Human SFRP1 gene, its coded protein and application

A gene and protein technology, applied in the field of new human genes and proteins, can solve the problems such as the absence of public reports on the use of SFRP1 protein to inhibit tumors

Inactive Publication Date: 2007-05-16
CHINESE NAT HUMAN GENOME CENT AT SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Before the present invention, there was no published report related to the use of the human SFRP1 protein of the present invention to suppress tumors

Method used

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  • Human SFRP1 gene, its coded protein and application
  • Human SFRP1 gene, its coded protein and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] After treating 15 liver cancer cell lines with the demethylation drug DAC (final concentration: 2000nM), the gene expression level difference before and after treatment was detected by gene chip, and it was found that the expression of SFRP1 gene was up-regulated in 8 cell lines. Among them, the gene chip experiment mainly includes the following four steps: chip preparation, sample preparation, hybridization reaction and signal detection, and result analysis.

[0047] 1. Chip preparation At present, glass or silicon wafers are mainly used as carriers for preparing chips. The target gene is arranged on the carrier as a probe in order by the spotting method, and the target gene can be divided into genomic DNA, cDNA (or artificially synthesized DNA).

[0048] 2. Sample preparation The extraction steps of total RNA in the sample to be tested are as follows: take 15 liver cancer cell lines treated with demethylation drug DAC and 15 liver cancer cell lines without demethylati...

Embodiment 2

[0061] RT-PCR assay was used to detect the expression of SFRP1 gene in liver tissue.

[0062] 1. Tissue isolation The tissues used in the experiment were derived from 72 HBV-positive primary liver cancer patients who expressed alpha-fetoprotein (RT-PCR confirmed that AFP was positive in the liver cancer and negative in the adjacent tumor). Once the surgically resected liver was isolated from the body, the lesion and surrounding paracancerous tissues 5 cm away were quickly excised and stored in liquid nitrogen (-80°C). The diagnosis of cancer and paracancer is based on pathological diagnosis. In addition, 2 cases of normal liver and fetal liver tissue were also taken for the same treatment, that is, stored in liquid nitrogen (-80°C).

[0063] 2. Total RNA extraction kit The RNA extraction reagent uses TRIzol reagent (GIBCO / BRL), which is produced based on the one-step extraction method with acidic phenol. The utensils and water used for RNA extraction must be RNase-free to en...

Embodiment 3

[0074] Overexpression of SFRP1 gene by transgenic technology can inhibit the growth of liver cancer cells.

[0075] 1. PCDNA3.1-SFRP1 plasmid construction PCR amplifies the ORF region of SFRP1 with a total of 314 amino acids, and introduces restriction sites BamHI and EcoRI at both ends. After digestion, it is ligated with PCDNA3.1 digested with the same enzyme, and transformed into Escherichia coli Single clones were picked and sequenced.

[0076] The primer sequences used are as follows, and the primers for SFRP1 are (the stop codon is not removed and his-myc is not included):

[0077] Sfrpl 3.1B(-): F: 5'-CGGGATCCATGGGCATCGGG-3':

[0078] R: 5'-CGGAATTCCGTCACTTAAACACGGACT-3'.

[0079] The plasmid was transfected into the cell line, and the expression of SFRP1 protein was detected by western detection with anti-SFRP1 antibody.

[0080] 2. Transfection

[0081] (1) On the first day, the cell (35mm) density reaches 80%, shake well at 37°C, and culture overnight with 5% CO2...

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Abstract

The invention discloses a human SFRP1 gene sequence and coding protein and application in the drug to inhibit tumour from growing, which is characterized by the following: treating or lessening the loss, non-functional or abnormal disease of human SFRP1 protein; making human SFRP2 gene express in the cancer cell through gene-transmitting technology or supplementing human SFRP1 protein in vitro; controlling cancer from breeding and killing cancer cell.

Description

technical field [0001] The present invention relates to a novel human gene and protein, in particular to a nucleic acid sequence of human SFRP1 gene and its encoded protein; in addition, the present invention also relates to the application of the gene encoded protein. Background technique [0002] SFRP, secreted frizzled-related proteins (secret frizzled-related proteins), contains a cysteine-rich domain (cysteine ​​rich domain, CRD), but lacks seven transmembrane domains, it may be related to frizzled, Frz) competes for binding to Wnt protein, thereby inhibiting the overexpression of Wnt protein. Wnt is a kind of secreted glycoprotein, which regulates and promotes the proliferation of normal human cells. Recent experiments have found that when the gene SFRP, which inhibits the function of this protein, is abnormal, the function of Wnt protein will be abnormal, which will make the cancer cells continue to proliferate, leading to colorectal cancer. When the normal SFRP gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/63A61K38/17A61P35/00
Inventor 黄健韩泽广张云丽
Owner CHINESE NAT HUMAN GENOME CENT AT SHANGHAI
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