CN103882015B - A kind of recombinant bacterial strain of high expression human growth hormone and construction process and application - Google Patents
A kind of recombinant bacterial strain of high expression human growth hormone and construction process and application Download PDFInfo
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Abstract
A kind of recombinant bacterial strain of high expression human growth hormone and construction process and application.The invention provides and a kind ofly express the restructuring E. coli operator of hGH, the expression plasmid containing this operon sequence and to recombinate the engineering bacteria QJSW-SZ01(CGMCC of hGH with transforming the secreting, expressing obtained containing the expression plasmid of this operon? No:7258).Its inventive point is the encoding sequence by transformation intestinal bacteria heat-stable toxin signal peptide, sudden change is fallen intestinal bacteria rare codon wherein and is avoided the formation of secondary structure as far as possible, change an amino acid at its end makes it be more conducive to guide people hGH secreting, expressing simultaneously, and combine using this signal peptide and escherichia coli alkaline phosphatase promotor (phoA promotor) and intestinal bacteria T7 terminator and make restructuring hGH in intestinal bacteria, be able to soluble form efficient secretory expression as expression regulation element, for final developing low-cost hGH pharmaceutical preparation lays the foundation.
Description
Technical field
The present invention relates to medical bioengineering field, especially a kind of recombinant bacterial strain of high expression human growth hormone and construction process and application.
Background technology
Human growth hormone (humangrowthhormone, hGH), be also called somatotropin (somatotorpin), the wetting ability strand sphaeroprotein be made up of 191 amino acid, its relative molecular mass is at about 22kD, iso-electric point P1 is 4.9, there are two pairs of disulfide linkage in molecule.HGH is a kind of important non-glycosylated protein matter hormone secreted by human pituitary's frontal lobe eosinophil.Pituitary gland, just can growth hormone releasing under the stimulation of hypothalamus somatotropin releasing factor, and Somat then can suppress its release.Tethelin is by blood transportation to target tissue, affects nearly all organization type and cell, even comprises immuning tissue, cerebral tissue and hemopoietic system.Its Main Function is the growth and differ entiation stimulating bone, chondrocyte; Function protein matter, sugar and fatty metabolism etc., be mainly used in treating nanism clinically, and Recent study finds that hGH also can treat the various diseases such as burn, wound, fracture, osteoporosis.Large quantity research confirms, growth hormone deficiency is one of factor causing children growth obstacle important.The acceptor of hGH is throughout the whole body of people.The mechanism of action of hGH is commonly considered as being undertaken by cAMP, after specific receptors is combined on target cell membrane, changes the transhipment of amino acid and meta-bolites, induces the synthesis of some specific protein and nucleic acid.
The species specificity of tethelin is very strong, and the difference of the tethelin of different plant species is very large.The tethelin of traditional clinical application extracts from the hypophysis of human brain, expensively cannot to promote, can only extract at most the human growth hormone of 3 ~ 5mg in the hypophysis of a people.After the eighties in 20th century, molecular biological appearance has driven the development of Restruction albumen, makes to utilize engineered method scale operation HGH to become possibility.Similar to Native human growth's factor or on all four genetically engineered drug can be obtained like this.
Within 1985, U.S. FDA ratifies the restructuring hGH of gene engineering production first for clinical treatment and use, and its demand and sales volume rank first in genetically engineered drug.The nineties, people start with cells of mamma animals synthesizing recombined human tethelin (191 peptide), and aminoacid sequence and the naive tethelin of said preparation are completely the same, because it synthesizes in mammalian cell, but not intestinal bacteria, therefore free of contamination danger, decrease antigenicity simultaneously.In recent years, people constantly attempt again utilizing multiple expression system such as insect cell, silkworm, transgenic mice, rabbit, goat, ox etc. to express human growth hormone, but it is not high to there is expression amount, complex process, fermentation aftertreatment loaded down with trivial details, be difficult to the problems such as suitability for industrialized production.Pichia spp, as lower eukaryotes, has the feature of efficient secretory expression, can utilize the feature of the cutting of native signal peptide simultaneously, obtains the human growth hormone without Met.
Traditional utilizes escherichia coli expression restructuring hGH, has the technological approaches that two kinds different, and a kind of is the hGH producing secretor type, and one is then produce born of the same parents' inner mold.Through practical proof, produce the hGH of secretor type, although step is simple, foreign protein interference is less, and the environment of oxidized form is beneficial to protein folding, and its expression amount is very low, generally will will differ from more than 1 order of magnitude with born of the same parents' inner mold.But but there is processing step complexity in the albumen producing born of the same parents' inner mold, foreign protein disturbs the shortcomings such as more.In addition, also there is the shortcomings such as activity is low, purity is low in the human growth hormone adopting above-mentioned two kinds of methods to obtain.
Summary of the invention
Technical problem to be solved by this invention is to overcome existing the recombinate method of hGH of escherichia coli expression that utilizes and all there is shortcomings different separately and the problem such as the hGH activity that obtains is low, purity is low, the recombinant bacterial strain providing a kind of high expression to recombinate hGH and construction process and application.
The present invention adopts following technical scheme to realize: design improvement intestinal bacteria heat-stable toxin signal peptide, and combine using this signal peptide and escherichia coli alkaline phosphatase promotor (phoA promotor) and intestinal bacteria T7 terminator and make restructuring hGH be able to efficient secretory expression in intestinal bacteria as expression regulation element, for final developing low-cost hGH pharmaceutical preparation lays the foundation.
In a first aspect of the present invention, provide a kind of E. coli operator of expressing restructuring hGH, its structure comprises: escherichia coli alkaline phosphatase promotor (phoA promotor), the encoding sequence (SeqIDNO:1) through the intestinal bacteria heat-stable toxin signal peptide (heat-stableenterotoxinsignalpeptide) of Optimizing Reconstruction, the encoding sequence (SeqIDNO:2) of recombinant human hGH optimized and intestinal bacteria T7 terminator (T7terminater).
Build the method for described E. coli operator, comprise the following steps: according to hGH full length sequence design primer, and the codon replacing the unfavorable expression of intestinal bacteria is to obtain the gene order of optimization; Transformation intestinal bacteria heat-stable toxin signal peptide also guides the expression of hGH using the signal peptide of the intestinal bacteria heat-stable toxin selected as leading peptide.In above-mentioned steps, the optimized gene sequences h GH that the codon replacing the unfavorable expression of intestinal bacteria in hGH complete sequence obtains is as shown in SEQIDNO:2;
GCATTCCCAACTATACCACTAAGTCGACTATTCGATAACGCTATGCTTCG
GGCCCATCGTCTTCATCAGCTAGCCTTTGACACCTACCAGGAGTTTGAAG
AGGCCTATATCCCCAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCT
CCCTCTGTTTCTCAGAATCGATTCCGACACCCTCCAATCGCGAGGAAACACAACAGAAAT
CCAACCTAGAGCTCCTCCGCATAAGCTTGCTGCTCATCCAGTCGTGGCTCGAGCCCGTGC
AGTTCCTGAGGAGTGTCTTCGCCAACAGCCTGGTCTACGGCGCCTCTGATTCGAACGTGT
ACGACCTGCTGAAGGACCTAGAGGAAGGGATCCAAACGCTGATGGGGAGGCTGGAAGATG
GCAGCCCGCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCAC
ACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGG
ACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCT
TCTAG(SeqIDNO：2)
The aminoacid sequence of the restructuring hGH of its coding is as follows:
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPT
PSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEG
IQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIV
QCRSVEGSCGF
The sequence of the intestinal bacteria heat-stable toxin signal peptide optimized is as shown in SEQIDNO:1:
ATGAAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTTTTCTATTGCTACAAAT
GCCTAT(SeqIDNO：1)；
The encoding sequence of signal peptide-hGH fusion rotein is as shown in SEQIDNO:3:
ATGAAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTTTTCTATTGCTACAAAT
GCCTATGCATTCCCAACTATACCACTAAGTCGACTATTCGATAACGCTATGCTTCG
GGCCCATCGTCTTCATCAGCTAGCCTTTGACACCTACCAGGAGTTTGAAG
AGGCCTATATCCCCAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCT
CCCTCTGTTTCTCAGAATCGATTCCGACACCCTCCAATCGCGAGGAAACACAACAGAAAT
CCAACCTAGAGCTCCTCCGCATAAGCTTGCTGCTCATCCAGTCGTGGCTCGAGCCCGTGC
AGTTCCTGAGGAGTGTCTTCGCCAACAGCCTGGTCTACGGCGCCTCTGATTCGAACGTGT
ACGACCTGCTGAAGGACCTAGAGGAAGGGATCCAAACGCTGATGGGGAGGCTGGAAGATG
GCAGCCCGCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCAC
ACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGG
ACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCT
TCTAG(SeqIDNO：3)
Add pPIC9K carrier self signal α peptide cleavage site Lys-Arg-Glu-Ala between two sections of aminoacid sequences of signal peptide-hGH fusion rotein, for the L-glutamic acid that connects and phenylalanine, during secreting, expressing, cutting obtains gene recombination hGH.
In a second aspect of the present invention, above-mentioned operon is cloned in the e. coli plasmid vector carrying any replicon (comprising pMB1, P15A, ColE1 and pSC101 replicon) effect playing efficient secretory expression restructuring hGH by us.We are inserted into after intestinal bacteria heat-stable toxin signal peptide after the hGH encoding sequence application NcoI of optimization and HindIII enzyme being cut, and the benefit of design is like this that we can obtain the protein sequence identical with natural hGH aminoacid sequence and not worry there are other impurity amino acid incorporations.
In a third aspect of the present invention, provide a kind of expression vector for method of the present invention and host cell, it contains the expression plasmid of the above-mentioned E. coli operator of the present invention.Described host cell comprises DH5 α, JM109, JM109 (DE3), BL21, BL21 (DE3) and derivative strain, Rosetta, Rosetta (DE3) and derivative strain, W3110, YK537, C41 (DE3) and C43 (DE3).Preferred described host cell is e. coli bl21 (DE3).The cell walls of this thalline is more fragile, just can break under the condition of multigelation, discharges the albumen in periplasmic space.
Intestinal bacteria of the present invention (Escherichiacoli) bacterial strain QJSW-SZ01, on February 26th, 2013 is stored in China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center, and deposit number is CGMCCNo.:7258.
In a fourth aspect of the present invention, provide a kind of method of Restruction hGH, it comprises step:
A () carries out activation and the amplification of engineering bacteria by LB substratum and improved seed substratum, substratum and feed profile in suitable tank, expresses restructuring hGH under culture parameters control condition;
B () centrifugal or ultrafiltration mode obtains the thalline containing people hGH by low-temperature and high-speed.In another preference, described expression vector is expression vector pET22b (+)-rhGH of the promotor containing IPTG induction, or described host cell is intestinal bacteria.
In another preference, in step (a), carry out high density fermentation.
In another preference, described step (b) comprises step:
I () adopts the broken fermentation thalli of freeze thawing, the centrifugal precipitation of abandoning of low-temperature and high-speed receives supernatant;
(ii) by saltout and or ultrafiltration preliminary purification is carried out to broken bacterium liquid supernatant liquor;
(iii) column chromatography adopts: cation-exchange chromatography, affinity chromatography, gel permeation chromatography and combination thereof.
The present invention relates to and a kind ofly express the restructuring E. coli operator of hGH, the expression plasmid containing this operon sequence and to recombinate the engineering bacteria of hGH with transforming the secreting, expressing obtained containing the expression plasmid of this operon.Its advantage is, by the encoding sequence of transformation intestinal bacteria heat-stable toxin signal peptide, sudden change is fallen intestinal bacteria rare codon wherein and is avoided the formation of secondary structure as far as possible, change an amino acid at its end makes it be more conducive to guide people hGH secreting, expressing simultaneously, protein expression by escherichia coli alkaline phosphatase promoter regulation, utilizes the translation efficiency that intestinal bacteria T7 terminator comes in Enhanced expressing process simultaneously.
Feature of the present invention is a kind of E. coli operator of the escherichia coli alkaline phosphatase signal peptide crossed with E. coli tryptophan promoter and Optimizing Reconstruction.The expression vector utilizing above-mentioned operon to build transforms the host that resistance is applicable to, can obtain can efficient secretory expression restructuring hGH engineering bacteria, obtain good secreting, expressing effect, solve small molecules heterologous protein and be easily degraded when expression in escherichia coli and the too low problem of the expression amount caused.Method of the present invention can take into account expression output, and purifying process, its lytic activity and final amino acid form, and have very high using value.
The invention has the advantages that:
(1) expression process is simple.
(2) expression amount is high.
(3) purifying process is easy, and the rate of recovery is high.Expressing because albumen can be secreted in periplasmic, because this simplify purification procedures, improve yield rate, make scale operation rhGH become possibility.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example.
Accompanying drawing explanation
Fig. 1 is the electrophorogram (rhGH electrophorogram) of purified product rhGH;
Purified product and standard substance (middle inspection institute) carry out electrophoresis and contrast and determine molecular weight and electrophoresis purity.
Fig. 2 is that rhGH enzyme cuts high performance liquid phase identification chromatography figure (rhGH enzyme cuts spectrogram);
Get recombinant human somatropin's reference substance, the solution containing 2mg in every 1ml is made with tris buffer (pH7.5), get this liquid 300 μ l, [trypsinase of TPCK process of learning from else's experience is appropriate for trypsin solution, the solution containing 2mg in every 1ml is made with tris buffer (pH7.5)] 20 μ l and tris buffer (pH7.5) 300 μ l, mixing, to put in 37 DEG C of water-baths 4 hours, put-20 DEG C of termination reactions immediately, in contrast product solution.Get the application rhGH, make the solution containing 2mg in every 1ml with tris buffer (pH7.5), get this liquid 300 μ l, standby with legal system, as need testing solution; Cutting peptide figure by the enzyme of high performance liquid chromatography to the application rhGH and reference substance, to carry out contrast basically identical.
Fig. 3 is the high performance liquid phase purity detecting result figure (rhGH high-efficient liquid phase chromatogram) of purified product rhGH;
Carry out purity detecting by high-efficient liquid phase technique to purified product, calculate by area normalization method, total correlation protein content must not cross 6.0%.The product of purifying is more than 95%.
Fig. 4 is the mass spectrometric detection result figure D1 (rhGH mass spectrum-D1) of qualification purified product rhGH accurate molecular weight;
Fig. 5 is the mass spectrometric detection result figure D2 (rhGH mass spectrum-D2) of qualification purified product rhGH accurate molecular weight.
D1, D2 are all that to be configured to volumetric molar concentration with water (containing 0.1%TFA) be 10umol/L; Matrix HCCA or CHCA TA30 (50%ACN, 50%H
2o, 0.1%TFA) to be configured to mass concentration be 10mg/ml.
MALDI-TOFMS condition:
Instrument: AutoflexspeedTOF/TOF (BrukerDaltonics, Germany)
Laser source: Nd:YAG355nm
Acceleration voltage: 19.5kV.
Energy of lasers: 75-80%
Time of lag: 500ns
Testing method: linear positive ion mode, actual molecular weight sweep interval is 5000-30000.
Mass spectrometric detection is carried out to purified product accurate molecular weight.
Embodiment
The present inventor is through further investigation, express with preferred carrier pET22b (+), this carrier contributes to hGH eukaryotic gene correctly folding in protokaryon, and be secreted in periplasmic space with soluble form, only just can discharge target protein by multigelation thalline, thus simplify separation purifying technique.
In another preference, the e. coli bl21 (DE3) that the hGH optimized with the present invention is Coding Sequence Transformed, cultivate through high density fermentation, IPTG induces, rhGH is present in periplasmic with solvable, activity form, simultaneously easy purifying process, namely can broken thalline by the method for multigelation, obtains high expression level, high yield pulp1, has a kind of method preparing rhGH of industrialization value.
Carrier of the present invention comprises expression plasmid, such as, containing the plasmid expression vector of the promotor of IPTG induction, and in described expression vector, inserts the encoding sequence of hGH.A kind of preferred expression vector is carrier pET20b (+), pET22b (+), pET26b (+) and pET27b (+).Being applicable to Host Strains of the present invention is prokaryotic host cell, especially e. coli bl21 (DE3).
In the present invention, the fermentation condition of engineering bacterium expression rhGH is not particularly limited.The fermentation condition of this area routine can be adopted.Usually, engineering bacteria is with peptone, yeast powder, NH
4cl etc. are nitrogenous source, and glycerine, glucose, molasses etc. are carbon source, and phosphoric acid salt etc. are inorganic salt, add substratum based on electrolytes and minerals; And pass through adjustment and the fed-batch mode (soda acid and nutritive element) of controling parameters (pH, temperature, tank pressure, stirring velocity etc.), regulate dissolved oxygen (DO), the factors such as specific growth rate, reduce the accumulation of acetic acid, improve thalline output and expression level.
For the selection of substratum, generally include the selection of nitrogenous source, carbon source, inorganic salt, VITAMIN, trace element etc.
A () nitrogenous source is containing organic nitrogen source and inorganic nitrogen-sourced, wherein organic nitrogen source is the mixture of peptone, yeast powder, total concn 1.5-4%; Inorganic nitrogen-sourced as ammoniacal liquor or NH
4the ammonium salts such as Cl, concentration 0.4-1%.
B () inorganic salt comprise phosphoric acid salt, magnesium salts, calcium salt etc.Preferably phosphate buffer concentration 20-50mM, pH6.5-7.5; Magnesium salts, calcium concentration are 0.1-1mM.
C () adds vitamins B in the medium as growth factor, concentration 1-50 μ g/L.
D () trace element comprises Fe
3+, Mn
2+, Cu
2+, Co
2+, Zn
2+, boric acid etc., concentration is 10
-8-10
-6mM.
E () carbon source comprises glycerine, glucose, molasses etc.Can be single carbon source, also can be mixed carbon source.Preferably glycerol concentration is 3-6%; Glucose concn is 3-5%.
F () inductor uses IPTG, preferably concentration is at 0.5-0.8mM;
(g) cultivation stage dissolved oxygen (DO) more than 30%, induction period is more than 50%; PH cultivation stage controls at 6.8-7.0, and induction period controls at 7.2-7.4; Temperature cultivation stage controls at 35-37 DEG C, and induction period controls at 30-32 DEG C;
After fermentation expression rhGH, remove fermented liquid supernatant with centrifugation, obtain thalline.The mode of application multigelation carries out brokenly bacterium, after buffered soln suspension thalline, carries out centrifugal to crude extract, goes precipitation, results supernatant liquor.Crude extract by saltouing, the method such as ultrafiltration carries out chromatography purification after carrying out preliminary purification again, also directly can carry out chromatography purification.
Be applicable to chromatographic technique of the present invention and comprise cation-exchange chromatography, gel permeation chromatography, affinity chromatography.
(a) ion chromatography.Select CMSepharoseFastF gel media, salt gradient elution.
(b) affinity chromatography: select the affine HeparinSepharoseCL-6B gel media of heparin, salt gradient elution.Through two step purifying, the rhGH albumen of purity more than 95% can be obtained.
After purifying, rhGH can make various formulation with routine techniques.
In an example of the present invention, have selected the secretor type plasmid vector contributing to eukaryotic gene correct translation in prokaryotic cell prokaryocyte: containing the expression vector of the promotor of IPTG induction, and in described expression vector, insert the encoding sequence of rhGH, build rhGH engineering bacteria (intestinal bacteria) that is efficient, stably excreting expression.Cultivate through high density fermentation, IPTG induces, and rhGH is present in periplasmic with soluble form; Expression level is high, accounts for bacterial protein about 20%.Because expression level is higher, rhGH can be secreted in periplasmic again, and has natural radioactivity, not only avoids and slightly carries the destruction of process to protein, and improve purification yield, reduce cost.By easy, purification process fast, namely obtain high-quality hGH sterling.
Embodiment 1:rhGH expresses the structure of engineering bacteria
1, the acquisition of optimized gene
According to the natural acid sequence of known hGH, also consider that elimination hairpin structure etc. is unfavorable for the secondary structure expressed according to e. coli codon preferences, under the condition not changing aminoacid sequence, the encoding sequence primer of design hGH.The full length sequence of rhGH is obtained by PCR method.The encoding sequence of intestinal bacteria heat-stable toxin signal peptide is by precious biosynthesizing.
2, expression plasmid pET22b (+)-rhGH structure with transform Host Strains
Expression plasmid is pET22b (+) expression vector (this expression vector contains promotor and the peIB secreting signal peptide of IPTG induction) purchased from InVitrogen company, and Host Strains is e. coli bl21 (DE3).
The escherichia coli alkaline phosphatase signal peptide of synthesis and the encoding sequence of restructuring hGH is processed respectively with restriction enzyme NcoI and HindIII double digestion, and expression vector pET22b (+), 37 DEG C of enzymes are cut and are reclaimed corresponding fragment in 2 hours, to connect spend the night with T4DNA ligase enzyme at 16 DEG C.Connect product conversion and enter intestinal bacteria, positive colony selected by LB flat board containing penbritin (100ug/mL) and carries out plasmid enzyme restriction qualification: the plasmid DNA restriction enzyme of extracting and NcoI+HindIII, 37 DEG C of reactions 3 hours, obtain recombinant plasmid pET22b (+)-rhGH inserting rhGH gene in pET22b (+).
Bacterium colony PCR is utilized to carry out positive identification.Application NcoI+HindIII double digestion qualification sequence is correctly in insertion vector pET22b (+).In addition, undertaken by ordinary method forward and inverse to sequential analysis, entrust the precious biotech firm in Dalian to carry out the mensuration of sequence, confirm cloned sequence and implementation sequence completely the same.
Shake flask test, after this project bacterium is cultivated, after IPTG induces 2 hours, target protein expression amount accounts for total protein about 20%.
The selection of the 2-in-1 suitable expression vector of embodiment
According to the method that embodiment 1 is similar, hGH sequence is inserted several different expression vector respectively, if pET28a (+) (purchased from invitorgen company), pET29a (+) (purchased from invitrogen company), pET-30Ek/LIC are purchased from invitorgen company) identical restriction enzyme site (NcoI/HindIII), obtain plasmid pET28a (+)-rhGH, pET29a (+)-rhGH, pET-30Ek/LIC-rhhGH.
Plasmid pET28a (+)-rhGH, pET29a (+)-rhGH, pET-30Ek/LIC-rhGH are transformed corresponding Host Strains respectively, picks out resistant strain.Shake flask test is carried out together with the engineering bacteria containing plasmid pET22b (+)/rhGH, after IPTG (or pectinose) induces 2 hours, target protein expression amount containing the engineering bacterium expression of plasmid pET22b (+)-rhGH accounts for total protein about 20%, and containing plasmid pET28a (+)-rhGH, pET29a (+)-rhGH, the target protein expression amount of the engineering bacterium expression of pET-30Ek/LIC-rhGH accounts for total protein 10%, 12% and 13% respectively.This shows, pET22b (+)-rhGH expression vector is better than other expression vectors.
The selection of embodiment 3 optimal medium
Choose e. coli bl21 (DE3) mono-clonal proceeding to pET22b (+)-rhGH of preparation in embodiment 1, be inoculated in the 250ml Erlenmeyer flask containing 50mlLB seed liquor, cultivate 3-4h, treat OD
600reach 0.8-1.0, the ratio in 1: 10 is inoculated in the 1000ml Erlenmeyer flask containing 250ml improved seed liquid, cultivates about 8-10h, treats OD
600reach 6-8, by tank fermentation on 1: 10, stream adds carbon source (glycerine), magnesium salts, ammoniacal liquor regulates pH6.8-7.0, temperature 37 DEG C, DO > 30%, treat OD
600reach 15-18, add 0.5-0.8mMIPTG and start induction, suitably add carbon source (glycerine), nitrogenous source, phosphate buffered saline buffer regulate pH7.2-7.4, DO > 50%, temperature 30-32 DEG C, induction 4-5h terminates.Sample carries out SDS-PAGE detection, measures protein content.
The structure of embodiment 4rhGH secreting, expressing engineering bacteria and high density fermentation
With empty host BL21 (DE3) of plasmid pET22b (+)-rhGH transformation of E. coli, Rosseta (DE3), W3110 and C43 (DE3), the engineering bacteria obtained is cultivated by AP5 culture media shaking vase, SDS-PAGE electroresis appraisal expression of results.AP5 medium component: 0.15% glucose, 1.1% acid hydrolyzed casein, 0.06% yeast extract, 0.019%MgSO
4, 0.107%NH
4cl, 0.373%KCl, 0.12%NaCl, 0.12mol/L trolamine pH7.4.
High density fermentation: by strain inoculation to containing in the 250ml Erlenmeyer flask of 50mlLB seed liquor, cultivate 3-4hr, treat OD
600reach 0.8-1.0, the ratio in 1: 10 is inoculated in the 1000ml Erlenmeyer flask containing 250ml improved seed liquid, cultivates about 8-10h, treats OD
600reach 6-8, by tank fermentation on 1: 10, stream adds carbon source (glycerine), magnesium salts, ammoniacal liquor regulates pH6.8-7.0, temperature 37 DEG C, DO > 30%, treat OD
600reach 15-18, add 0.5-0.8mMIPTG and start induction, suitably add carbon source (glycerine), nitrogenous source, phosphate buffered saline buffer regulate pH7.2-7.4, DO > 50%, temperature 30-32 DEG C, induction 5h terminates.Sample carries out SDS-PAGE detection, obtains clear band (Fig. 1) at about 24kD.
The purifying of embodiment 5rhGH
Fermented liquid is the centrifugal 10min of 5000rpm at 4 DEG C, obtains thalline, and after thalline-80 DEG C of quick-frozen 24h, room temperature is slowly melted, then-20 DEG C of room temperatures after freezing 7 days are slowly melted.Microscopic examination, somatic cells wall breaks, and becomes spheroplast.The centrifugal 30min of 20000rpm, obtains supernatant.Ultrafiltration and concentration and exchange buffering liquid: use Millipore ultra-fine filter, the ultra-filtration membrane molecular weight that dams is 10KD, leaves and takes concentrated solution (small molecular weight impurity and salt are removed in effect) during ultrafiltration.
1. chromatography 1 (positively charged ion chromatography)
Chromatography media: CMSepharoseFastF
Damping fluid: solution A: PBS (PH7.4,20mM)
Solution B: PBS (PH7.4,20mM)+1MNaCl
Loading: by the rhGH solution loading of ultrafiltration and concentration.
Cleaning: with the solution A cleaning chromatography column of 6CV after loading.
Gradient: with 10CV, solution B is risen to 100% from 0% after cleaning.
Collect: collect rhGH sample peak.
2. chromatography 2 (affinity chromatography):
Chromatography media: HeparinSepharoseCL-6B
Damping fluid: solution A: PBS (PH7.4,20mM)
Solution B: PBS (pH7.4,20mM)+1MNaCl
Loading: ion is handed over column chromatography protein solution loading.
Cleaning: with the solution A cleaning chromatography column of 6CV after loading.
Gradient: with 10CV, solution B is risen to 100% from 0% after cleaning.
Collect: collect rhGH sample peak.
Sample is through this two steps purifying, and namely after ion chromatography, affinity chromatography, purity is increased to more than 95%.According in " the Chinese medicine examination criteria working specification of Chinese Pharmacopoeia 2010 editions ", the measuring method of growth hormone biological activity detects, hypophysis big white mouse body weight method and method two is gone to go hypophysis big white mouse shin bone method to detect according to method one respectively, by calculating, the application rhGH purified product and standard substance have identical biological activity.
The invention provides the preparation method of a kind of excreting and expressing recombinant human hGH, this method provide the expression vector of a kind of hGH that recombinates, it contains secretion signal peptide-coding sequence, can express in hGH protein excretion to periplasmic, host cell containing this recombinant plasmid is through low temperature multigelation, recombinant protein can be made when not destroying bacterial inner membrane to be discharged in damping fluid, not destroy the activity of albumen.
Claims (1)
1. a method of Restruction hGH, comprises the following steps:
A () carries out activation and the amplification of engineering bacteria by LB substratum and improved seed substratum, treat OD
600reach 6-8, by tank fermentation on 1: 10, stream adds carbon source glycerine, magnesium salts, ammoniacal liquor regulates pH6.8-7.0, temperature 37 DEG C, DO > 30%, treat OD
600reach 15-18, add 0.5-0.8mMIPTG and start induction, suitably add carbon source glycerine, nitrogenous source, phosphate buffered saline buffer regulate pH7.2-7.4, DO > 50%, temperature 30-32 DEG C, induction 5hr terminates, and express restructuring hGH;
The purifying of (b) rhGH
Fermented liquid is the centrifugal 10min of 5000rpm at 4 DEG C, obtains thalline, and after thalline-80 DEG C of quick-frozen 24h, room temperature is slowly melted, then-20 DEG C of room temperatures after freezing 7 days are slowly melted; Microscopic examination, somatic cells wall breaks, and becomes spheroplast; The centrifugal 30min of 20000rpm, obtains supernatant; Ultrafiltration and concentration and exchange buffering liquid: use Millipore ultra-fine filter, the ultra-filtration membrane molecular weight that dams is 10KD, leaves and takes concentrated solution during ultrafiltration;
Chromatography 1 positively charged ion chromatography:
Chromatography media: CMSepharoseFastF,
Damping fluid: solution A: pH7.420mMPBS, solution B: pH7.420mMPBS+1MNaCl,
Loading: by the rhGH solution loading of ultrafiltration and concentration,
Cleaning: with the solution A cleaning chromatography column of 6CV after loading,
Gradient: with 10CV, solution B is risen to 100% from 0% after cleaning,
Collect: collect rhGH sample peak;
Chromatography 2 affinity chromatography:
Chromatography media: HeparinSepharoseCL-6B,
Damping fluid: solution A: pH7.420mMPBS, solution B: pH7.420mMPBS+1MNaCl,
Loading: ion is handed over column chromatography protein solution loading,
Cleaning: with the solution A cleaning chromatography column of 6CV after loading,
Gradient: with 10CV, solution B is risen to 100% from 0% after cleaning,
Collect: collect rhGH sample peak;
Described engineering bacteria is e. coli bl21 (DE3), and it contains the carrier of the E. coli operator of expressing recombinant human somatropin rhGH; The structure of the E. coli operator of described expression recombinant human somatropin rhGH comprises escherichia coli alkaline phosphatase promotor, through the encoding sequence of the intestinal bacteria heat-stable toxin signal peptide of Optimizing Reconstruction as shown in SEQIDNO:1, the encoding sequence of recombinant human hGH optimized is as shown in SeqIDNO:2 and intestinal bacteria T7 terminator; The encoding sequence of signal peptide-hGH fusion rotein is as shown in SEQIDNO:3; Described carrier is pET22b (+)-rhGH.
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| CN107723290A (en) * | 2017-08-14 | 2018-02-23 | 蔡祥胜 | One kind restructuring human oocyte's oolemma ZP3 albumen and preparation method thereof |
| EA035207B1 (en) * | 2017-10-08 | 2020-05-15 | Илья Владимирович ДУХОВЛИНОВ | Strain of escherichia coli bl21 (de3)/pet-21a(+) bacteria - producer of predominantly monomeric somatotropine |
| CN109988233B (en) * | 2017-12-13 | 2021-01-26 | 江苏众红生物工程创药研究院有限公司 | Recombinant human growth hormone and its coding gene, preparation method and application |
| CN109402134A (en) * | 2018-11-29 | 2019-03-01 | 湖南百尔泰克生物科技有限公司 | A kind of preparation method and applications of the engineering bacteria of high efficient expression growth hormone |
| CN111303275A (en) * | 2018-12-12 | 2020-06-19 | 江苏众红生物工程创药研究院有限公司 | Recombinant human growth hormone, preparation method and pharmaceutical application thereof |
| CN111763680A (en) * | 2019-05-22 | 2020-10-13 | 杭州杰迪生物科技有限公司 | Preparation method of human insulin-like growth factor-I or analogue thereof, coding gene, expression vector and host cell |
| CN112266924A (en) * | 2020-01-03 | 2021-01-26 | 浙江理工大学绍兴生物医药研究院有限公司 | Method for efficiently expressing and secreting human growth hormone by using escherichia coli |
| CN112239760B (en) * | 2020-09-18 | 2022-02-11 | 深圳科兴药业有限公司 | Recombinant engineering bacterium for efficiently expressing recombinant hGH (human growth hormone) and construction method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1618968A (en) * | 2003-11-21 | 2005-05-25 | 上海新生源医药研究有限公司 | Production method of recombination human growth hormone |
| CN101663046A (en) * | 2007-03-30 | 2010-03-03 | Ambrx公司 | Modified FGF-21 polypeptides and its purposes |
| CN103173440A (en) * | 2012-12-28 | 2013-06-26 | 吉林修正药业新药开发有限公司 | Operon, carrier and engineering bacterium of recombination human growth hormone with efficient secretory expression as well as preparation method of carrier and engineering bacterium |
-
2013
- 2013-12-17 CN CN201310699346.0A patent/CN103882015B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1618968A (en) * | 2003-11-21 | 2005-05-25 | 上海新生源医药研究有限公司 | Production method of recombination human growth hormone |
| CN101663046A (en) * | 2007-03-30 | 2010-03-03 | Ambrx公司 | Modified FGF-21 polypeptides and its purposes |
| CN103173440A (en) * | 2012-12-28 | 2013-06-26 | 吉林修正药业新药开发有限公司 | Operon, carrier and engineering bacterium of recombination human growth hormone with efficient secretory expression as well as preparation method of carrier and engineering bacterium |
Non-Patent Citations (1)
| Title |
|---|
| High-level secretion of human growth hormone by Escherictria coli;Clung Nan Chang et al.;《Gene》;19871231;第55卷;摘要，材料与方法，图1 * |
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