Active segment of HER2/neu with Herstatin interaction, coding gene and application thereof

An active fragment, coding gene technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of inhibition of cell proliferation, reduced ability of clone formation, unclear Herstatin, etc.

Inactive Publication Date: 2006-11-15
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, Herstatin acts on HER2 overexpressed SKOV-3 cells and NIH3T3/HER2 cells, which can reduce its clone formation ability sever

Method used

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  • Active segment of HER2/neu with Herstatin interaction, coding gene and application thereof
  • Active segment of HER2/neu with Herstatin interaction, coding gene and application thereof
  • Active segment of HER2/neu with Herstatin interaction, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, HER2 / neu interacts with Herstatin active fragment

[0053] 1. Cloning and expression of Herstatin gene

[0054] Transfection reagent Lipofectamine TM and pcDNA3.1 / Myc-His (-) are Invitrogen products; Restriction enzymes are purchased from New England Biolabs; T 4 DNA ligase was purchased from Promega; Pyrobest DNA polymerase and DNA molecular weight standard DL2000 were TaKaRa products; DNA fragment recovery kit and plasmid recovery kit were purchased from Biotech; primers were synthesized by Borun Biological Co., Ltd.

[0055] 1. Construction of pcDNA3.1(-)myc-his / Hst vector

[0056] According to the method introduced by Doherty et al. (Doherty JK, Bond C, Jardim A, Adelman JP, Clinton GM.1996.The ER-2 / neu receptor tyrosine kinase gene encodes a secreted autoinhibitor.Proc Natl Acad Sci USA 96:10869-10874) The full-length cDNA of Herstatin (clone number gi|10181232|gb|AF177761.2|AF177761 ) was cloned between the restriction endonuclease XhoI and Hin...

Embodiment 2

[0092] Example 2. Co-immunoprecipitation experiments verify the active fragments of interaction between HER2 / neu and Herstatin

[0093] In order to further verify the correctness of computer modeling. This model was further verified by co-immunoprecipitation of Herstatin and HER2 / neu mutants.

[0094] Material

[0095] Transfection reagent Lipofectamine TM For Invitrogen products; pcDNA3.1(-)myc-his / Hst, vectors pEGFP / ECDt, pEGFP / mE1 and pEGFP / mE2 according to literature (Wang JN, Feng JN, YuM, Xu M, Shi M, Zhou T, Yu XD , Shen BF, Guo N.2004. Structural analysis of the epitopes on erbB2 interacted with inhibitory or non-inhibitory monoclonal antibodies. Mol Immunol 40: 963-969) method construction. proteinA / G plusagarose beads, Santa Cruz products. Cocktail, Roche product. anti-Herstatin, mouse monoclonal antibody CW001, upstate product; GFP rabbit polyclonal antibody, product of BioVision.

[0096] will be 3×10 6 CHO cells were plated on a 10 cm culture plate and tran...

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Abstract

An active fragment for the mutual action between HER2/neu and Herstation is the polypeptide with the amino acid residue sequence shown by SEQ ID No.1 or the polypeptide whose amino acid residue sequence has one or more substituted or lost, or added residues. It is also a sensitive site, where HER2/neu molecule can be effectively suppressed. It can play its role in preparing the HER2/neu antagonistic medicines.

Description

technical field [0001] The invention relates to an active fragment interacting with HER2 / neu and Herstatin, its coding gene and application. Background technique [0002] HER2 / neu is one of the members of the EGFR family, which includes EGFR (epidermal growth factor receptor, also known as c-c-erbB-1, HER1), HER2 / neu (c-erbB-2), HER3 (c-erbB-3) and HER4 (c-erbB-4) and other members. HER2 / neu gene encodes 1255 amino acids with a molecular weight of 185KD. The extracellular region is divided into four functional domains, the L1 and L2 regions, and the S1 and S2 regions. The two S regions are cysteine-rich regions. The tyrosines at C-terminal 1139, 1196 and 1246 in the cytoplasm are tyrosine phosphorylation regions. HER2 / neu is activated by forming homologous or heterodimers with EGFR family members, accompanied by intracellular tyrosine phosphorylation of HER2 / neu, causing intracellular sites that can bind to proteins containing SH2, SH3, and PTB regions Activation, there...

Claims

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Application Information

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IPC IPC(8): C07K14/435A61K38/17A61P35/00C07K14/47C12N15/12C12N15/63
Inventor 胡品良郭宁冯建男王嘉宁沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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