Polynucleotide affecting SRE activity and its coding polypeptides and use

A technology of polynucleotide and nucleotide sequence, applied in the direction of medical preparations containing active ingredients, applications, peptide sources, etc., can solve the problems of reduced ability of SRF to bind DNA, aging of SRF, and reduced ability of gene induction

Inactive Publication Date: 2007-03-21
SINOGENOMAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More importantly, in vitro experiments found that SRFΔ4, 5 can inhibit the expression of full-length SRF, thereby inhibiting the expression of genes regulated by SRF
[0010] 3. SRF and Aging
This difference in SRF may contribute to the obs

Method used

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  • Polynucleotide affecting SRE activity and its coding polypeptides and use
  • Polynucleotide affecting SRE activity and its coding polypeptides and use
  • Polynucleotide affecting SRE activity and its coding polypeptides and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1, two-step method flux quantification RT-PCR technique amplifies target gene

[0086] gene name

Upstream primer (5'-3')

Downstream primer (5'-3')

SREIF1

SREIF2

SREIF3

SREIF4

SREIF5

SREIF6

SREIF7

SREIF8

SREIF9

ctgctcgcacaggactcgg

cagacgccacggagtttgtg

gtgcgatgagcttcttcggc

gtgatatgagcaacaatggaccag

caccatgatgcaaatctgcga

aggacctgggtttcagtgatgag

gctcctctggctgatggcat

ggactttccttacctgtttttccag

cgcttgccattcaacatcatg

gggctataactcagactgggatctg

tggcaccagaaaaacccatctc

tgcaagtaacgggtcaactgagc

cactatattactcactcgtcaccactctg

tgggtcgaagaaggtgctgg

cctgatagccagtcgagttctcttatatc

tcctgggacagagccactaagtc

ggctatgtggctgttgtgtgc

ggaaaataaactgagcctactgaggag

[0087] (2) Using the above primers, select a template from the existing cDNA library and tumor library according to the expression profile of the target gene, and perform ini...

Embodiment 2

[0097]Embodiment 2, the construction of target gene eukaryotic expression vector

[0098] The purified product of secondary amplification and the eukaryotic expression vector pcDNA3.1 / V5-His-TOPO (Invitrogen, abbreviated as pcDT) were ligated according to the conditions suggested by the kit manufacturer. The ligation product was transformed into Escherichia coli DH5α by electric shock method, and the transformant was grown on a solid LB plate medium containing ampicillin, and the grown single clone colony was selected, the plasmid was extracted, digested with EcoRI, and the digested product was identified by agarose gel electrophoresis. Positive clones with inserts were selected, and the correct positive insert clones were selected by sequencing (ABI PRISM 3700 DNA analyzer), named SREIF1, 2, 3, 4, 5, 6, 7, 8, and 9 respectively.

[0099] At the same time, the culture fluid was collected, and the precipitated protein was analyzed by SDS-PAGE to obtain SREIF1, 2, 3, 4, 5, 6, 7,...

Embodiment 3

[0101] Example 3. The dual-luciferase reporter gene method was used to determine the inhibitory effect of the target gene on PMA-induced SRE activation.

[0102] Human embryonic kidney 293T cells were co-transfected with the target gene and the reporter gene pSRE-luc, pRL-TK, and the activity of SRE was determined by detecting the two luciferase activities respectively. The method of co-transfection was cationic non-lipid transfection method, which was carried out by Wiglass Biotechnology (Beijing) Co., Ltd., as described in the product manual.

[0103] The transfection steps are as follows:

[0104] (1) Cell culture: use 293T cells (ATCC Number: CRL-11268) (2.0×104) with DMEM (Dulbecco's modified Eagle's medium) medium (Hyclone, SH0022.02) containing 10% fetal bovine serum Spread on a 96-well cell culture plate (Costar, 3599) with a seeding density of 12,000 / well, a medium volume of 100ul / well, and cultivate in an incubator at 5% CO2 for 18-24 hours at 37°C.

[0105] (2) Pr...

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PUM

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Abstract

The present invention discloses one new kind of polynucleotide encoding human protein with function of affecting SRE activity, and its coding polypeptide and the antibody of the polypeptide. The present invention also discloses the application of the exogenous expression of the polynucleotide in host cell in affecting SRE activity. The present invention also discloses the use of the polypeptide and antibody in preparing medicine for preventing and treating muscle system diseases or tumors related with proliferation and muscle differentiation.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the field of gene expression regulation. Specifically, the invention relates to a new class of polynucleotide encoding a human protein with the function of affecting SRE activity, its encoded polypeptide, and an antibody to the polypeptide. The present invention also relates to the application of the effect of exogenous expression of this new polynucleotide in host cells on SRE activity. The present invention also relates to the use of the polypeptide and antibody in the preparation of drugs for preventing and treating muscular system diseases or tumors related to proliferation or muscle differentiation. Background technique [0002] Serum response element SRE (serum response element) is a short cis-acting element mainly present in the promoter regions of two types of genes. One of them is immediate early genes, such as c-fos, fosB, junB, egr-1 and-2, etc. Can mediate activation of im...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C07K16/18A61K38/17A61P21/00A61P35/00G01N33/53C12Q1/68
Inventor 高霞贺鹏飞石太平高鹏张晨颖程华玲邓唯唯李娜马进京郭金海童郁蓉蔡恬静陆阳王平章王欣宇王峰马大龙
Owner SINOGENOMAX
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