128]

[0129]

[0130] *In the table, the volume less than 20μl is supplemented with water.

[0131] **The preparation of the binding buffer (EMSA bu...

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Abstract

The invention relates to a DNA sequence regulating eukaryotic gene transcription, and its binding proteins. The inventor firstly identifies and separates the non-coding DNA sequence for transcription silencing of a mediated var gene family from Plasmodium falciparum, and its binding proteins actins. The interaction between the DNA sequence and the actins makes the var gene be anchored in a perinuclear heterochromatin zone to lead to gene silencing; and substances adjusting the var gene transcription and then adjusting the virulence of Plasmodium falciparum can be screened based on the new discovery of the invention.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a DNA sequence and a binding protein for regulating eukaryotic gene transcription. Background technique [0002] Plasmodium falciparum erythrocyte membrane surface protein (PfEMP1) is an encoded product of the var gene family, and is one of the most important virulence factors of Plasmodium falciparum, mediating processes such as adhesion between infected erythrocytes and host cell receptors and immune evasion. The var gene family has the characteristics of mutually exclusive expression, and in about 60 var genes, generally only one of them is expressed. During the life cycle cycle of Plasmodium in erythrocytes, the only expressed member is constantly switching (Switching), resulting in a high degree of antigenic variation. Studies have shown that this process is regulated at the transcriptional level: all var genes are located in the nucleus, silent genes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12Q1/68
CPCY02A50/30
Inventor 潘卫庆张青锋
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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