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Sequence and application of a promoter of a human autophagic gene beclin 1

A promoter and gene technology, applied in the field of molecular biology, can solve problems such as expression regulation research

Inactive Publication Date: 2011-06-22
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little research has been done on the expression regulation of this gene

Method used

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  • Sequence and application of a promoter of a human autophagic gene beclin 1
  • Sequence and application of a promoter of a human autophagic gene beclin 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] PCR amplification of promoter sequence of human beclin 1 gene

[0076] First, the genomic DNA was extracted from the commercially available human normal liver cell line L02 (purchased from the Cell Bank of the Chinese Academy of Sciences), that is, in the collected cultured cell samples (5×10 7 ), add 10 times volume of extraction buffer (10mM Tris-HCl pH 8.0, 0.1M EDTA pH 8.0, 20μg / mL RNase, 0.5% SDS), react at 37°C for 1 hour, add proteinase K to a final concentration of 100μg / ml in a water bath at 50°C for 3 hours, stirring occasionally during this period. Cool to room temperature, add an equal volume of Tris saturated phenol, mix well and centrifuge at 12,000 rpm for 5 minutes, collect the supernatant, extract once more with phenol: chloroform and chloroform, and precipitate genomic DNA with 2 times the volume of absolute ethanol , the resulting genomic DNA was fully dissolved with an appropriate amount of TE and stored at 4°C.

[0077] After obtaining the genomi...

Embodiment 2

[0081] Construction of human beclin 1 gene promoter eukaryotic expression vector

[0082] The beclin 1 promoter -644 / +197 fragment amplified by PCR was forwardly or reversely inserted into the pMD18-T vector (purchased from TaKaRa Company) to obtain the plasmid pMD18-T-beclin1 containing appropriate restriction sites (-644 / +197) and pMD18-T-beclin1-rev (+197 / -644).

[0083] Use BglII and HindIII to digest pMD18-T-beclin1 (-644 / +197) to obtain a fragment containing 841bp promoter sequence (SEQ ID NO: 1), and then insert the fragment into eukaryotic through BglII and HindIII restriction sites Expression vector pGL3-Basic (purchased from Promega Company) of the 5' end upstream of the luciferase reporter gene, thus completing the p(-644 / +197) plasmid (that is, containing the positively inserted SEQ ID NO: 1 sequence construction of the plasmid).

[0084] To complete the construction of the p(+197 / -644) plasmid (i.e., the plasmid containing the sequence of SEQ ID NO: 1 inserted i...

Embodiment 3

[0091] Activity identification of human beclin 1 gene promoter in L02 human immortalized normal liver cells

[0092] The preparation of ultrapure plasmids for cell transfection uses QIAGEN Plasmid Midi Kit and carries out plasmid column purification according to the method provided by the supplier:

[0093] 1. Take 25-50ml of the cultured bacteria solution overnight (that is, the aforementioned transformed E. coli), centrifuge at 6,000rpm for 10 minutes, and collect the cells;

[0094] 2. Resuspend the pellet in 4ml Buffer P1 (provided by QIAGEN Plasmid Midi Kit), add 4ml Buffer P2, mix by inverting gently, let stand at room temperature for 5 minutes, add 4ml pre-cooled BufferP3 (provided by QIAGEN Plasmid Midi Kit), and invert to mix Evenly, place on ice for 15 minutes;

[0095] Centrifuge at 20,000g for 30 minutes at 3.4°C, and immediately remove the supernatant containing plasmid DNA;

[0096] Centrifuge at 20,000g for 15 minutes at 4.4°C, and immediately remove the super...

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Abstract

The invention belongs to the field of molecular biology and discloses a promoter from a promoter region of an autophagic gene beclin 1 and application thereof. The invention also discloses an expression vector containing the promoter and a host cell. The invention also discloses a method for the high-level expression of target genes by using the same. The promoter of the invention has wide activities and can direct the high-level expression of the target gens in various host cells.

Description

technical field [0001] The invention belongs to the field of molecular biology; more specifically, the invention relates to a human autophagy gene promoter and the application of the promoter. Background technique [0002] Autophagy is a widespread physiological mechanism, which mainly combines macromolecular substances in the cytoplasm (such as proteins, glycogen, etc.) Organelles) are degraded in large quantities in vesicles wrapped in unit membranes to achieve recycling to maintain the process of cell self-stabilization. Autophagy has a variety of physiological functions, such as: ①Provide nutrients for cells when nutrients are scarce; ②Cell differentiation; ③Cell death and aging; ④Prevent the occurrence of cancer. Autophagy is also related to the pathological process of various diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and heart failure. [0003] The human autophagy gene beclin 1 is the first gene involved in autophagy found in ma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/79C12N1/21C12N5/10
Inventor 赵慕钧唐红答亮李栋毛怡李颖徐振华李载平
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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