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Human testicle specificity protein 50 gene expression regulator screening system and method of screening its gene expression regulator

A technology of gene expression and regulator, applied in gene therapy, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2006-08-30
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recent studies have shown that abnormally high expression of TSP50 is found in more than 90% of breast cancer tissues, and this expression is limited to malignant epithelial cells (Shan, J., Yuan, L, Xiao, Q., Chiorazzi, N., Budman, D., Teichberg, S., and Xu, HP, TSP50, A possible protease in human testis, is activated in breast cancer repithelial cells. Cancer Res. 2002, 62, 290-294)

Method used

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  • Human testicle specificity protein 50 gene expression regulator screening system and method of screening its gene expression regulator
  • Human testicle specificity protein 50 gene expression regulator screening system and method of screening its gene expression regulator
  • Human testicle specificity protein 50 gene expression regulator screening system and method of screening its gene expression regulator

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Experimental program
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Embodiment 1

[0052] Extraction of Human Genomic DNA

[0053] Take 3ml of heparin-anticoagulated human peripheral blood, centrifuge at 3000rpm at 4°C for 20 minutes, absorb the upper layer of plasma, add 5 times the volume of sterile double-distilled water, mix well, and place at room temperature for 5-10 minutes. 4000rpm, 4°C, centrifuge for 20 minutes, discard the supernatant. Add 5ml of normal saline to restore the white blood cells to an isotonic environment. Centrifuge at 4000rpm at 4°C for 15 minutes, discard the supernatant, and obtain the white blood cell layer. Add 5ml of digestion buffer (TES solution) (15mM Tris-cl, 15mM EDTA, 15mM Nacl, 0.5% SDS) to the white blood cells, add proteinase K to a final concentration of 0.1mg / ml, mix well, and place in a 50°C water bath for 3- 5 hours (4.5h). After cooling, add an equal volume of Tris phenol and extract twice with chloroform:isoamyl alcohol (24:1). Aspirate the supernatant carefully, add 1 / 10 volume of 3M sodium acetate (pH5.2),...

Embodiment 2

[0055] Fishing of the TSP50 promoter region

[0056] 1. Primer Design

[0057] Search the nucleotide sequence of the TSP50 promoter region from GenBank according to literature reports, and design primers for PCR amplification according to the sequence, so as to capture the TSP50 promoter. The primer sequences are as follows: the upstream primer is: 5'-GGGGTACCCCCAAGCAGTCC-3'; the downstream primer is: 5'-GAAGATCTTCCCGGGGTGGC-3'. The 5' end has a KpnI restriction site, and the 3' end has a BglII restriction site.

[0058] 2. PCR amplification, electrophoresis and recovery

[0059] Using human genomic DNA as a template, the primers synthesized above were used for PCR amplification. The PCR reaction conditions were: 94°C, 5min; 94°C, 1min, 62°C, 45sec, 72°C, 1min, 30 cycles; 72°C, 10min. The above PCR product was electrophoresed with 1% agarose gel and a 1.7Kb band was recovered.

[0060] 3. TA cloning and screening of PCR products

[0061] Ligate the above recovered produc...

Embodiment 3

[0063] Construction of TSP50-p-pGL3 and TSP50-p-pEGFP recombinant plasmids

[0064] Amplify the above-mentioned TA clone bacteria and extract the plasmid, digest with KpnI and BglII, and recover a 1.7Kb fragment; at the same time, digest pGL3 with KpnI and BglII, recover the digested plasmid, and combine the recovered fragment and pGL3 plasmid Use TAKARA ligase I for ligation. When constructing the TSP50-p-pEGFP recombinant plasmid, the positive TA cloning plasmid and the pEGFP vector were respectively digested with EcoRI and the fragments were recovered for ligation. The ligation method was carried out according to the instructions of the TAKARA Ligation Kit, at 16°C After 4 hours of ligation, the ligation product was treated as above and then electrotransformed into DH5α. The bacteria were spread on LB medium with ampicillin and cultured overnight in a 37°C incubator. Pick the colonies and inoculate them into 2ml of LB medium, shake and culture overnight at 37°C, extract the...

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Abstract

The present invention relates to a screening model of human testicular specific protein 50 (TSP50) expression regulator and method for screening TSP 50 gene expression regulator. Said screening system contains a recombinant carrier containing exogenous polynucleotide and host cell transformed or transfected by said recombinant carrier. Said recombinant carrier contains original carrier and human TSP 50 gene promoter, in which the described original carrier contains a report gene and contains no promoter, the described human TSP 50 gene promoter is connected to upstream of report gene, and the activity of human TSP 50 gene promoter has correlation with expression of report gene. Said invention also discloses a method for constructing said screening system and its application.

Description

Technical field: [0001] The invention discloses a screening system for a human testis-specific protein 50 gene expression regulator, and also provides a method for screening a human testis-specific protein 50 gene expression regulator, which belongs to the technical field of biomedicine. Background technique: [0002] Breast cancer is a common malignant tumor in women, and the incidence rate is increasing year by year. At present, the treatment of breast cancer is still based on surgery, supplemented by radiotherapy and chemotherapy. With the development of society, radical mastectomy will be replaced by breast-conserving surgery, which is more prone to postoperative recurrence and metastasis, and the existing chemotherapeutic drugs do not specifically act on tumor cells, while killing tumor cells, normal cells It also has a killing effect, so the side effects are relatively large, and it is very easy to develop drug resistance, and the pain it brings to patients is obvious ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/12C12N5/10C12N15/63A61K48/00
Inventor 鲍永利李玉新孟祥颖乌垠徐浩鹏单继东王淼易静雯
Owner NORTHEAST NORMAL UNIVERSITY
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