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 exp

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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Example Embodiment

[0025] Example 1

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

[0027] Log in to GeneBank to find 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 the subsequent research and development will be expressed in prokaryotic cells, the coding code of the above hEGF eukaryotic polypeptide sequence is changed. Comparative analysis of codons and E. coli preferred codons. According to the codon compatibility and without changing the amino acid sequence of hEGF active peptide, the nucleic acid sequence of hEGF is analyzed with reference to the biological software Vector NTI Suitor 7.0, and the known hEGF is genetically modified and optimized. In the gene sequence, the rare codons of E. coli are changed to preferred codons to improve the high-level expression of the target gene in E. coli. After transgene verification, the optimized sequence of ...

Example Embodiment

[0029] Example 2

[0030] 2.1 Obtaining expression strains

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

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

[0033] The recombinant positive monoclonal colony was inoculated into LB liquid medium containing 50ug / mL kanamycin, cultured overnight at 37°C and 220rpm with shaking, and inoculated the bacterial solution from the overnight culture at a ratio of 1% to 50ug / mL In a fresh liquid medium of kanamycin, culture at 37°C and 220 rpm until the concentration of the bacterial solution OD600 is about 0.9. Immediately add the inducer IPTG to 0.5 Mm. The induction group and the control group are cultured for 3 hour...

Example Embodiment

[0037] Example 3

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

[0039] Pass the purchased Balb / c 3T3 cells (purchased from ATCC) to 3-4 generations, count them, and then pave a 96-well plate with 1×105 cells per well. After culturing in a 96-well plate DMEM complete medium (10% serum) for 24 hours, adding maintenance medium (0.4% serum) to continue culturing for 24 hours.

[0040] hEGF (final concentration of 20ng / mL), plus control NC and EGF standard (final concentration of 20ng / mL) small peptides to stimulate mouse embryonic fibroblasts Balb / c 3T3 cells 72 hours, 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 is 6.1×107IU. The results are shown in Table 1 and Figure 4 The proliferation effect of the hEGF treatment group was significantly better tha...

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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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