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Human epidermal growth factor hEGF gene optimization sequence, method for preparing same and application of human epidermal growth factor hEGF gene optimization sequence

An epidermal growth factor, gene optimization technology, applied in epidermal growth factor, growth factor/inducing factor, biochemical equipment and methods, etc., can solve the problems of low expression, low biological activity, high cost, and achieve high expression, The effect of high biological activity

Inactive Publication Date: 2017-10-17
江苏迈健生物科技发展股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Technical problem to be solved: the purpose of the present invention is to solve the technical problems of the existing prokaryotic system expressing recombinant hEGF (rhEGF) with low expression, low biological activity, cumbersome operation and high cost, and provide a human epidermal growth factor hEGF gene optimization Sequences and their preparation methods and applications

Method used

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  • Human epidermal growth factor hEGF gene optimization sequence, method for preparing same and application of human epidermal growth factor hEGF gene optimization sequence
  • Human epidermal growth factor hEGF gene optimization sequence, method for preparing same and application of human epidermal growth factor hEGF gene optimization sequence
  • Human epidermal growth factor hEGF gene optimization sequence, method for preparing same and application of human epidermal growth factor hEGF gene optimization sequence

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Experimental program
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Effect test

Embodiment 1

[0026] Gene optimization method of human epidermal growth factor hEGF

[0027] Log in to GeneBank to search for the gene sequence of the human epidermal growth factor hEGF active peptide. The nucleic acid length is 159bp. hEGF is a eukaryotic polypeptide sequence. Considering that subsequent research and development will be expressed in prokaryotic, the coding code of the above hEGF eukaryotic polypeptide sequence The codons were compared with the preferred codons of Escherichia coli. According to the degeneracy of codons and without changing the amino acid sequence of hEGF active peptide, the nucleic acid sequence of hEGF was analyzed with reference to the biological software Vector NTI Suitor 7.0, and the known hEGF was genetically modified and optimized. The rare codons of E. coli in the gene sequence are changed to preferred codons to improve the high-level expression of the target gene in E. coli. And the transgene verification was carried out, and finally the optimized ...

Embodiment 2

[0030] 2.1 Acquisition of expression strains

[0031] The prokaryotic expression vector pET-30a-hEGF obtained in Example 1 was transferred into Escherichia coli BL21 (DE3) (purchased from Tiangen Biological Company) by the heat shock method, and 50 μg of kanamycin was added to the LB plate medium / mL for screening, and single clones were selected for sequencing confirmation and identification.

[0032] 2.2 Induced expression of human epidermal growth factor hEGF gene optimized sequence

[0033] Inoculate recombinant positive monoclonal colonies into LB liquid medium containing 50ug / mL kanamycin at a concentration of 37°C and shake at 220rpm overnight. In the fresh liquid medium of kanamycin, cultivate at 37°C and 220rpm until the OD600 of the bacterial solution is about 0.9, immediately add the inducer IPTG to 0.5Mm, continue to culture the induction group and the control group for 3h, centrifuge at 12000rpm for 10min and collect bacteria.

[0034] 2.3 Extraction and identi...

Embodiment 3

[0038] Biological activity verification of human epidermal growth factor hEGF

[0039] The purchased Balb / c 3T3 cells (purchased from ATCC Company) were transferred to passage 3-4, counted, and then plated in a 96-well plate with 1×105 cells per well. After culturing in DMEM complete medium (10% serum) in a 96-well plate for 24 hours, add maintenance medium (0.4% serum) to continue culturing for 24 hours.

[0040] hEGF (final concentration: 20ng / mL), add control group NC and EGF standard (final concentration: 20ng / mL) small peptide to stimulate mouse embryonic fibroblast Balb / c 3T3 cells for 72 hours, then detect hEGF by MTT method Promote cell proliferation. The microplate reader detects the absorbance at a wavelength of 450nm, and the absorbance value is directly proportional to the cell proliferation activity. The calculated specific activity was 6.1×107IU. The results are shown in Table 1 and Figure 4 , the proliferation effect of the hEGF treatment group was signific...

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Abstract

The invention discloses a human epidermal growth factor hEGF gene optimization sequence, a method for preparing the same and application of the human epidermal growth factor hEGF gene optimization sequence. The human epidermal growth factor hEGF gene optimization sequence is shown as SEQIDNO.1. The human epidermal growth factor hEGF gene optimization sequence, the method and the application have the advantages that cDNA [complementary DNA (deoxyribonucleic acid)] structures of EGF (epidermal growth factor) genes are changed, alkali bases of individual genes on nucleotide sequences are replaced by synonym codons, genes with the optimal codon usage bias are selected to synthesize EGF optimization sequences, prokaryotic expression vectors pET-30a-hEGF are constructed and can be expressed in Escherichia coli hosts in a high-level manner, and accordingly the human epidermal growth factor hEGF gene optimization sequence, the method and the application have important significance on EGF industrial production and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an optimized human epidermal growth factor (hEGF) gene sequence and its preparation method and application. Background technique [0002] EGF is the second growth factor discovered after the nerve growth factor. It was first isolated from the subcollar gland of mice by Chone in 1959, because it can promote the eruption of mouse incisors and the opening of eyelids. It was originally called "dental lid factor". ". It was later discovered that this polypeptide can promote epidermal growth, and it was named epidermal growth factor (ie, mouse epidermal growth factor, mEGF) 121. In 1975, Gergoyr extracted a substance that inhibits gastric acid secretion from human urine, called urine Urogastrone was later confirmed to be human EGF (hEGF). [0003] hEGF contains 53 amino acid residues, the molecular weight is 6200Da, the sedimentation coefficient is 1.255, the isoelectric poin...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C07K14/485C12N15/70C12N1/21A61K8/64A61Q19/00C12R1/19
CPCA61K8/64A61K2800/86A61Q19/00C07K14/485C12N15/102C12N15/70C12N2800/22
Inventor 张超庞玉红段云飞王丹常菁虞强
Owner 江苏迈健生物科技发展股份有限公司
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