Hepatocyte growth factor receptor key structural domain fusion protein and application thereof

A technology of hepatocyte growth factor and fusion protein, which is applied in the field of fusion protein, can solve the problem of no anti-C-Met neutralizing antibody drug, etc., and achieve the effect of good application prospect, low cost and simple construction method

Inactive Publication Date: 2015-05-20
SUZHOU RES INST OF TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are no FDA-approved neutralizing antibody drugs against C-Met

Method used

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  • Hepatocyte growth factor receptor key structural domain fusion protein and application thereof
  • Hepatocyte growth factor receptor key structural domain fusion protein and application thereof
  • Hepatocyte growth factor receptor key structural domain fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Construction of lentiviral expression vector for ED-His fusion protein

[0027] Using pCR-Blunt-EGFR containing the full-length sequence of human EGFR as a template, primers P1 and P2 (synthesized by Suzhou Jinweizhi Co., Ltd.) were designed and synthesized according to the signal peptide sequence of EGFR gene, and an enzyme cutting site Age I was added to the upstream primer to amplify A 78bp signal peptide was added. Using pCR-Blunt-C-Met containing the full-length human C-Met gene as a template, PCR primers P3 and P4 (synthesized by Suzhou Jinweizhi Co., Ltd.) were designed and synthesized according to the sequence of the extracellular region of the human C-Met gene registered on GenBank, A restriction site SalI and a His tag were added to the downstream primers, and the target fragment ED of about 2800 bp was amplified. Using these two PCR products as templates and using P1 and P4 as primers, a second PCR amplification reaction was performed, and the two PCR produc...

Embodiment 2

[0036] Construction of IPT34-His fusion protein lentiviral expression vector

[0037] Using pCR-Blunt-EGFR containing the full-length sequence of human EGFR as a template, primers P1 and P2 (synthesized by Suzhou Jinweizhi Co., Ltd.) were designed and synthesized according to the signal peptide sequence of EGFR gene. Using pCR-Blunt-C-Met as a template, primers P5 and P6 (synthesized by Suzhou Jinweizhi Co., Ltd.) were designed and synthesized according to the sequence of the IPT34 region of the human C-Met gene. The first PCR amplification reaction was performed separately to obtain two PCR products. Using these two PCR products as templates and using P1 and P6 as primers, a second PCR amplification reaction was performed, and the two PCR products were joined together by overlap extension PCR. The amplified product was first cloned into the pCR-Blunt cloning vector, followed by digestion, ligation and other steps, and finally the lentiviral expression vector pRRL-CMV-IPT34 w...

Embodiment 3

[0040] Expression and purification of recombinant C-Met-ED and C-Met-IPT34 fusion proteins

[0041] 293T cells in the logarithmic growth phase were inoculated, and when the cells grew to 50%-60% confluence, the calcium phosphate method was used to mix pRRL-CMV-C-Met-ED with the control vector containing green fluorescent protein (pRRL-CMV- GFP) co-transfected 293T cells to express C-Met-ED protein. Meanwhile, in order to express C-Met-IPT34 protein in 293T cells, pRRL-CMV-C-Met-IPT34 and pRRL-CMV-GFP were co-transfected into 293T cells. 293T cells transfected with pRRL-CMV-GFP were used as positive control, and 293T cells without transfection were used as blank control. After 48 h of cell culture, if image 3 As shown, the cells of the C-Met-ED group, the C-Met-IPT34 group and the positive control group all expressed green fluorescent protein through fluorescence microscope observation, while the cells of the negative control group that were not transfected with the plasmid ...

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Abstract

The invention relates to a hepatocyte growth factor receptor key structural domain fusion protein and an application thereof. The fusion protein comprises an epidermal growth factor receptor strong signal peptide, a hepatocyte growth factor receptor key structural domain and a His tag. The fusion protein is used for the preparation of an antibody. The hepatocyte growth factor receptor key structural domain fusion protein is stably expressed by a lentiviral vector in 293T cells in a long-acting manner and can be specifically bound with the hepatocyte growth factor. The fusion protein has an important role in research and development on neutralizing antibody drugs for screening and blocking a C-Met / HGF signal transduction pathway to further inhibit tumor growth.

Description

technical field [0001] The invention belongs to the field of fusion proteins, in particular to a hepatocyte growth factor receptor key domain fusion protein and application thereof. Background technique [0002] C-Met is the only known receptor for hepatocyte growth factor (HGF), and it is also a kind of transmembrane receptor with autophosphorylation activity, which plays an important role in the occurrence and development of various malignant tumors. Clinical studies have found that overexpression of C-Met can be found in lung cancer, liver cancer, colorectal cancer, and breast cancer, which have a high incidence in my country, and is positively correlated with tumor malignancy, metastasis, and prognosis. C-Met mainly includes three structural domains with different functions: extracellular region, transmembrane region and intracellular region. The C-Met extracellular domain (Extracellular domain, ED) contains a semaphorin axial protein-like domain, a Cys-rich PSI region,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/63C07K16/28
Inventor 房健民郭佳蒋明尹衍新于丽华蒋韵
Owner SUZHOU RES INST OF TONGJI UNIV
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