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Gene sequence and polypeptide of FGFR2b ectodomain and application thereof

An extracellular segment, base sequence technology, applied in the field of genetic engineering, can solve the problem of unclear mechanism, and achieve the effect of inhibiting redness and sebum secretion.

Inactive Publication Date: 2014-04-30
GUANGDONG YANZHIBAO BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When mesenchymal cells interact with epithelial cells, they activate EGF, FGF, IGF and other signaling pathways, which eventually lead to inflammation, but the specific mechanism is still unclear

Method used

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  • Gene sequence and polypeptide of FGFR2b ectodomain and application thereof
  • Gene sequence and polypeptide of FGFR2b ectodomain and application thereof
  • Gene sequence and polypeptide of FGFR2b ectodomain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Expression of wild-type and mutant FGFR2b extracellular segment polypeptide genes in Escherichia coli

[0042] This example describes the preparation methods of wild-type and mutant FGFR2b extracellular segment genes (SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5), and their production in Escherichia coli (E.coli) A method for expressing and preparing FGFR2b extracellular segment polypeptides (SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6).

[0043] 1. Preparation of FGFR2b extracellular segment polypeptide gene:

[0044] 1. Extract the total mRNA of wild-type FGFR2b extracellular segment from human placenta tissue by Trizol method, and establish a cDNA library;

[0045] (1) Extract mRNA by Trizol method:

[0046] When the cells are cultured to a confluence of 90%, add Trizol solution to lyse the cells; transfer the resulting cell lysate to an EP tube, add 1 / 5 volume of chloroform, and pipette the upper colorless aqueous phase into a new EP tube , add 1 / 2 Trizol volume of ...

Embodiment 2

[0104] The extracellular segment of embodiment 2FGFR2b is expressed in mammalian cells

[0105] This example describes the preparation of potentially glycosylated forms of FGFR2b by recombinant expression of wild-type and mutant FGFR2b extracellular domain genes (SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5) in mammalian cells The method of the extracellular segment polypeptide.

[0106] 1. Primer design: SEQ ID NO.13 and 14

[0107] Upstream primer: ATATGGATCC GCCGCCACC ATG GCACCATACTGGACCAAC;

[0108] Downstream primers: GCGCAAGCTT TCATTA CAGGATGACTGTTACCAC including enzyme cutting sites BamH Ⅰ and Hind Ⅲ.

[0109] 2. Carrier construction:

[0110] The vector pCDNA3.1 (-) (purchased from Invitrogen, USA) was used as the expression vector;

[0111] FGFR2b (SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5) were respectively linked to pCDNA3.1 (the method is the same as in Example 1), and the resulting vector was called pCDNA3.1-FGFR2b.

[0112] 3. pCDNA3.1-FGFR2b transfected 293 cells

[...

Embodiment 3

[0122] Example 3 FGFR2b extracellular segment expressed in CHO cells

[0123] This example describes the expression of wild-type and mutant FGFR2b extracellular segment genes (SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5) in CHO cells to prepare FGFR2b extracellular segment polypeptides (SEQ ID NO. .2, the method of SEQ ID NO.4, SEQ ID NO.6), the preparation of the gene sequence used is shown in Example 1.

[0124] 1. Primer design:

[0125] Upstream primer: ATAT GGATCC GCCGCCACC ATG GCACCATACTGGACCAAC;

[0126] Downstream primers: GCGCGAATTC TCATTA CAGGATGACTGTTACCAC including restriction sites BamH Ⅰ and EcoRI.

[0127] 2. Carrier construction:

[0128] The vector pIRESneo3 (purchased from Clontech) was used as an expression vector;

[0129] FGFR2b (SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5) were respectively linked to pIRESneo3 (the method is the same as in Example 1), and the three vectors thus generated were all called pIRESneo3-FGFR2b.

[0130] 3. pIRESneo3-FGFR2b transfected ...

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Abstract

The invention provides gene sequences of wild type, S252W mutant type and P253R mutant type FGFR2b ectodomains and polypeptides coded thereby respectively. A preparation system of the polypeptide comprises a recombinant vector, a host cell and a preparation method, an antibody of FGFR2b ectodomain and a preparation method thereof as well as a fusion polypeptide, an antagonist and the like of the FGFR2B ectodomain and Fc domain. The invention also provides an application of the polypeptide of FGFR2b ectodomain and a composition in treating eczema, acne, psoriasis, skin allergy, seborrheic dermatitis or alopecia seborrheica; an FGF signal channel is inhibited through the FGFR2b ectodomain, the symptoms such as swelling, pruritus and the like of inflammatory skin diseases are inhibited, and an inhibitory effect is also realized on the sebum secretion.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the gene sequence and polypeptide of the extracellular segment of FGFR2b and its application. Background technique [0002] Inflammatory skin disease refers to skin redness and swelling, papules, pustules, etc., and keratinocytes proliferate and protrude, sometimes accompanied by itching, which brings great troubles to patients. Eczema is a common inflammatory skin disease of the epidermis and superficial dermis caused by various internal and external factors. It is generally believed that it has a certain relationship with allergies (ie, allergic reactions). Acne is a chronic inflammatory skin disease involving the pilosebaceous glands, which is commonly known as "acne" in adolescence. Acne mostly occurs in seborrhea areas such as the face, chest and back. Foreign studies have shown that the incidence of acne among adolescents aged 12-24 is as high as 85%. In recent years, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/71C12N15/70C12N15/85C12N15/866C12N15/81C12N1/21C12N5/10C12N1/19C07K19/00A61K38/17A61K47/48A61K48/00A61K39/395A61P17/00A61P17/04A61P17/06A61P17/08A61P17/10A61P17/14A61P37/08
CPCA61K38/179A61P17/00A61P17/02A61P17/04A61P17/06A61P17/08A61P17/10A61P17/14A61P37/08C07K14/71C12N2501/113C12N2510/02A61K47/42A61K48/00C07K2319/30C12N15/63A61K45/06
Inventor 汪炬王宜
Owner GUANGDONG YANZHIBAO BIOTECHNOLOGY CO LTD
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