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Gene of restraining activation NF-kB and NFAT, and coded polypeptide

A gene and amino acid technology is applied in the field of genes that inhibit the activation of NF-kB and NFAT and their encoded polypeptides, which can solve the problems of poor selectivity and large toxic and side effects.

Inactive Publication Date: 2005-11-16
SINOGENOMAX +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of these drugs can inhibit CaN in non-immune cells, with poor selectivity and large toxic and side effects. Therefore, it is necessary to develop reagents that have no toxic effects and can directly target NFAT, which will help to develop new, safe and effective drugs with strong selectivity. immunomodulator

Method used

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  • Gene of restraining activation NF-kB and NFAT, and coded polypeptide
  • Gene of restraining activation NF-kB and NFAT, and coded polypeptide
  • Gene of restraining activation NF-kB and NFAT, and coded polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Cloning of embodiment 1, NFIF1 gene cDNA

[0072] Searched the predicted genes with unknown human functions in the nr database of NCBI, and obtained the sequence of human unknown functional genes (Ref: XM_114657; Locus ID: 203245; UniGene: Hs.373606). Methods Sequence correction was performed, and the final sequence was set as sequence 4. Design NFIF1 gene-specific primers according to sequence sequence 4:

[0073] 5' Primer: 5'-GGGGGTGGGTGAGGGG-3'

[0074] 3' Primer: 3'-ACCCCTGCCCTCACTGGATG-3'

[0075] Use the above primers to carry out PCR amplification reaction with mixed human tissue cDNA library (brain, lung, pancreas, testis, Clonetech K1420-1, K1241-1) as template, and the reaction conditions are as follows:

[0076] The reaction volume is 50 μl, which contains:

[0077] 2 μl of mixed human tissue cDNA template (0.5 μl each for brain, lung, colon, and testis)

[0078] The final concentration of primer 5' primer and 3' primer is 0.2 μM each

[0079] dNTP fin...

Embodiment 2

[0085] Embodiment 2, RT-PCR method detects the transcription product of NFIF1 gene in human tissue with the specific primer of NFIF1 gene:

[0086] 5' Primer: 5'-TGTGTCCGTCGCCATGACAG-3'

[0087] 3' Primer: 5'-TGGTTGAGTGGCAGGTGAGG-3'

[0088] 12 kinds of human normal tissues (heart, pancreas, testis, ovary, prostate, colon, small intestine, skeletal muscle, thymus, lymph nodes, tonsils, white blood cells); 6 kinds of human tumor tissues (lung cancer, pancreatic cancer, ovarian cancer, prostate cancer) , colon cancer, breast cancer); and cDNA libraries of 8 kinds of fetal components (fetal lung, fetal heart, fetal liver, fetal spleen, fetal kidney, fetal brain, fetal skeletal muscle, fetal thymus) (Clonetch, K1420-1, 1241-1) was used as template for PCR amplification.

[0089] The PCR amplification conditions are as follows:

[0090] The reaction volume is 50 μl, which contains:

[0091] 2 μl of mixed human tissue cDNA template (0.5 μl each for brain, lung, colon, and testis...

Embodiment 3

[0099] Embodiment 3, the construction of NFIF1 gene eukaryotic expression vector

[0100] The cDNA fragment of NFIF1 gene was excised from the pGEM-T Easy vector (Promega, A1360) with EcoRI (Promega), and the eukaryotic expression vector pcDNA3.1 / mycHis(-)B (Invitrogen, V85520) was digested with EcoRI at the same time, According to the method described in J.Sambrook et al., Molecular Cloning Experiment Guide 2nd Edition, the digested NFIF1 was connected to the carrier overnight at 16°C, transformed into Escherichia coli DH5α, and the transformant was grown on LB plate medium containing ampicillin , select the grown colony, extract the plasmid, digest with EcoRI, identify the digested product by agarose gel electrophoresis, select positive clones with inserts, and select the correct positive clones by sequencing (using ABI PRISM3700 DNA analyzer, as above). The insert clone was named pcDNA3.1B-NFIF1.

[0101] At the same time, the culture solution was collected, and the precip...

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Abstract

A human gene NFIF1 for suppressing the activation of NF-kB and NFAT, its cDNA sequence, the polypeptide coded by it, the process for testing its functions by dual-leuciferinase report gene method, and its application in preparing the medicines for preventing and treating inflammation, anaphylactia, autoimmunopathy, tumor etc are disclosed.

Description

technical field [0001] The invention relates to the field of gene expression regulation, in particular to a gene that inhibits the activation of human nuclear factor NF-κB and NFAT, a preparation method of the gene, an expression vector containing the gene, a polypeptide encoded by it, an antibody to the polypeptide, and the polypeptide , antibodies in the prevention and treatment of diseases related to human nuclear factor NF-κB and NFAT, especially drugs for inflammation, allergic diseases, autoimmune diseases and tumors, and in the development of drugs that regulate lymphocyte activation, proliferation and apoptosis use. Background technique [0002] NFAT and NF-κB proteins are members of the transcription factor superfamily, and although they are expressed in many types of cells, they play key roles in regulating the activation, proliferation and activation-induced cell death of lymphoid cell lineages. NFAT and NF-κB have many commonalities, including similar DNA bindin...

Claims

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Application Information

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IPC IPC(8): C07K14/475
Inventor 石太平马大龙吕冰峰武春晓童郁蓉高鹏娄雅欣王露
Owner SINOGENOMAX
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