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New human cell factor VSTM1-v2 and application thereof

A technology of host cell and genetic engineering, applied in human new cytokine VSTM1-v2 and its application field, can solve problems such as complex functions

Inactive Publication Date: 2011-04-27
PEKING UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

It is generally believed that IL-6 and TGF-β provide the initiation factors for the differentiation of initial T cells into Th17 cells, and IL-23 plays a very important role in maintaining the differentiation into Th17 cells; TNF-α, IL-1β and other inflammatory Cytokines can promote the differentiation of Th17 cells; Th17 cells can secrete IL-21, which has a self-feedback effect; while IL-25, IL-27, and IL-35 show more inhibitory effects on Th17 cell differentiation, but their effects may be more complicated

Method used

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  • New human cell factor VSTM1-v2 and application thereof
  • New human cell factor VSTM1-v2 and application thereof
  • New human cell factor VSTM1-v2 and application thereof

Examples

Experimental program
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Embodiment 1

[0071] Cloning and bioinformatics analysis of the cDNA of embodiment 1, VSTM1 gene

[0072] First, the following method was used to prepare the granulocyte cDNA library: use TRIzol (invitrogen) to extract the total RNA of granulocytes (operate according to the instructions), and use Reverse transcript TM Kit (Invitrogen) was used to synthesize a single-stranded cDNA library by reverse transcription (operated according to the instructions). The method for collecting the above-mentioned peripheral blood mononuclear cells and granulocytes is as described in the literature (Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1g. Scandinavian Journal of Clinical and Laboratory Investigation. Supplement (Oslo) 97, 77-89).

[0073] The cDNA sequence and alternative splicing bodies were analyzed by comparing with NCBI's Hurnan_est da...

Embodiment 2

[0083] Example 2, Expression profile analysis of VSTM1-v2 in tissues and cells

[0084] In order to analyze the mRNA expression level of VSTM1-v2 in normal tissues, in this example, the purchased clontech human normal tissue cDNA library was used to amplify VSTM1 using the nested PCR amplification conditions in Example 1. Using 5' primer (5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'SEQ ID No: 15) and 3' primer (5'-CATGTGGGCCATGAGGTCCACCAC-3'SEQ ID No: 16) to amplify GAPDH as an internal reference, the amplification condition was 94°C (5 minutes) → 94°C (40 seconds), 58°C (40 seconds), 72°C (40 seconds), 20 cycles of amplification → 72°C (7 minutes). PCR amplified products were subjected to agarose gel electrophoresis, stained with EB, and detected by GENE Snap gel imaging system.

[0085] For experimental results, see Figure 2A with Figure 2B As shown, among them, Figure 2A To represent 16 human normal tissue libraries, each lane is: 1, brain; 2, heart; 3, kidney; 4, liver; 5, lung...

Embodiment 3

[0086] Example 3, construction of eukaryotic cell expression plasmid pcDB-VSTM1-v2-myc-his

[0087]In order to detect the function of VSTM1-v2, a eukaryotic expression plasmid containing VSTM1-v2 cDNA was constructed in this example: pcDNA3.1B-VSTM1-v2-myc-his (pcDB-VSTM1-v2-myc-his). Use an upstream primer (5'-CGAGCGGCCGCATGACCGCAGAATTCCTCTC-3'SEQ ID No: 17) with a Not I (TaKaRa) restriction site and a downstream primer (5'-CTTGGTACCGACACTTTCAGTGCCGCATATT-3') with a Kpn I (TaKaRa) restriction site SEQ ID No: 18) Perform PCR amplification on the pGEM-T-VSTM1-v2 vector (refer to the preparation in Example 1) (reaction temperature and time: 94°C, denaturation for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C 30 seconds, 72°C extension for 1 minute, 35 cycles of amplification; finally 72°C extension for 10 minutes), to obtain the full-length ORF fragment of the cDNA of VSTM1-v2, and then digest the PCR product with Not I and Kpn I, Simultaneously digest ...

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Abstract

The invention relates to a new human cell factor BSTM1-v2 and application thereof, in particular to a gene or a protein of a transcript VSTM1-v2 of VSTM1 or immune fragments of the gene and the protein, and application of the gene or protein of the VSTM1-v2 or immune fragments of the gene and the protein in promoting Th17 differentiation and the function of killing CD8+ lymphocytes, and in preparing medicinal compositions for preventing and / or treating immune related diseases. The invention also relates to an antagonist of the VSTM1-v2, such as a monoclonal antibody or a polyclonal antibody, which comprises a vector, a host cell or a composition of the VSTM1-v2, a reagent for detecting the VSTM1-v2 or immune fragments thereof, and application of the antagonist and the reagent.

Description

technical field [0001] The present invention relates to a new human cytokine and its application, in particular to the gene or protein of VSTM1-v2 or their immune fragments, the gene or protein of VSTM1-v2 or their Immune fragments promote Th17 differentiation and CD8 + The application in the killing function of T lymphocytes and the application in the preparation of pharmaceutical compositions for the treatment of immune-related diseases, also relates to the antagonist of VSTM1-v2, the vector, host cell or composition comprising VSTM1-v2, and detection Reagents for VSTM1-v2 or immunological fragments thereof, and uses thereof. Background technique [0002] The immune system plays a vital role in the body. Externally, it can defend against the invasion of pathogenic microorganisms, and internally, it can remove aging, diseased and dead cells in time to maintain the stability of the internal environment. The immune system accomplishes the above functions through the immune ...

Claims

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Application Information

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IPC IPC(8): C07K14/52C12N15/19C12N15/63C12N1/21C12N1/15C12N1/19C12N5/10C07K16/24C12N15/11A61K38/19A61K39/395A61K48/00A61P31/00A61P37/00A61P35/00G01N33/53C12Q1/68
CPCC07K16/24A61K38/00C07K14/52A61P31/00A61P35/00A61P37/00A61P37/02
Inventor 马大龙韩文玲郭晓欢王平章李婷黄晶郭金海付伟伟张岩飞石太平
Owner PEKING UNIV
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