CN1854294A - Escherichia expression system of secretory expression recombinant human epidermal growth factor - Google Patents

Escherichia expression system of secretory expression recombinant human epidermal growth factor Download PDF

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CN1854294A
CN1854294A CNA2005100252893A CN200510025289A CN1854294A CN 1854294 A CN1854294 A CN 1854294A CN A2005100252893 A CNA2005100252893 A CN A2005100252893A CN 200510025289 A CN200510025289 A CN 200510025289A CN 1854294 A CN1854294 A CN 1854294A
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rhegf
pbl10egf
gene
phoaspl10
expression plasmid
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CN100489096C (en
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甘人宝
钱悦
朱俊
冯宝山
丁红珍
张倩
叶勤
张海毅
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SHANGHAI HAOHAI BIOLOGICAL TECHNOLOGY Co Ltd
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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

The escherichia expression system of secretory expression recombinant human epidermal growth factor
Technical field
The invention belongs to bioengineering field, be specifically related to prepare recombinant human epidermal growth factor (rhEGF) by gene engineering method.
Background technology
Human epidermal growth factor's (Human epidermal growth factor is called for short hEGF) is a kind of glycosyl of not being with, and by the single chain polypeptide that 53 amino acid are formed, molecular weight is 6,216 dalton.HEGF is a kind of important polypeptide growth factor in the human body, it has the various biological function, can with special receptors bind on the epidermic cell, information is transmitted into cell, change intracellular potential of hydrogen and free calcium concentration, promote glycolysis-and proteinic synthetic, increase some specificity genetic transcription, thereby promote dna replication dna and cell fission.EGF can impel the various kinds of cell division, can promote the epithelial cell growth of epidermic cell, neurocyte and organ-tissue.In organism grew, EGF also can promote the growth of newborn infant's tooth, bone and each organ.Clinical test results shows, hEGF can impel ectoderm, mesoblastema growth, promote the healing of burn, wound and surgical wound, quicken to transplant the growth of epidermis and corneal transplant, therefore, the treatment to wound burn treatment, skin and corneal transplantation, skin ulcerous, keratohelcosis, gastrointestinal ulceration and irritability gastric mucosa injury all plays an important role.In recent years, hEGF is used for the treatment of squamous cell carcinoma, skin carcinoma abroad, has obtained significant curative effect.
HEGF extensively is present in people's the body fluid and some body of gland and tissue, but content is extremely low, can not prepare in a large number for research and clinical usefulness in the tissue extraction mode.In the research formerly, present inventors adopt engineered method, hEGF gene, intestinal bacteria alkaline phosphatase promotor and the signal peptide sequence of synthetic are cloned among the plasmid pTZ18R, are built into secreting, expressing plasmid pAE-8 and obtain engineering strain EE-8.This project bacterial strain is secreting, expressing rhEGF successfully, and high expression level amount is 150mg/l (patent application CN00127953.X).
Intestinal bacteria alkaline phosphatase promotor is a kind of promotor of medium tenacity, utilizes above-mentioned promotor to make the expression amount of rhEGF reach 150mg/l, if use stronger promotor instead, such as P LCan strong promoter make the secreting, expressing amount of rhEGF go another step? be with this idea, present inventors have begun experimental design, simultaneously, consider P LPromotor causes the movement system in the Escherichia coli cell to have little time rhEGF is secreted to born of the same parents, and rhEGF is accumulated in the born of the same parents in a large number in case startup may make the instantaneous great expression of rhEGF.In order to improve the saturating film secernment efficiency of rhEGF, present inventors set about from the modelled signal peptide, have finished the structure and the Optimizing Conditions of Fermentation of expression plasmid and engineering strain smoothly, thereby have finished the present invention.
Therefore, an object of the present invention is to provide signal peptide (phoAspL10) sequence of a brand-new design.
Another object of the present invention provides the expression plasmid pBL10EGF of secretory expression recombinant human epidermal growth factor (rhEGF).
A further object of the present invention provides expression plasmid pBL10EGF transformed host cells.
Of the present invention also have a purpose to provide genetic engineering bacterium BE-2.
Further purpose of the present invention provides signal peptide phoAspL10 and the application of expression plasmid pBL10EGF on the rhEGF that the preparation direct secretion is expressed.
Further purpose of the present invention provides direct secretion and expresses the rhEGF preparation method of rhEGF to substratum.
Summary of the invention
The invention provides signal peptide (phoAspL10) gene (SEQ ID NO:1) of a brand-new design:
5’ATG?AAA?CAA?AGC?ACT?CTG?CTG?CTG?CTG?CTG?CTT?CTG?CTG?CTG
CTG?ACC?CCT?GTG?ACA?AAA?GCG?3’
The present invention also provides the expression plasmid pBL10EGF of secretory expression recombinant human epidermal growth factor, and it comprises the phoAspL10 gene of described brand-new design, and is positioned at the temperature sensitive promotor P of described upstream region of gene LWith the hEGF gene that is positioned at described gene downstream.Described temperature sensitive promotor P LThe source see Giladi H, Goldenberg D, Koby S, Oppenheim AB.:Enhanced activity of the bacteriophage lambda PL promoter at lowtemperature.FEMS Microbiol Rev.1995 Aug; 17 (1-2): 135-40.
Among the expression plasmid pBL10EGF of the present invention, contained recombinant protein gene comprises the polypeptide and the protein gene of all known human bodies at present.
Host cell provided by the invention is to have e. coli bl21 (DE3) cell amicillin resistance, that expression plasmid pBL10EGF transforms.
The present invention also provides the engineering strain BE-2 of secreting, expressing rhEGF, and described engineering strain is the highest bacterial strain of expression amount that goes out from e. coli bl21 (DE3) cell screening that expression plasmid pBL10EGF transforms.
Direct secretion provided by the invention is expressed rhEGF and is comprised the steps: to the rhEGF preparation method of substratum
(1) chemosynthesis phoAspL10 sequence;
(2) be initial plasmid construction expression plasmid pBL10EGF with pBLGlu2;
(3) make up and filter out engineering strain BE-2;
(4) fermentation culture of genetic engineering bacterium BE-2;
(5) separation and purification of expression product.
The signal peptide phoAspL10 of present inventor's design is a kind of brand-new sequence, and its length is 21 amino acid, and wherein the 6th to the 15th amino acid is designed to nonpolar amino acid-leucine.Signal peptide phoAspL10 is stronger than the hydrophobicity of intestinal bacteria alkaline phosphatase signal peptide, is more conducive to saturating film and the secretion of rhEGF.
Present inventors have successfully finished the structure of expression plasmid and engineering strain according to above-mentioned experimental design.In the secreting, expressing plasmid pBL10EGF that the present invention makes up, with P LThe phoAspL10 sequence and the hEGF gene of strong promoter, brand-new design are connected.The largest benefit of this phraseology is that direct efficient secretory expression rhEGF is to substratum, kept its natural space structure, product does not need through protein renaturation, thereby the biological activity of expression product is very high, makes that also follow-up purification procedures is simplified.And other adopts P LThe proteic method of promoter expression, or expression product forms inclusion body in born of the same parents, product also will carry out protein renaturation behind purifying, or expression product expressed to the pericentral siphon of bacterium, brings very big trouble for follow-up separation and purification.
On the basis that the fermentation condition and the inducing temperature of engineering strain are groped, present inventors find, when inducing for 38 ℃, 42 ℃ of will adopt more usually of the secreting, expressing amount of rhEGF are high when inducing.Under the fermentation and inductive condition of this optimization, the maximum amount that engineering strain BE-2 expresses rhEGF reaches 384mg/l, through separation and purification, obtain purity greater than 95%, specific activity is greater than 1.0 * 10 6The proteic rhEGF product of IU/mg produces a desired effect fully.
Above result of study provides multiple choices when expressing other recombinant polypeptide or albumen from now on: adopt P LThe placed in-line expression system of phoAspL10 sequence of promotor, brand-new design or intestinal bacteria alkaline phosphatase expression system.
Description of drawings
Fig. 1 makes up synoptic diagram for expression plasmid pBL10EGF.
The positive cloning nucleic acid electrophorogram of Fig. 2.Wherein, the 1st hurdle is a dna marker; The negative contrast in the 2nd hurdle; The positive contrast in the 3rd hurdle; The 4th hurdle is blank; The positive clone in the 5th hurdle.Fig. 3 is engineering strain screening SDS-PAGE electrophorogram.The negative contrast in the 1st hurdle (the empty bacterium of YK537); The 2nd hurdle is the hEGF contrast; The 3rd hurdle is BE-1; The 4th hurdle is BE-2; The 5th hurdle is BE-3; The 6th hurdle is BE-4; The 7th hurdle is BE-5; The 8th hurdle is BE-6.
Fig. 4 A is the leaven line chart of engineering strain BE-2.Fig. 4 B is the abduction delivering electrophorogram of engineering strain BE-2.Wherein, the 1st hurdle is the hEGF contrast; The 2-6 hurdle is respectively 20,22,25,26 and 27 hours supernatants of inducing culture.
Fig. 5 is hydrophobic chromatography figure.
Fig. 6 is anionresin figure.
Fig. 7 is gel-filtration figure.
Fig. 8 is a SDS-PAGE electrophoresis rating diagram.Wherein, the 1st hurdle is that the molecular weight of albumen mark (is followed successively by 10K from bottom to top, 20K, 30K ... 100K); The 2nd hurdle is contrast hEGF; The 3rd hurdle is the rhEGF product.Fig. 9 is the reversed-phase HPLC rating diagram.
Figure 10 is molecular weight determination figure.
Figure 11 is a N terminal amino acid sequence determination data.
Figure 12 is biological activity determination figure.
Describe the present invention with embodiment below.These embodiment only illustrate, and the present invention are not constituted any restriction.
Embodiment 1 chemosynthesis signal peptide phoAspL10 sequence and human epidermal growth factor's (hEGF) tandem gene (SEQ ID NO:2)
↓ initiator codon 5 ' GAT ACC
Figure A20051002528900061
AAA CAA AGC ACT CTG CTG CTG CTG CTG CTT CTG CTG
| (underscore partly is the signal peptide sequence of ← brand-new design CTG CTGContinuous ten leucines of ACC CCT GTG ACA AAA GCG AAT TCC GAC TCT GAA TGC CCG) → | ← CTG TCT CAC GAC GGT TAC TGC CTA CAC GAT GGT GTT TGC ATG TAT
Human epidermal growth factor (hEGF) gene A TC 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
Figure A20051002528900071
When we synthesize above-mentioned tandem gene in design, fully followed following principle: the codon of promptly selecting the intestinal bacteria preference for use; AT, GC content near and be evenly distributed; Avoid the generation of secondary structure in the fragment.
At the downstream end of above-mentioned tandem gene, we have designed the restriction enzyme site of restriction enzyme HindIII, are convenient to gene clone (gene and primer are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).
The structure (Fig. 1) of embodiment 2 expression plasmids
2.1 the enzyme of initial vector pBLGlu2 is cut processing
2.1.1 the EcoRI enzyme is cut
Be formulated as follows reaction mixture:
10 μ g plasmid pBLGlu2;
20 μ l, 10 * H damping fluid (TaKaRa company);
5 μ l EcoRI restriction enzymes (15U/ μ l, TaKaRa company);
The sterilization distilled water is supplied 200 μ l.
Above-mentioned reaction mixture was reacted 4 hours in 37 ℃.
2.1.2 enzyme is cut the recovery of back carrier
In the reaction mixture of 2.1.1 gained, add 20 μ l 3M sodium acetates and 400 μ l dehydrated alcohols, behind the abundant mixing, in 12, centrifugal 5 minutes of 000rpm, abandon supernatant, add 400 μ l, 70% ethanol again, abundant mixing, 12, centrifugal 2 minutes of 000rpm abandons supernatant, and vacuum is drained.
2.1.3 mung-bean nuclease (Mung Bean Nuclease) enzyme is cut
Be formulated as follows reaction mixture:
2.1.2 the carrier of middle recovery;
10 μ l, 10 * mung-bean nuclease damping fluid (TaKaRa company);
1 μ l Mung Bean Nuclease (45U/ μ l, TaKaRa company);
The sterilization distilled water is supplied 100 μ l.
Above-mentioned reaction mixture was reacted 30 minutes in 30 ℃.
2.1.4 the recovery of dna fragmentation
The dna fragmentation of gained among the 2.1.3 is reclaimed by the operation steps on the test kit specification sheets with UNIQ-10 pillar PCR product purification test kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), with 50 μ l elutriant wash-outs.
2.1.5 insulation
The elutriant of gained among the 2.1.4 is incubated 10 minutes in 65 ℃.
2.1.6 the HindIII enzyme is cut
Be formulated as follows reaction mixture: the elutriant among the 2.1.5;
6 μ l, 10 * M damping fluid (TaKaRa company);
2 μ l HindIII restriction enzyme (15U/ μ l, TaKaRa companies; )
The sterilization distilled water is supplied 60 μ l.
Above-mentioned reaction mixture is spent the night in 37 ℃ of reactions.
2.1.7 the big segmental recovery of DNA
Enzyme among the 2.1.6 is cut the reaction solution that spends the night carry out 1% agarose electrophoresis, under ultraviolet lamp, downcut big band, the operation steps of pressing on the test kit specification sheets with UNIQ-10 pillar DNA glue recovery test kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) reclaims the big fragment of DNA, with 50 μ l elutriant wash-outs.
2.2 the enzyme of phoAspL10, hEGF tandem gene is cut processing
The above-mentioned tandem gene of 1OD of getting chemosynthesis is with the dissolving of 200 μ l sterilization distilled water, gets 10 μ l and makes the HindIII enzyme and cut processing.
Be formulated as follows reaction mixture:
10 μ l phoAspL10, hEGF tandem gene;
4 μ l, 10 * M damping fluid (TaKaRa company);
2 μ l HindIII restriction enzymes (15U/ μ l, TaKaRa company);
The sterilization distilled water is supplied 40 μ l.
Above-mentioned reaction mixture is spent the night in 37 ℃ of reactions, carry out 1% agarose electrophoresis then, under ultraviolet lamp, downcut big band, the operation steps of pressing on the test kit specification sheets with UNIQ-10 pillar DNA glue recovery test kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) reclaims the big fragment of DNA, with 50 μ l elutriant wash-outs.
2.3 connect
Be formulated as follows reaction mixture:
After treatment the big fragment of carrier among the 5 μ l 2.1.7;
The phoAspL10 that cuts through the HindIII enzyme among the 12 μ l 2.2, hEGF tandem gene;
2 μ l, 10 * T 4Dna ligase damping fluid (TaKaRa company; )
1 μ l T 4Dna ligase (350U/ μ l, TaKaRa company).
Above-mentioned reaction mixture is spent the night in 16 ℃ of reactions.
2.4 the preparation of competent cell
10 μ l TOP10 glycerol stocks are seeded in the 3ml LB substratum, and 37 ℃ of 200rpm overnight incubation are got 100 μ l bacterium liquid and are inserted in the 3ml LB substratum again, and 37 ℃ of 200rpm are cultured to A 6000.3～0.4, then 4, centrifugal 10 minutes of 000rpm abandons supernatant, thalline is with calcium solution (the 0.1mol/l CaCl of precooling 2) washing, 4, centrifugal 10 minutes of 000rpm, thalline are resuspended in the calcium solution of 200 μ l standby.
2.5 transform
Get the connection product in 2.3, add in the competent cell of preparation in 2.4, ice bath 30 minutes, in 42 ℃ of heat-shockeds 90 seconds, add 800 μ l LB substratum subsequently, then in 37 ℃, 100rpm, the LB flat board (agar content is 1.5%) that 100 μ l coating contains 100 μ g/ml penbritins is got in jolting 30 minutes, is inverted overnight incubation for 37 ℃.
2.6 screening positive clone
The upstream primer of synthetic phoAspL10, hEGF tandem gene and downstream primer (the above-mentioned primer of 1OD is standby with the dissolving of 400 μ l sterilization distilled water respectively) are used for PCR reaction screening positive clone, and primer sequence is as follows:
Upstream primer (21bp): 5 ' GAT ACC ATG AAA CAA AGC ACT, 3 ' (SEQ ID NO:3);
Downstream primer (18bp): 5 ' CCC AAG CTT ATT AAC GCA, 3 ' (SEQ ID NO:4).
In 3ml LB (wherein the concentration of penbritin is 100 μ g/ml), at 37 ℃, the 200rpm overnight incubation is carried out the PCR reaction with the bacterium colony nutrient solution as template with the some bacterium colonies that grow in the aseptic toothpick picking 2.5.Each bacterium colony nutrient solution sample carries out the PCR reaction by following combination and reaction conditions.
Be formulated as follows reaction mixture:
0.25 μ l Ex Taq (5U/ μ l, TaKaRa company);
5 μ l, 10 * Ex Taq damping fluid (TaKaRa company);
4 μ l dNTP Mixture (TaKaRa company);
1 μ l upstream primer (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is synthetic);
1 μ l downstream primer (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is synthetic);
1 μ l bacterium colony nutrient solution;
The sterilization distilled water is supplied 50 μ l.
Reaction conditions: 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 40 seconds, totally 30 circulations.
The negative control of PCR reaction replaces the bacterium colony nutrient solution with 1 μ l sterilization distilled water, and positive control is with dissolved 1 μ l phoAspL10, hEGF tandem gene replacement bacterium colony nutrient solution in 2.2.Get PCR reaction solution 20 μ l and carry out 1.5% agarose electrophoresis, under ultraviolet lamp, what the PCR reaction product of bacterium colony nutrient solution sample located to occur band in about 240bp is the positive colony (see figure 2).
2.7 a small amount of extracting of plasmid
The nutrient solution 10 μ l that get positive colony are seeded in the 3ml LB substratum (wherein the concentration of penbritin is 100 μ g/ml), 37 ℃ of 200rpm overnight incubation, with UNIQ-10 pillar plasmid a small amount of extraction agent box (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), press the plasmid of the operation steps extracting positive colony on the test kit specification sheets.
2.8 determined dna sequence
We have synthesized sequencing primer (5 ' AGC ACA TCA GCA GGA CGC A 3 ', 19bp, SEQID NO:5), and positive colony is carried out determined dna sequence (entrusting Shanghai Bo Ya Bioisystech Co., Ltd).Sequencing result shows that in the expression plasmid pBL10EGF that we make up, it is in full accord that the dna sequence dna of phoAspL10, hEGF tandem gene and our design.
By above-mentioned steps, we have finished the structure work of expression plasmid pBL10EGF.
The structure and the screening of embodiment 3 engineering strains
3.1 the structure of engineering strain
The competent cell for preparing e. coli bl21 (DE3) by the method in 2.4, expression plasmid pBL10EGF is transformed in the competent cell of BL21 (DE3) by the method in 2.5, get the LB flat board (agar content is 1.5%) that 100 μ l coating contains 100 μ g/ml penbritins, be inverted overnight incubation for 37 ℃.
3.2 the screening of engineering strain
3.2.1 the abduction delivering of engineering strain
The single bacterium colony that obtains in the picking 3.1 is put into 3 milliliters of LB substratum (wherein the concentration of penbritin is 100 μ g/ml), 37 ℃ of 200rpm incubated overnight, transfer in the LB substratum in 1: 10 ratio then, continue again to cultivate after 4 hours and be warming up to 42 ℃, cultivate end after 6 hours.Get 1ml nutrient solution supernatant as sample, adding trichoroacetic acid(TCA) to final concentration is 10%, 12, and centrifugal 5 minutes of 000rpm abandons supernatant, adds 1ml acetone in 12, and centrifugal 5 minutes of 000rpm abandons supernatant, and it is standby to dry precipitation.
3.2.2 SDS-PAGE gel electrophoresis analysis
The prescription of SDS-PAGE gel is as follows:
Concentrate glue (5%) Separation gel (15%)
Water 2ml Water 2.7ml
0.75M?Tris·HCl(pH6.8) 0.4ml 1.5M?Tris·HCl(pH8.8) 2ml
30% acrylamide 0.5ml 30% acrylamide 5ml
10％SDS 30μl 10％SDS 100μl
10% ammonium persulphate 30μl 10% ammonium persulphate 100μl
10％TEMED 30μl 10％TEMED 100μl
The supernatant liquor sample precipitation for preparing among the 3.2.1 is added 1 * load sample damping fluid, in boiling water bath, place after 5 minutes and carry out SDS-PAGE gel electrophoresis analysis (200 volts of voltages, 45 minutes), according to electrophoresis result, this bacterial strain that the rhEGF expression amount is the highest is the engineering strain that screening obtains, called after BE-2 (see figure 3).
1 * load sample damping fluid: 10%SDS 1.5ml, mercaptoethanol 250 μ l, glycerine 500 μ l, 0.75MTrisHCl (pH6.8) 330 μ l, bromjophenol blue is an amount of, and water is settled to 20ml.
1 * electrophoretic buffer: 15.1g Tris, the 94g glycine, 50ml 10%SDS, transferring behind the pH to 8.3 again with concentrated hydrochloric acid, water is settled to 5,000ml.
The fermentation culture of embodiment 4 genetic engineering bacterium BE-2
Seed culture medium is the LB substratum.
Fermentative medium formula is as follows:
Yeast extract 10g/l, peptone, 20g/l, sodium-chlor 10g/l, glucose 5g/l.
Get the genetic engineering bacterium BE-2 glycerol stock that screening obtains among the 700 μ l embodiment 3, insert in the 25ml LB substratum, cultivated 12 hours in 30 ℃, 250rpm, be transferred in the 50ml LB substratum with 2.8% inoculum size again, similarity condition was cultivated 10 hours down, was linked in 5 liters of fermentor tanks that 2.5 liters of fermention mediums are housed by 6% inoculum size then and fermented.Culture temperature is set at 30 ℃, and air flow is 4ml/min, and pH is 7.0, and initial mixing speed is 400rpm, and dissolved oxygen is controlled at 20%.Begin after glucose has consumed to add 250g/l glucose with permanent pH mode stream, control pH value is 7.0.Cultivate cell concentration OD after 12 hours 600Be approximately 36, be warming up to 38 ℃ and begin to induce.Abduction delivering stage stream adds the mixed solution that contains glucose (250g/l) and yeast extract (200g/l), and the initial flow rate of acceleration is 12.6ml/h, is incremented to 25.4ml/h with step-wise manner gradually.Cultivated 27 hours, the high expression level amount of rhEGF is the 384mg/l (see figure 4) in the substratum.
The separation and purification of embodiment 5 expression products
5.1 ultrafiltration
With the medium supernatant molecular weight cut-off that obtains among the embodiment 7 is 5,000 ultra-filtration membrane carries out ultrafiltration and concentration, remove molecular weight by this step and concentrate less than 5,000 impurity and to supernatant liquor, the volume after final the concentrating is about 1/10 of original volume.
5.2 hydrophobic chromatography
Adding solid ammonium sulfate to final concentration in the ultrafiltrated is 10% (mass percent), after the filtration, (Φ 35 * 300mm) for last PhenylSepharose 6 Fast Flow posts, wash with 10% ammoniumsulphate soln earlier, use 50mM phosphoric acid buffer (pH7.0) to carry out wash-out again, collect the component (see figure 5) that contains rhEGF.
5.3 anionresin
(Φ 35 * 300mm) with going up QSepharose Fast Flow post after ten times of the component dilutions of collecting in 5.2 with 50mM TrisHCl (pH7.2) damping fluid, earlier with the flushing of 50mM TrisHCl (pH7.2) damping fluid, in three column volumes, carry out gradient elution with 2M NaCl again, collect the component (see figure 6) that contains rhEGF from 0-100%.
5.4 gel-filtration
RhEGF component in 5.3 concentrated with vacuum freeze-drying method go up Superdex 30 prep grade posts again behind the volume (Φ 35 * 1200mm), and moving phase is 50mM phosphoric acid buffer (pH7.0), and collection contains the component (see figure 7) of rhEGF.
The calibrating of embodiment 6 purified products
6.1 purity calibrating
6.1.1SDS-PAGE gel electrophoresis scanning
Machine scanning analysis as calculated, the purity of rhEGF product reaches 96.8% (see figure 8) greater than 95%.
6.1.2 reversed-phase HPLC
The HPLC analysis condition is as follows:
Analytical column: (Φ 2.1 * 30mm) for RP-300 C8 post.
Buffer A: 0.1%TFA.
Buffer B: 0.1%TFA, 90% acetonitrile.
Gradient: 0%～100%B is in 30 minutes.
Flow velocity: 0.4ml/min.
Machine integral analysis as calculated, the purity of rhEGF product reaches 98.3% (see figure 9) greater than 95%.
6.2 molecular weight determination
Entrust proteome research analytic centre of Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences to measure, the molecular weight of rhEGF product sample is 6,216 dalton's (see figure 10)s.
6.3N terminal amino acid sequence analysis
Entrust proteome research analytic centre of Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences to analyze, 15 amino acid of rhEGF product sample N end have been measured altogether, its N terminal sequence is: Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His-Asp-Gly-Tyr-Cys-Leu (SEQ IDNO:6), and with natural human Urogastron sequence of N terminal amino acid sequence consistent (seeing Figure 11).
6.4 titration (biological activity determination)
Adopt Balb/c3T3 cell mtt assay to measure.The Balb/c3T3 cell of taking the logarithm vegetative period behind tryptic digestion, makes 6 * 10 with the RPMI1640+10%FBS nutrient solution 4/ ml cell suspension is inoculated in 96 orifice plates (0.1ml/ hole), puts 37 ℃, 5%CO 2Training case 24 hours; Former training base is removed in suction, adds RPMI1640+0.4%FBS and trains basic 0.1ml/ hole, 37 ℃, 5%CO 2The hungry culturing cell of training case 24 hours; Former training base is removed in suction, standard substance and sample 0.1ml/ hole that adding is diluted with RPMI1640+0.4%FBS training base: standard substance are diluted to earlier 50IU/ml in advance, carry out a series of 4 times of dilutions again, sample is diluted to 50ng/ml equally in advance, carry out a series of 4 times of dilutions again, each extent of dilution is done two repetitions, puts 37 ℃, 5%CO 2Training case 72 hours; Every hole adds 5mg/ml MTT solution 10 μ l, puts 37 ℃, 5%CO 2Training case 4 hours; Every hole is inhaled and is removed 80 μ l supernatants, adds DMSO 100 μ l, and mixing is measured OD 570Value.Concentration with sample is X-coordinate, OD 570Value is carried out data processing for the ordinate zou mapping with computer sigmoid curve regression Calculation method, calculates the ED of sample and standard substance 50Calculate the X that tires of sample according to following formula 1:
X 1＝X 0×(D 1/D 0)×(E 0/E 1)
Wherein: X 0Be tiring of standard substance, D 1Be the pre-extension rate of sample, D 0Be the pre-extension rate of standard substance, E 1ED for sample 50, E 0ED for standard substance 50
After measured, the specific activity of rhEGF sample is greater than 1.0 * 10 6IU/ milligram albumen reaches 1.21 * 10 6IU/mg albumen (seeing Figure 12).
Embodiment described above is intended to set forth preferred forms of the present invention rather than limits the present invention in any form.Those skilled in the art, all drop in the claim scope of patent application of the present invention in conjunction with the various changes that the general knowledge of this area is done according to enlightenment of the present invention.
Sequence table
＜110〉Gan Renbao, money please, Zhu Jun, Feng Baoshan, Ding Hongzhen, Zhang Qian, Ye Qin, Zhang Haiyi
＜120〉escherichia expression system of secretory expression recombinant human epidermal growth factor
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<400>5
agcacatcag?caggacgca 19
<210>6
<211>15
<212>PRT
＜213〉synthetic
<400>6
Asn?Ser?Asp?Ser?Glu?Cys?Pro?Leu?Ser?His?Asp?Gly?Tyr?Cys?Leu
1 5 10 15

Claims (9)

1. signal peptide gene phoAspL10, it has sequence shown in the SEQ ID NO:1 in the sequence table.
2. the expression plasmid pBL10EGF of secreting, expressing rhEGF, it comprises the described phoAspL10 gene of claim 1, and is positioned at the temperature sensitive promotor PL of described upstream region of gene and is positioned at the hEGF gene in described gene downstream.
3. e. coli bl21 (DE3) host cell that transforms of the described expression plasmid pBL10EGF of claim 2.
4. host cell as claimed in claim 3 is characterized in that having amicillin resistance.
5. engineering strain BE-2, described engineering strain are the highest bacterial strains of rhEGF secreting, expressing amount that goes out from e. coli bl21 (DE3) cell screening that expression plasmid pBL10EGF transforms.
6. signal peptide gene phoAspL10 and the expression plasmid pBL10EGF application on the rhEGF that the preparation direct secretion is expressed.
7.rhEGF the preparation method, it is characterized in that comprising the steps:
(1) chemosynthesis phoAspL10 sequence;
(2) be initial plasmid construction expression plasmid pBL10EGF with pBLGlu2;
(3) make up and filter out engineering strain BE-2;
(4) fermentation culture of genetic engineering bacterium BE-2;
(5) separation and purification of expression product.
8. preparation method as claimed in claim 7, wherein said fermentation culture cultivate engineering strain at 30 ℃ earlier, and then are warming up to 38 ℃ of methods of inducing the rhEGF secreting, expressing.
9. preparation method as claimed in claim 7, wherein said separation and purification is the array mode that adopts ultrafiltration, hydrophobic chromatography, anionresin and gel-filtration.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102212596A (en) * 2010-04-01 2011-10-12 孙民富 Preparation method of human epidermal growth factor
CN102952817A (en) * 2011-08-16 2013-03-06 上海昊海生物科技股份有限公司 Method for production of recombinant human epidermal growth factor by temperature induction
CN103060249A (en) * 2012-06-06 2013-04-24 浙江普洛康裕制药有限公司 Escherichia coli and method for efficiently secreting and expressing human epidermal growth factor by using same
CN103834676A (en) * 2014-02-28 2014-06-04 中国科学院福建物质结构研究所 Plasmid vector of escherichia coli secretory expression heterologous protein and establishment method of plasmid vector
CN107987150A (en) * 2017-12-19 2018-05-04 江苏艾迪药业有限公司 A kind of method that hEGF can be prepared in the slave urine of industrialized production
CN110564661A (en) * 2019-09-23 2019-12-13 杭州纽龙生物科技有限公司 Method for culturing engineering bacteria for expressing recombinant human epidermal growth factor
CN112175063A (en) * 2020-10-28 2021-01-05 宁波博睿瀚达生物科技有限公司 Process for preparing high-purity recombinant epidermal growth factor by high performance liquid chromatography
CN112209999A (en) * 2020-10-28 2021-01-12 宁波博睿瀚达生物科技有限公司 Method for rapidly separating pigment in recombinant epidermal growth factor fermentation liquid
CN116555310A (en) * 2023-03-28 2023-08-08 广州汉方合成生物研究中心(有限合伙) Method for high-throughput screening of heterologous constitutive promoter and application thereof

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102212596A (en) * 2010-04-01 2011-10-12 孙民富 Preparation method of human epidermal growth factor
CN102952817A (en) * 2011-08-16 2013-03-06 上海昊海生物科技股份有限公司 Method for production of recombinant human epidermal growth factor by temperature induction
CN102952817B (en) * 2011-08-16 2014-07-09 上海昊海生物科技股份有限公司 Method for production of recombinant human epidermal growth factor by temperature induction
CN103060249A (en) * 2012-06-06 2013-04-24 浙江普洛康裕制药有限公司 Escherichia coli and method for efficiently secreting and expressing human epidermal growth factor by using same
CN103834676A (en) * 2014-02-28 2014-06-04 中国科学院福建物质结构研究所 Plasmid vector of escherichia coli secretory expression heterologous protein and establishment method of plasmid vector
CN107987150A (en) * 2017-12-19 2018-05-04 江苏艾迪药业有限公司 A kind of method that hEGF can be prepared in the slave urine of industrialized production
CN110564661A (en) * 2019-09-23 2019-12-13 杭州纽龙生物科技有限公司 Method for culturing engineering bacteria for expressing recombinant human epidermal growth factor
CN112175063A (en) * 2020-10-28 2021-01-05 宁波博睿瀚达生物科技有限公司 Process for preparing high-purity recombinant epidermal growth factor by high performance liquid chromatography
CN112209999A (en) * 2020-10-28 2021-01-12 宁波博睿瀚达生物科技有限公司 Method for rapidly separating pigment in recombinant epidermal growth factor fermentation liquid
CN116555310A (en) * 2023-03-28 2023-08-08 广州汉方合成生物研究中心(有限合伙) Method for high-throughput screening of heterologous constitutive promoter and application thereof
CN116555310B (en) * 2023-03-28 2024-03-29 广州市金因源生物技术有限公司 Method for high-throughput screening of heterologous constitutive promoter and application thereof

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