Human cytokine and use thereof

A technology of cytokines and host cells, applied in the field of small interfering RNA and polypeptide preparation, can solve problems such as unsatisfactory curative effect, large toxic and side effects, and impact on clinical application

Inactive Publication Date: 2008-06-04
SINOGENOMAX +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

IBD treatment is currently dominated by 5-aminosalicylic acid and glucocorticoids, but these two types of drugs not only have unsatisfactory curative effects, but also have large toxic

Method used

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  • Human cytokine and use thereof
  • Human cytokine and use thereof
  • Human cytokine and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1, the cloning of the cDNA of NS128 gene

[0115] Through bioinformatics analysis, it was found that UniGene: Hs.474991, whose corresponding gene is FLJ22349, Ref: NM_024821, GeneID: 79879, is a new human gene with unknown function, and the sequence was corrected using the Human_est database through the BLASTn method, and the final sequence was obtained Set as SEQ ID NO:1. Design specific primers for the full-length reading frame of the NS128 gene based on the sequence:

[0116] Forward primer 5'-CTGCTCGCACAGGACTCGG-3'

[0117] Reverse primer 5'-CAGATCCCAGTCTGAGTTATAGCCC-3'

[0118] Using the above primers, the human normal lung tissue cDNA library (Clontech: K1420-1) was used as a template for PCR amplification reaction, and the reaction conditions were as follows:

[0119] The reaction volume is 50 μl, which contains:

[0120]

[0121] Reaction temperature and time: 94°C, denaturation for 5 minutes; then denaturation at 94°C for 30 seconds, annealing a...

Embodiment 2

[0123] Example 2. Detection of the inhibitory effect of NS128 on the trans-acting factor Elk1

[0124] In this example, the dual-luciferase reporter gene method was used to detect the inhibitory function of the NS128 gene on the trans-acting factor Elk1. This method used an in vivo signal transduction pathway trans-reporter system, and the pFR-luc reporter plasmid used was loaded with firefly Luciferase gene, the expression of this gene is regulated by a synthetic promoter, which contains the basic promoter assembly (TATA box), and the upstream activation region (UAS) of GAL4, and the pFA2-Elk1 plasmid contains the DNA binding site of GAL4 and The binding site of transcription factor Elk1, the structures of pFR-luc, pFA2-Elk1 and pRL-TK reporter plasmids are shown in Figure 2A , 2B and 2C. When Elk1 in the cell is activated through a certain signal transduction pathway, Elk1 will bind to the enhancer component of the reporter plasmid pFA2-Elk1, thereby initiating the transc...

Embodiment 3

[0139] Example 3. Detecting the expression level of NS128 in cell lines and tissues

[0140] In order to prove the mRNA expression level of NS128 in cell lines and tissues, the mRNA in cell lines and tissues was extracted in this example, and detected by RT-PCR and Northern Blot.

[0141] 1. Northern Blot detection of mRNA expression levels in tissues

[0142] Using NS128-specific upstream primer 5'-CTGCTCGCACAGGACTCGG-3' and downstream primer 5'-CTCAGACTGGGATCTGGAG-3', NS128 ORF was amplified by PCR, recovered and purified by agarose gel electrophoresis, and labeled with fluorescein using a random primer labeling kit as probe. Similarly PCR amplified and labeled GAPDH probe. Total RNA was extracted from normal human brain, heart, testis, placenta, muscle, and lung tissues using Trizol (Invitrogen). 20 μg of each tissue sample was separated by electrophoresis and transferred to a nylon membrane. After pre-hybridization, the above-mentioned probes were added and hybridized ...

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Abstract

The invention discloses a new secretive cytokine NS128 of human beings, which relates to a coding amino acid sequence of the cytokine or the polynucleotide of the cytokine fragment, containing a genetic engineering carrier and a host cell of the polynucleotide. The invention also relates to a production method of the amino acid sequence of the cytokine or the polypeptide of the cytokine fragment and the production method of the polypeptide as well as a small interference RNA, which suppresses the expression of the cytokine. The invention also relates to a salt which contains the polynucleotide or the salt of acceptable drugs, or a medical composition of the polypeptide or the salt of acceptable drugs. The invention further relates to applications of the polynucleotide of the cytokine, the polypeptide or the small interference RNA in preparation of prevention and/or treatment medicines for tumor, infection, hematopoietic disorder and autoimmune disease, etc., in particular having clinical implications for tumor, inflammation and nervous system disease and so on related to MAPK signal pathway. The invention also relates to a method of in vitro testing whether the expression of the polynucleotide or the polypeptide changes. The invention also relates to a monoclonal or polyclonal antibody which is specifically bound with the polypeptide or an active fragment of the polypeptide.

Description

technical field [0001] The invention relates to the fields of molecular biology, genetic engineering, oncology and cell biology. Specifically, the present invention relates to a novel cytokine polynucleotide and polypeptide, a vector or host cell containing the polynucleotide, and a preparation method and use of the polypeptide. The invention also relates to a small interfering RNA that inhibits the expression of the cytokine. The present invention also relates to the application of the cytokine or small interfering RNA in the preparation of drugs for the prevention and / or treatment of tumors, infections, hematopoietic dysfunction, autoimmune diseases, etc., especially the tumors, inflammations and nervous system diseases related to the MAPK signaling pathway Wait. Background technique [0002] Cytokines are small molecule polypeptide factors synthesized and secreted by immune cells and non-immune cells of the body, and they regulate various cellular physiological function...

Claims

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Application Information

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IPC IPC(8): C07K14/715C12N15/12A61K31/7088A61K48/00A61P35/00A61P37/00A61P31/00C12Q1/68G01N33/68C07K16/18
Inventor 邱晓彦黄晶马大龙石太平贺鹏飞刘伟高霞石婵奇陆阳张晨颖
Owner SINOGENOMAX
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