Escherichia expression system of secretory expression recombinant human epidermal growth factor

A technology of secretion expression and Escherichia coli, applied in the field of bioengineering, can solve problems such as low content

Active Publication Date: 2006-11-01
SHANGHAI HAOHAI BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] hEGF widely exists in human body fluids and some glands and tissues, but the content is extremely low, and it is impossible to prepare a large amount by tissue extraction for research and clinical use

Method used

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  • Escherichia expression system of secretory expression recombinant human epidermal growth factor
  • Escherichia expression system of secretory expression recombinant human epidermal growth factor
  • Escherichia expression system of secretory expression recombinant human epidermal growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Chemically synthesized signal peptide phoAspL10 sequence and human epidermal growth factor (hEGF) tandem gene (SEQ ID NO: 2)

[0040] ↓Start codon 5’GAT ACC AAA CAA AGC ACT CTG CTG CTG CTG CTG CTG CTT CTG CTG

[0041] | ← Newly designed signal peptide sequence (the underlined part is CTG CTGACC CCT GTG ACA AAA GCG AAT TCC GAC TCT GAA TGC CCG ten leucines in a row) →|← CTG TCT CAC GAC GGT TAC TGC CTA CAC GAT GGT GTT TGC ATG TAT

[0042] Human epidermal growth factor (hEGF) gene ATC GAA GCT CTG GAC AAA TAC GCG TGC AAC TGT GTT GTT GGT TACATC GGT GAA CGT TGC CAG TAC CGT GAC CTG AAA TGG TGG GAA CTG

[0043]

[0044] When designing and synthesizing the above-mentioned tandem genes, we fully followed the following principles: the codons preferred by Escherichia coli were selected; the contents of AT and GC were close and evenly distributed; the generation of secondary structures in the fragments was avoided.

[0045] At the downstream ...

Embodiment 2

[0046] The construction of embodiment 2 expression plasmid ( figure 1 )

[0047] 2.1 Enzyme digestion treatment of the starting vector pBLGlu2

[0048] 2.1.1 EcoRI digestion

[0049] Prepare the following reaction mixture:

[0050] 10 μg of plasmid pBLGlu2;

[0051] 20 μl 10×H buffer (TaKaRa);

[0052] 5 μl EcoRI restriction endonuclease (15U / μl, TaKaRa company);

[0053] Make up 200 μl with sterilized double distilled water.

[0054] The above reaction mixture was reacted at 37° C. for 4 hours.

[0055] 2.1.2 Recovery of the vector after digestion

[0056] Add 20μl 3M sodium acetate and 400μl absolute ethanol to the reaction mixture obtained in 2.1.1, mix well, centrifuge at 12,000rpm for 5 minutes, discard the supernatant, then add 400μl 70% ethanol, mix well, 12,000rpm Centrifuge for 2 minutes, discard the supernatant, and vacuum dry.

[0057] 2.1.3 Digestion with Mung Bean Nuclease

[0058] Prepare the following reaction mixture:

[0059] The carrier recovered i...

Embodiment 3

[0115] Example 3 Construction and screening of genetically engineered strains

[0116] 3.1 Construction of genetically engineered strains

[0117] Prepare competent cells of Escherichia coli BL21(DE3) according to the method in 2.4, transform the expression plasmid pBL10EGF into the competent cells of BL21(DE3) according to the method in 2.5, and take 100 μl of LB coated with 100 μg / ml ampicillin Plates (agar content 1.5%) were cultured upside down at 37°C overnight.

[0118] 3.2 Screening of genetic engineering strains

[0119] 3.2.1 Induced expression of genetically engineered strains

[0120] Pick the single colonies obtained in 3.1 and put them into 3 milliliters of LB medium (the concentration of ampicillin is 100 μg / ml), culture overnight at 37°C at 200 rpm, then transfer them into LB medium at a ratio of 1:10, and then After continuing to cultivate for 4 hours, the temperature was raised to 42° C., and the cultivation was terminated after 6 hours. Take 1ml of the cu...

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Abstract

This invention includes signal peptide gene phoAspL10, expression plasmid pBL10EGF, host cell of bacillus coli BL21 (DE3) inverted by expression plasmid pBL10EGF, engineering strain BE-2 screened from the host cell and preparation method of rhEGF. The strain BE-2 can excrete and express rhEGF directly into culture after fermentation and the quantity is 384mg/l.The purity is more than 95% and specific activity more than 1.0*106IU/mg protein. The product rhEGF reserves crude space configuration and shows high biological activity.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to the preparation of recombinant human epidermal growth factor (rhEGF) through a genetic engineering method. Background technique [0002] Human epidermal growth factor (Human epidermal growth factor, referred to as hEGF), is a single-chain polypeptide composed of 53 amino acids without sugar groups, with a molecular weight of 6,216 Daltons. hEGF is an important polypeptide growth factor in the human body. It has a variety of biological functions. It can bind to specific receptors on epidermal cells, transmit information into cells, change intracellular pH and free calcium concentration, and promote glycolysis. Decomposition and protein synthesis, increase the transcription of certain specific genes, thereby promoting DNA replication and cell division. EGF can promote the division of various cells, and can promote the growth of epithelial cells of epidermal cells, nerve c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C12N15/11C12N15/12C12N15/63C12N15/70C12P21/02
Inventor 甘人宝钱悦朱俊冯宝山丁红珍张倩叶勤张海毅
Owner SHANGHAI HAOHAI BIOLOGICAL TECH
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