Novel human tumor inhibiting gene KIAA0157 and application thereof

A gene and preparation technology applied in the field of new human tumor suppressor genes

Inactive Publication Date: 2010-05-26
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Novel human tumor inhibiting gene KIAA0157 and application thereof
  • Novel human tumor inhibiting gene KIAA0157 and application thereof
  • Novel human tumor inhibiting gene KIAA0157 and application thereof

Examples

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Effect test

Embodiment 1

[0021] Example 1 Obtaining human KIAA0157 cDNA

[0022] Using the THAP11 gene as a bait, using the yeast two-hybrid technology commonly used in the field to study its interacting protein, a DNA sequence was obtained, and the DNA sequence was used for BLAST search. The present invention found that it has high homology with the inferred human gene KIAA0157 . The total RNA of human bone marrow cells was proposed, and reverse transcription was performed according to the instructions of the reverse transcription kit (Promega). Take 1μg of quantified RNA, add water to make up to 10.5μl, 70℃, 10min, add 2μl 10×buffer, 2μl MgCl 2 , 2μl dNTP, 1μl oligo dT, 1μl rRNasin, 1.5μl AMV (15U), total reaction system 20μl; 42℃, 1h; 94℃, 5min; 4℃, 5min. RNA is reverse transcribed into cDNA. Use 5'-CGGAATTCAATGTCCTACAG AGAGCAGGTT-3'; 5'-GGGGATCCTTAAATCTGGGAGG TCTGAGTG-3' as primers for PCR amplification of the target fragment. The PCR conditions are:

[0023] Cycle 1(1x)

[0024] 94℃ 5min

[0025] Cy...

Embodiment 2

[0033] Example 2 KIAA0157 subcellular location

[0034] To observe the subcellular localization of KIAA0157, we constructed KIAA0157 cDNA into the EcoRI / KpnI site of pEGFP vector to form a KIAA0157-GFP fusion gene expression vector. The expression vector was transfected into NIH3T3 cells, and laser scanning confocal microscopy showed that the fluorescence signal was distributed in whole cells ( figure 1 ).

Embodiment 3

[0035] Example 3 Prokaryotic expression and purification of KIAA0157 protein and preparation of polyclonal antiserum

[0036] Using the KIAA0157-Flag plasmid as the template, and 5’-CGGAATTCCCAAGGAACAAGAAAGAAGATT-3’; 5’-CCGCTCGAGTTAAATCTGGGAGG TCTGAGT-3’ as the primers, PCR was used to amplify the C-terminal 161 amino acids of KIAA0157. The PCR product and PGEX-4T-2 vector were digested with EcoRI and Xholl and then ligated, transformed into E.coli BL21(DE3) competent strain, picked a single clone, and identified by digestion, the sequence analysis was correct. Inoculate E.coli BL21(DE3) transformed with GST-KIAA0157 plasmid into LB medium, culture at 37℃ to OD: 0.6~1.0, add 1mM IPTG, induce 4h at 30℃, collect bacteria by centrifugation, and detect EDAG protein expression by SDS-PAGE Happening. Purified by B-PER GST Spin Purification Kit to obtain a relatively pure GST-KIAA0157 fusion protein. The purified GST-KIAA0157 fusion protein was used to immunize New Zealand large ear w...

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Abstract

The invention provides a novel human tumor inhibiting gene and an encoded protein thereof. The gene and the encoded protein can inhibit the growth of tumor cells, are down-regulated in various tumor tissues, and have a close relation with the generation of tumors. The gene and the encoded protein and derivatives thereof can be used for the diagnosis, treatment and drug screening of various tumors.

Description

Technical field [0001] The present invention relates to a new human tumor suppressor gene, and also relates to the DNA constituting the gene and the protein encoded by the DNA and the application in tumor diagnosis and treatment. Background of the invention: [0002] Tumor occurrence is the result of the interaction between environmental carcinogenic factors and host genes, which gradually leads to and continuously accumulates a series of gene abnormalities, resulting in the imbalance of the dynamic balance of cell proliferation and death. From normal cells to life-threatening aggressive malignant tumors, most of them need to go through the stage of precancerous lesions. The occurrence of malignant tumors is a multi-stage gradual evolution process, which can be roughly divided into several stages, such as excitation, promotion, progression and metastasis. In the multi-stage evolution of cancer, mutations of a series of genes are often accumulated, which may involve changes in mu...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/11C12N15/86C07K14/47C07K16/18C12Q1/68G01N33/577G01N33/574A61K38/17A61P35/00
Inventor 杨晓明李长燕胡德庆许望翔詹轶群
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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