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NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour

A tumor, protein peptide technology, applied in the direction of DNA/RNA fragments, antineoplastic drugs, peptide/protein components, etc., can solve the problem of no tumor cell growth and tumor formation.

Inactive Publication Date: 2007-03-14
NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although the current research has considered that the downstream regulatory gene (human N-mycdownstream regulated gene, hNDRG) N-myc in human myc family genes may be related to the occurrence of tumors, and listed it as a tumor suppressor candidate gene (Deng YC et al., Shengwu Huaxue Yu Shengwu Wuli Jinzhan, 2001, Volume 28, Phase 1, Pages 72-76), related to the occurrence and development of tumors in the nervous system and respiratory system, but the above studies were only conducted on isolated cell lines, so far no Reports about NDGR2 and NDRG4 proteins inhibiting tumor cell growth and tumor formation in animals or humans

Method used

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  • NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour
  • NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour
  • NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Cloning and sequencing of ndrg2 and ndrg4 genes

[0051] 1. Transformation of the double enzyme cutting site of the target fragment

[0052] Transform the plasmid pMD18-T (product of Dalian Bao Biology Co., Ltd.) containing the target fragment into the host strain JF1125 (provided by Shanghai Medical University). The double restriction site of the target fragment is BamHI at the 5' end and EcoRI at the 3' end. If an expression vector is used For pQE30 (product of QIAGEN), the restriction site needs to be transformed into BamHI at the 5' end and HindIII at the 3' end, and the designed PCR primers are:

[0053] Forward: 5'-CG GGATCC GCGGAGCTGCAGGAGGT-3'

[0054] Reverse: 5'-CC AAGCTT TTAACAGGAGACCTCCATGGTG-3'

[0055] The PCR amplification reaction system is:

[0056] 10×pfuDNA polymerase reaction buffer 5ul

[0057] Forward primer (25pmol / ul) 1ul

[0058] Reverse primer (25pmol / ul) 1ul

[0059] dNTPs (10Mm) 1ul

[0060] DNA template 3ul

[0061] pfu...

Embodiment 2

[0089] Example 2: Preparation and purification of NDRG2 and NDRG4 proteins

[0090] 1. Investigation of the amount of expression

[0091] Insert pQE30 / M15 positive clones into 5ml ampicillin-resistant LB liquid medium, culture at 200rpm at 37°C for 16 hours, take 5ml into 300ml ampicillin-resistant LB liquid medium, and culture in a 1000ml Erlenmeyer flask at 37°C For about 3 hours, when the OD value of the culture medium is 0.5, take out 1ml and centrifuge at 5000rpm for 10min, and take the precipitate as a blank control; add IPTG to the final concentration of 1.5mmol / L in the Erlenmeyer flask, and continue to culture NDRG2 at 37°C for 5 hours. NDRG4 was further cultured at 39°C for 4 hours, removed and centrifuged at 5000rpm for 10min, and the precipitate was collected to investigate the expression level.

[0092] The results of induced expression showed that the expression level of NDRG2 was 42.85%, and the molecular weight was 39977Da; the expression level of NDRG4 was 20...

Embodiment 3

[0138] Example 3 Inhibition of NDRG2 and NDRG4 Proteins on Tumor Cell Gastric Cancer 7901 and Liver Cancer HHCC

[0139] Gastric cancer 7901 cells and liver cancer HHCC cells were diluted to 7×10 with 1640 medium containing 10% serum. 4 / ml, NDRG2 and NDRG4 proteins were added respectively, and observed after 30 and 40 hours of culture. At the same time, the 1640 medium containing 10% serum was diluted to 7×10 4 Gastric cancer 7901 cells / ml and liver cancer HHCC cells were used as controls to investigate the inhibitory effect of NDRG2 and NDRG4 proteins on tumor cells gastric cancer 7901 and liver cancer HHCC.

[0140] The results showed that no addition of NDRG2 and NDRG4 proteins had no effect on the growth of gastric cancer 7901 and liver cancer HHCC tumor cells, while the growth of gastric cancer 7901 and liver cancer HHCC tumor cells was significantly inhibited after adding NDRG2 and NDRG4 proteins for 30 and 40 hours, respectively. As the culture time prolongs, the inhi...

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Abstract

The present invention adopts gene engineering method in mass expression of N-myc downstream regulating gene subtype ndrg2 and ndrg4 coded NDRG2 and NDRG4 protein, and researching the physical and chemical properties of NDRG2 and NDRG4 protein and their biological functions on tumor cell. Flow cell technology results show that NDRG2 protein has the influence on gastric cancer 7901 cell of prolonging G1 stage and shortening G2 stage and S stage, the influence on liver cancer HHCC cell of shortening G1 stage and G2 stage and prolonging S stage; while NDRG4 protein has the influence on both gastric cancer 7901 cell and liver cancer HHCC cell of prolonging G1 stage and shortening G2 stage and S stage. Animal experiment shows that both NDRG2 and NDRG4 protein have obvious suppression on tumor cell growth and tumor cell formation and the great dosage has even obvious tumor suppressing effect.

Description

technical field [0001] The present invention relates to N-myc downstream regulation gene subtype 2 and / or 4 protein and its application in diagnosing and suppressing tumors. Background technique [0002] Cancer is the product of activation of proto-oncogenes and / or deletion or inactivation of tumor suppressor genes. Oncogenes are genes present in cells or viruses that induce transformation in normal cells and allow them to acquire one or more new biological characteristics. This gene is related to the occurrence and development of tumors. Oncogenes are highly conserved and limitedly expressed in normal cells, and play an important role in cell differentiation and proliferation. When an oncogene is activated, it can cause cell cancer. Usually, the inherent oncogene in the cell is called a cellular oncogene (c-onc), and the inactive c-onc is called a proto-oncogene (proto-onc). [0003] The main sign of oncogene activation is gene amplification, which refers to the duplicat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C07K14/435C07K14/47A61K38/16A61P35/00
Inventor 王军志张翊药立波饶春明陈杰裴德宁
Owner NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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