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Polynucleotide related to mitochondrial membrane potential decrease and coded polypeptide and application thereof

A technology of mitochondrial membrane potential and polynucleotides, applied in the field of polypeptides

Inactive Publication Date: 2010-11-24
SINOGENOMAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The model based on JC-1 dye to screen inducers and blockers of mitochondrial membrane potential decline has been proposed several years ago. In recent years, many researchers have proposed to combine JC-1 with other dyes for screening. method, but there is no report on the real application of JC-1 for large-scale screening

Method used

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  • Polynucleotide related to mitochondrial membrane potential decrease and coded polypeptide and application thereof
  • Polynucleotide related to mitochondrial membrane potential decrease and coded polypeptide and application thereof
  • Polynucleotide related to mitochondrial membrane potential decrease and coded polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1. Amplification of target gene by two-step flux RT-PCR technology

[0074] (1) Search the NCBI refseq database for unknown human function prediction genes to obtain the unknown human function gene sequence, and use the Human_est database to perform sequence correction by the BLASTn method. The final sequence is set as the following sequence: SEQ ID NO:1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7. Design specific primers for genes MMPRG1, 2, 3, 4 based on such sequences:

[0075]

[0076] (2) Using the above primers, select templates from the existing cDNA library and tumor library according to the expression profile of the target gene, and perform initial amplification. The current library includes 12 normal human tissues (heart, pancreas, testis, ovary, prostate, colon, small intestine, skeletal muscle, thymus, lymph nodes, tonsils, white blood cells); 6 human tumor tissues (lung cancer, pancreatic cancer, ovarian cancer) , Prostate cancer, colon cancer, breast c...

Embodiment 2

[0088] Example 2. Construction of eukaryotic expression vector of target gene

[0089] The two-expanded purified product and the eukaryotic expression vector pcDNA3.1 / V5-His-TOPO (Invitrogen, abbreviated as pcDT) were ligated according to the conditions recommended by the kit manufacturer. The ligation product was transformed into E. coli DH5α by electroporation. The transformant was grown on a solid LB plate medium containing ampicillin. The single cloned colony was selected and the plasmid was extracted. The digested product was digested with EcoRI. The digested product was identified by agarose gel electrophoresis. Select positive clones with inserts, and select the correct forward insert clones by sequencing (ABI PRISM 3700 DNA Analyzer), and name them MMPRG1, 2, 3, and 4 respectively.

[0090] At the same time, the culture fluid was collected, and the precipitated protein was analyzed by SDS-PAGE to obtain MMPRG1, 2, 3, 4 polypeptide.

[0091] MMPRG1, 2, 3, 4 protein analysis r...

Embodiment 3

[0092] Example 3. Using JC-1 dye to determine the effect of the target gene on the decrease of mitochondrial membrane potential

[0093] After transfecting Hela cells with the target gene, use JC-1 dye to stain the cells, operate as described in the product manual, and observe the changes in cell morphology under a fluorescence microscope. The specific steps are as follows:

[0094] In this experiment, a 96-well cell culture plate was used, and each gene to be tested was set up with 3 parallel repetitive wells, and pcDT empty plasmid and Bax plasmid were used as empty vector and positive control respectively. The dosages in the following steps are all single-hole dosages.

[0095] (1) Cell culture: add 1.2×10 4 Two Hela cells (ATCC Number: CRL-11268) were plated on 96-well cell culture plates (Costar, 3599) with DMEM (Dulbecco's modified Eagle's medium) medium (Hyclone, SH0022.02) containing 10% fetal bovine serum ) (100μl culture medium / well), set at 37℃, 5% CO 2 Culture in a cell...

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Abstract

The invention discloses new polynucleotide coding the human protein with the function of causing mitochondrial membrane potential decrease and polypeptide coded by the polynucleotide and an antibody of the polypeptide. The invention also discloses application of the new polynucleotide in causing mitochondrial membrane potential decrease through exogenous expression in host cells.

Description

[0001] The present invention is a divisional application with an application number of 200610165851.7, an application date of December 14, 2006, and the title of the invention "Polynucleotides related to mitochondrial membrane potential decline and their encoded polypeptides and uses". Technical field [0002] The present invention belongs to the field of biotechnology and relates to the field of gene expression regulation. Specifically, the present invention relates to a new class of polynucleotides encoding human proteins with the function of causing a decrease in mitochondrial membrane potential, as well as the encoded polypeptides and antibodies of the polypeptides. The invention also relates to the application of the exogenous expression of such new polynucleotides in host cells to cause the mitochondrial membrane potential to drop. Background technique [0003] Apoptosis belongs to programmed cell death, which is a complex process involving many biochemical reactions in cells...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N15/11C07K14/47C07K16/18C12Q1/68G01N33/53A61K38/17A61K48/00A61K31/7088A61P43/00
Inventor 石太平骆叶马大龙高霞张晨颖袁劲松邓唯唯于鹏高鹏程华玲陆阳马进京王平章
Owner SINOGENOMAX
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