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Tumor-inhibiting protein and the use thereof

Inactive Publication Date: 2006-06-01
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] The aim of the present invention is to offer a novel tumor-inhibiting protein and its fragments, analogs and derivatives.

Problems solved by technology

Mutations including point mutation, loss or shift of DNA fragments of these two classes of genes can lead to uncontrolled cell growth and neoplasm.
These therapeutic methods are based on killing tumor cells directly, and it is difficult to completely eliminate all the cancer cells and is prone to injuring normal tissue, especially to impairing organism immune system and affecting native cellular immunity.
Deregulation of the dynamic balance, which exists between tumor and organism defense system under normal conditions, causes tumor proliferation and dissemination.
Tumor biological therapy refers to the treatment of organism by regulating it's biological reaction according to tumor host defense mechanism or using biological reagent, which results in the tumor cell inhibition or death.

Method used

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  • Tumor-inhibiting protein and the use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of Full-Length cDNA for HCRP1 Gene

[0083] According to the positional candidate cloning strategy and human genomic sequence database, primers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4) were designed to amplify the HCRP1 cDNA from the human liver cDNA library (GIBCO BRL). The polymerase chain reaction (PCR) was performed with pre-denaturing for 2 min and 35 cycles of 94° C. for 30 s, 50° C. for 30 s, and 72° C. for 2 min. The PCR product was purified by using low melting agarose gel (see Sambrook, J., Fritsh, E. F., and Maniais, T., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). Sequencing of the full-length cDNA was conducted by Bioasia, Co, Ltd (Shanghai, China).

[0084] The obtained 1917 bp full-length cDNA (SEQ ID NO:1) comprises a whole protein-encoding region (from 151 bp to 1341 bp), which codes for a protein consisting of 397 amino acids (SEQ ID NO:2).

example 2

RNA Blot Analysis of HCRP1 Expression in Multiple Human Tissues

[0085] Multiple-tissue RNA blot membrane fixed with 8 different normal human tissues was placed in to a hybridization tube. 5 mil pre-hybridization solution (Kingrace) was added and the mixture was pre-hybridized at 65° C. for 30 minutes. Then the denatured probe of HCRP1 was added, which was randomly labeled with 32P(Random Primer DNA Labeling Kit, Takara), and hybridized overnight at 65° C. The blot membrane was washed with resin buffer I (0.3M NaCl, 0.03M citrate sodium (PH7.0), 0.05% sodium dodecyl sulfate) at room temperature; and then washed twice (20 min each) at 50° C. with resin buffer II (15 mM NaCl, 1.5M citrate sodium (PH7.0), 0.1% sodium dodecyl sulfate (SDS)). After that, the hybridized film was exposed to X-ray and radioautographed at −70° C.

[0086] As shown in FIG. 1, HCRP1 gene was highly expressed in liver, moderately in lung, spleen, testis and muscle, yet minimally or not expressed in brain, heart an...

example 3

Construction of Eukaryotic Expression Vector of HCRP1 Gene and Expression and Measurement of HCRP1 Protein

[0087] Primer P3 (SEQ ID NO:5) and primer P4 (SEQ ID NO:6) were designed according to the cDNA sequence of HCRP1 with the restriction endonuclease site of XbaI and EcoRI added to 5′ site of the primers, respectively. PCR reaction was performed by using the verified sequence of HCRP1 as template. The PCR product was recovered from the low melting agarose gel and then was digested for an hour with restriction enzyme XbaI and EcoRI (Takara, Co, Ltd.), followed by purification again. The vector pCMV-Tag2A (Stratagene) was also digested for an hour with the same restriction enzyme XbaI and EcoRI, followed by purification on low melting agarose gel. The two recovered products were ligated with T4 DNA ligase (Takara) at 16° C. overnight and then were introduced into E. Coli DH5a. The recombinant HCRP1 plasmid was obtained by screening and designated as pcDNA3-Flag-HCRP1. Primer P5 (SE...

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Abstract

The invention has disclosed a new tumor suppressor protein HCRP1, the polynucleotide sequences encoding this polypeptides, and methods for production of the polypeptide using the recombinant technology. The tumor suppression protein, HCRP1, is obtained through the positional candidate cloning strategy. It locates in 8p22 region of human chromosome. The full length cDNA for HCRP1 is 1917bp, which encodes a protein of 397 amino acids. When introduced into liver cancer cells, HCRP1 can inhibit the malignant transformation of liver cancer cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation or International application number PCT / CN2004 / 000473, filed May 12, 2004 which claims priority to Chinese application No. CN 03116920.1 filed May 14, 2003, the contents of both are herein incorporated in their entirety by reference.FIELD OF THE INVENTION [0002] The present invention relates to the field of biological technology. More specifically, this invention is directed to a new tumor suppressor protein and the encoding polynucleotide thereof. This invention is also directed to the use and preparation of the polynucleotide and polypeptide. The polypeptide of this invention is a tumor suppressor protein that can inhibit malignant tumor proliferation. BACKGROUND OF THE INVENTION [0003] Malignant tumor, as a vital disease threatening the human health, is the second leading cause of mortality. Statistics shows that the top 5 malignant cancers contributing to death in China are stomach cancer, liver ca...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61K38/00C07K14/47C12N15/12
CPCA61K38/00C07K14/4703A61P35/00
Inventor ZHAO, MUJUNXU, ZHENHUALIANG, LIANGLI, ZAIPING
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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