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High expression production method of egf-fc fusion protein and mammalian expression system

A fusion protein and mammalian technology, applied in the field of genetic engineering, can solve the problem of high cost, achieve high purity and activity, reduce production cost, and achieve high biological activity

Active Publication Date: 2014-10-29
ABZYME BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is: in order to overcome the problem of high cost of Fc-EGF fusion protein (N-terminal Fc) in the prior art, the present invention provides a high-expression production method of EGF-Fc fusion protein and its mammalian expression system

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  • High expression production method of egf-fc fusion protein and mammalian expression system
  • High expression production method of egf-fc fusion protein and mammalian expression system
  • High expression production method of egf-fc fusion protein and mammalian expression system

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Artificially synthesized DNA sequence encoding EGF-Fc fusion protein

[0030] According to the human EGF gene sequence reported in the literature, the human EGF signal peptide (EGF signal peptide), the gene sequence encoding the EGF mature peptide (Asn971-Arg1023) and the gene sequence encoding human IgG1Fc were selected for artificial synthesis (as shown in SEQ ID NO: 2 shown); human IL-2 signal peptide (IL-2 signal peptide), gene sequence encoding EGF mature peptide (Asn971-Arg1023) and gene sequence encoding human IgG1Fc were artificially synthesized (as shown in SEQ ID NO: 3); human IL-2 signal peptide, artificial intron (Intron), gene sequence encoding EGF mature peptide (Asn971-Arg1023) and gene sequence encoding human IgG1Fc were artificially synthesized (as shown in SEQ ID NO: 4); During synthesis, a Hind III restriction site and a Kozak sequence are introduced upstream, and an Eco RI restriction site is introduced downstream of the gene. The above g...

Embodiment 2

[0031] Example 2. Construction of expression vectors

[0032] The synthetic gene and vector pcDNA3.1 were digested overnight at 37°C with restriction endonucleases Hind III and Eco RI (Fermentas). The digested products were subjected to agarose gel electrophoresis, and the DNA fragment recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used after cutting the gel. The vector and the target gene were mixed with the expression vector at a ratio of 1:9. The reaction system was 10 microliters, and ligated with T4DNA Ligase (Treasure Biotechnology, D6020A) at 16°C for 3 hours. The ligated products were transformed into competent cells DH5α by the calcium chloride method, spread on LB plates containing 50 mg / L ampicillin, and cultured upside down overnight at 37°C. Pick single clones on the plate and use universal primers CMVF and BGH to carry out colony PCR to identify positive clones. The screened positive clones were sent to the sequencing company for sequenci...

Embodiment 3

[0033] Example 3. Expression and purification of EGF-Fc fusion protein

[0034] 1. Expression experiment of EGF-Fc fusion protein in HEK293 cells

[0035] a) 24 hours before transfection, HEK293T cells were resuspended in DMEM complete medium containing 10% FBS, according to 1×10 6 Cells were plated in a 6-well cell culture plate per well, and cultured in a carbon dioxide incubator (37°C, 5% CO2) for 24 hours; Procelle cell transfection kit B (Jiangsu Procelle Biotechnology Co., Ltd., catalog number: AZ-r002 ) according to the instructions for transfection; 6 hours after transfection, the cells were washed twice with PBS, then replaced with a commercial serum-free medium, and cultured for 48 hours to collect the culture supernatant.

[0036] b) Centrifuge the cell culture supernatant at 12,000 rpm for 10 minutes, add an appropriate amount of Protein A purification medium, incubate for 1 hour, and collect the purification medium bound to the EGF-Fc fusion protein by centrifuga...

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Abstract

The invention relates to an EGF-Fc fusion protein and a high-expression production method of a mammal expression system of the EGF-Fc fusion protein. An EGF and IgG1Fc fusion protein expression cassette is constructed by gene engineering means; a mammal cell expression carrier is constructed further, mammal cells are transfected for high-expression production of the EGF-Fc fusion protein and finally purification, identification and biological activity test are carried out. According to the high-expression production method of the mammal expression system of the EGF-Fc fusion protein, HEK293T or CHO-K1 cell is transfected instantly for expression of EGF-Fc fusion protein by using the constructed EGF-Fc expression element combination, high yield of 25mg / L can be obtained, and the obtained fusion protein has high purity and activity. The EGF-Fc fusion protein produced by the method can be applied to scientific research, pharmacy, cosmetics activity additives, etc.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an EGF-Fc fusion protein and its high-expression production method in a mammalian cell expression system. Background technique [0002] In 1962, Dr. Cohen of the United States and Professor Montalcini, an Italian scientist, discovered the "epidermal growth factor" (epidermal growth factor, EGF), which determines skin age, for which they won the Nobel Prize in Physiology. EGF is a polypeptide molecule composed of 53 amino acid residues, with a molecular weight of 6 kilodaltons. There are six cysteine ​​disulfide bonds in the molecule, which form the receptor domain necessary for biological activity. Epidermal growth factor is mainly synthesized by the submandibular gland and duodenum, and widely exists in body fluids and various glands, among which the content in milk, urine and semen is high, but the concentration in serum is low. EGF has a wide range of promoting ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/79C12N5/071A61K38/18A61K8/64A61P17/02A61Q1/00
Inventor 江永海朱月珍陆雅臣
Owner ABZYME BIOTECH
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