Polypeptide-human transmembrane glycopeptide 25 and polynucleotide for encoding such polypeptide

A transmembrane glycoprotein and polynucleotide technology, applied in the direction of peptide/protein composition, anti-animal/human immunoglobulin, peptide source, etc., can solve problems such as cell differentiation

Inactive Publication Date: 2004-03-31
BIOWINDOW GENE DEV INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, overexpression of this protein in PC12 cells triggers cell differentiation

Method used

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  • Polypeptide-human transmembrane glycopeptide 25 and polynucleotide for encoding such polypeptide
  • Polypeptide-human transmembrane glycopeptide 25 and polynucleotide for encoding such polypeptide
  • Polypeptide-human transmembrane glycopeptide 25 and polynucleotide for encoding such polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1: Cloning of human transmembrane glycoprotein 25

[0117] Total RNA was extracted from human fetal brain by one-step method of guanidine isothiocyanate / phenol / chloroform. Poly(A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (product of Qiegene). 2ug poly(A) mRNA was reverse transcribed to form cDNA. Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multiple cloning site of the pBSK(+) vector (product of Clontech Company), transform DH5α, and the bacteria formed a cDNA library. The sequences of the 5' and 3' ends of all clones were determined using Dyeterminate cycle reaction sequencing kit (product of Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones, 1000A03, was a new DNA. The insert cDNA fragment contained in this clone was deter...

Embodiment 2

[0118] Example 2: Homology retrieval of cDNA clones

[0119] The sequence of the human transmembrane glycoprotein 25 of the present invention and the protein sequence encoded by it, using the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], Homology searches were performed in databases such as Genbank and Swissport. The gene with the highest homology to human transmembrane glycoprotein 25 of the present invention is a known mouse shc protein, and the accession number of the encoded protein in Genbank is U15784. The results of protein homology are shown in Figure 1. The two are highly homologous, with an identity of 52% and a similarity of 64%.

Embodiment 3

[0120] Example 3: Cloning of the gene encoding human transmembrane glycoprotein 25 by RT-PCR

[0121] The total RNA of fetal brain cells was used as a template, and oligo-dT was used as a primer to carry out reverse transcription reaction to synthesize cDNA. After purification with a Qiagene kit, PCR amplification was performed with the following primers:

[0122] Primer1: 5'-AACACATGGCTTACTGCCCCATAC-3'(3)

[0123] Primer2: 5'-CGAATTCAATGTCATATTTATTTT-3'(4)

[0124] Primer1 is the forward sequence starting from the 1bp at the 5' end of 1;

[0125] Primer2 is the 3' reverse sequence in 1.

[0126] The conditions of the amplification reaction: 50mmol / L KCl, 10mmol / L Tris-Cl, (pH8.5), 1.5mmol / L MgCl in a reaction volume of 50μl 2 , 200 μmol / L dNTP, 10 pmol primer, 1 U of Taq DNA polymerase (product of Clontech Company). On a PE9600 DNA thermal cycler (Perkin-Elmer), the reaction was performed for 25 cycles under the following conditions: 94°C for 30 sec; 55°C for 30 sec; 72°C...

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Abstract

The present invention provides a polypeptide-human transmembrane glycoprotein 25, polynucleotide for encoding the polypeptide and the process for producing the polypeptide by the DNA restructuring technique. The invention also discloses the method of use of the polypeptide for treating a plurality of diseases, e.g. malignant tumor, hematopathy, HIV affection, immunopathy and various types of inflammation. The invention also discloses the antagonist of the polypeptide and its therapeutic action. The invention also discloses the use of the polynucleotide for coding the new human transmembrane glycoprotein 25. íí

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention describes a polypeptide-human transmembrane glycoprotein 25, and a polynucleotide sequence encoding the polypeptide. The present invention also relates to the preparation method and application of the polynucleotide and polypeptide. Background technique [0002] Epidermal growth factor receptor is a transmembrane glycoprotein. The combination of epidermal growth factor and its outer region will activate complex tyrosine kinase activity, and the inner region will be autophosphorylated to activate cell signaling pathways and promote cell division. The epidermal growth factor receptor can stimulate the Ras-Raf-MAPK signaling pathway by interacting with the signaling protein containing the SH2 domain in the cell, and ultimately affect the process of gene transcription and expression in the nucleus. [0003] SH2 structures play an important role in directing protein interactio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C07K14/435C07K14/47C07K16/18C12N15/113C12N15/12C12N15/63C12N15/64C12P21/02
Inventor 毛裕民谢毅
Owner BIOWINDOW GENE DEV INC
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