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Experimental technique for large-scale production of recombinant soluble human stem cell factor and immunoglobulin Fc segment fusion protein (sSCF-Fc) in mammalian cell

A mammalian, large-scale technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, hybrid peptides, etc., can solve the problems of low glycosylation degree of protein products, activity and toxin content that are difficult to meet clinical diagnosis and treatment, etc. , to achieve high expression level, improve product yield and quality, and improve protein yield

Inactive Publication Date: 2012-02-08
ABZYME BIOTECH
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Problems solved by technology

[0015] At present, the sSCF-Fc recombinant protein produced in the world is basically obtained by Escherichia coli or yeast expression system, the protein product is not glycosylated or the degree of glycosylation is not high, and the activity and toxin content are difficult to meet the purpose of clinical diagnosis and treatment

Method used

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  • Experimental technique for large-scale production of recombinant soluble human stem cell factor and immunoglobulin Fc segment fusion protein (sSCF-Fc) in mammalian cell

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Embodiment Construction

[0021] 1. Preparation of Plasmids for Transfection

[0022] The expression plasmid pR-sSCF-Fc connected with the sSCF-Fc gene required for transfection was prepared with an endotoxin-removing plasmid purification kit (purchased from Qiagen). See the instruction manual for the method. Dissolve the plasmid with 200 μl TE buffer, and measure its concentration with a UV spectrophotometer.

[0023] 2. Preparation of PEI for transfection

[0024] 1) Pour 450ml of ultrapure water prepared by Milli-Q into a 500ml beaker;

[0025] 2) Weigh 500 mg linear PEI (purchased from Poly Science Company) on the balance, add it to the beaker, and stir it continuously with a magnetic stirrer;

[0026] 3) Add pre-prepared 12M concentrated HCl dropwise to make the pH<2.0 (about 800 μl is required). Stir the PEI solution for about 2-3 hours;

[0027] 4) Add pre-prepared 10M NaOH solution to adjust the pH to 7.0 (about 500 μl is required). Dilute the volume of PEI solution to 500ml with Milli-Q ...

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Abstract

The invention relates to a technique for transferring Fc segment fusion gene (sSCF-Fc) of human stem cell factor and human immunoglobulin G1 (IgG1) into a mammalian cell for large-scale production of a protein product of the gene, and aims to establish a method for large-scale production of sSCF-Fc protein with biological activity by using the mammalian cell. The experimental technique meets the overall requirements of reducing production cost, simplifying process flow and improving product yield and quality in industrial production. By adopting the method, a large amount of sSCF-Fc protein with high purity and high biological activity can be obtained through normal large-scale cell culture. The technique is applied to large-scale production of the sSCF-Fc protein; and the cost can be obviously reduced, the process flow can be simplified, and the product yield and quality can be improved.

Description

technical field [0001] The invention relates to a technique for transferring human stem cell factor and the Fc fragment fusion gene (sSCF-Fc) of human immunoglobulin IgG1 into mammalian cells to produce its protein product on a large scale. Background technique [0002] 1. The biological function and application prospect of SCF [0003] Stem cell factor (SCF), also known as KIT ligand (KIT Ligand, KITLG) or mast cell growth factor (Mast cell growth factor, MGF), is a glycoprotein that binds to the proto-oncogene c-Kit product (CD117). SCF can form transmembrane and soluble protein products through variable shearing, both of which have biological activity and play an important role in the process of body development, tumorigenesis, development and invasion, and the formation of other diseases effect. [0004] SCF is essential for hematopoiesis during embryonic development. SCF guides hematopoietic stem cells into the hematopoietic microenvironment, and maintains the viabi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C07K19/00
Inventor 柴笑梅朱月珍杨宣武周慧芳王小娟江永海
Owner ABZYME BIOTECH
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