CN103923937A - Method for soluble expression of recombinant protein of human brain natriuretic peptide and application - Google Patents
Method for soluble expression of recombinant protein of human brain natriuretic peptide and application Download PDFInfo
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Abstract
The invention provides a method for soluble expression of recombinant protein of a human brain natriuretic peptide (Human Brain Natriuretic Peptide, hBNP). The method comprises the following steps: 1) obtaining a recombinant gene, namely obtaining the recombinant gene His-DsbAmut-BNP of expressing the recombinant protein of the human brain natriuretic peptide, wherein the sequence of the recombinant gene comprises a purified tag gene sequence, a molecular chaperone protein gene sequence, a protease recognition site sequence and a human brain natriuretic peptide sequence, which are sequentially arranged along the direction from 5' to 3'; 2) obtaining recombinant plasmids, namely inserting the recombinant gene obtained in the previous step into a carrier vector, so as to obtain the recombinant plasmids containing the recombinant gene; 3) obtaining genetically engineered bacterium, transferring the recombinant plasmids obtained in the previous step into host cells to obtain the genetically engineered bacterium; 4) expressing exogenous genes, fermenting the genetically engineered bacterium, and expressing fusion protein containing the human brain natriuretic peptide; 5) purifying target protein, splitting thallus, collecting fusion protein, removing molecular chaperone and carrying out chromatographic purification by protease digestion, so as to obtain the recombinant protein of the human brain natriuretic peptide.
Description
Technical field
The invention belongs to technical field of bioengineering, relate in particular to a kind of methods and applications of solubility expression human brain natriuretic peptide recombinant protein.The invention still further relates to a kind of recombination of solubility expression human brain natriuretic peptide recombinant protein.
Background technology
Along with the rising of the common cardiovascular diseases sickness rate such as the quickening of aging population process and hypertension, coronary heart disease, the morbidity of heart failure raises just year by year.According to the statistical information of the U.S., show, the U.S. has 4,900,000 patients with heart failure at present, and has every year the newly-increased patient of 40-70 ten thousand, and the morbidity that people group center declines is about 1.5%-2.0%, and over-65s crowd can reach 6%-10%.In 8 years, the death toll causing due to heart failure has increased by 6 times in the past.In the cardiovascular diseases international cooperating research of 2000-2001 Asia, Chinese cardiovascular health multicenter joint study group result of study shows, the morbidity of China grownup heart failure is 0.9%, wherein in 35-74 year grownup, approximately has 4,000,000 patients with heart failure.
Congestive heart failure treatment at present mainly comprises: intravenous injection cardiac tonic (digitalis medicine, adrenomimetic drug class medicine, phosphodiesterase inhibitor etc.) and expansion blood vessel medicine (nitroglycerine, Sodium Nitroprusside etc.).But the side effect producing shows: neurohormone-hypertensin system that drug tolerance, tachycardia and other irregular pulse, ypotension and activation are relevant with pathologic process in heart failure.Recent study discovery, human brain natriuretic peptide, as the endogenous polypeptide of human secretory, can reduce the forward and backward load of heart, and the symptom while improving chronic cardiac patient acute attack has cardiac stimulant and vasodilation effect.For the Hemodynamics of improving congestive heart failure (CHF) patient, as artery and venectasia, increase sodium excretion and suppress Re-A-A and sympathetic nervous system has good effect, human brain natriuretic peptide does not forcibly strengthen myocardium contractility simultaneously, and there is no to expand at present blood vessel medicine and the side effect of diuretic to blood pressure and alteration in heart rate; No matter control of untoward reaction or curative effect etc. are all obviously better than to conventional medicament as medicine from the viewpoint of it.
Human brain natriuretic peptide (Human Brain Natriuretic Peptide, hBNP) claim again B-typeNatriuretic Peptide (B-type Natriuretic Peptide), be from pig brain, to be separated thereby gain the name in 1988 by Japanese scholars Sudoh at first, in fact it is mainly derived from ventricle.A kind of endogenous polypeptide as compensation of human secretory when BNP is heart failure, atrial natriuretic peptide (the Atrial Natriuretic Peptide that continues, ANP) the another member of natriuretic peptide family after, natriuretic peptide family mainly comprises atrial natriuretic peptide, brain natriuretic peptide, c-type natriuretic peptide (C-type Natriuretic Peptide, CNP), D type natriuretic peptide (D-type Natriuretic Peptide, DNP) and urine sodium peptide (Urodilati).In heart, BNP is mainly present in left atrium, and wherein right atrium content is the highest, but 60% secretion is from ventricle.Under physiological status, atrium, ventricle synthesize and secrete a small amount of BNP, so in normal people's blood circulation, only have the BNP of lower concentration, and under pathological state, while increasing as ventricle wall tension force, BNPYou cardiac muscular tissue is synthetic and release in a large number.Just because of the variation under this pathological state of brain natriuretic peptide, can come diagnosis of heart failure and judging prognosis by the BNP concentration in human body.There are some researches show, BNP is the diagnosis considerably beyond clinical expert to diagnostic value in heart failure.At present, BNP has become a biochemical indicator of diagnosis of heart failure.
Human brain natriuretic peptide and specific natriuretic peptide receptor (this receptor and guanylate cyclase phase lotus root connection) combination, causes the concentration rising of cGMP (cGMP) in cell and the diastole of smooth muscle cell.As second messenger, cGMP energy expansion artery and vein, reduce rapidly SAP, right atrial pressure and pulmonary capillary wedge and press, thereby reduce the forward and backward load of heart, and alleviate rapidly expiratory dyspnea degree and the constitutional symptom of patients with heart failure.
Brain natriuretic peptide is the natural agonist of renin-angiotensin-aldosterone system (RAAS), and it can antagonism myocardial cell, endothelin, norepinephrine and aldosterone in core fiber archeocyte and vascular smooth muscle cell.It can improve glomerular filtration rate(GFR, strengthens the excretion of sodium, reduces the secretion of feritin and aldosterone, also resists β-hypophamine and orthosympathetic guarantor's sodium water conservation, the effect of rising blood pressure.Brain natriuretic peptide participates in the adjusting of blood pressure, Q volume of blood and water salt balance, increases vascular permeability, reduces systemic vascular resistance and plasma volume, thereby has reduced the forward and backward load of heart, and increase cardiac output.Brain natriuretic peptide does not have positive inotropic action simultaneously, does not increase myocardium oxygen consumption.
The preparation of brain natriuretic peptide mainly contains chemical synthesis and gene engineering research, and wherein chemosynthesis brain natriuretic peptide cost is high, expensive, industrialization difficulty.The restructuring BNP preparation method that present stage generally adopts mainly adopt self fused in tandem express and with two kinds of other carrier proteins amalgamation and expressions, mostly at intracellular accumulation, easily form inclusion body, and there is bioactive albumen from renaturing inclusion bodies, need to be through the process of a series of sex change renaturation, complex process and yield are lower.Meanwhile, also have and adopt the chemosmosis fermentation process that adds triton x-100, improve protein outside fermenting process is released into born of the same parents.But add Triton thalline to be broken to meeting, the contradiction of Triton add-on and raising protein yield cannot solve.In order to address the above problem, the present invention's gene recombination technology, realizes the soluble overexpression of BNP in intestinal bacteria, preparation high purity, highly active human brain natriuretic peptide.
Molecular chaperones refers to that a class does not have dependency but has the protein of common function in sequence, they help other polypeptide or albumen to form specific conformation in cell, complete correct assembling, play a part in vivo to stablize new raw albumen, assist other protein correctly to fold, assemble, transport even degraded.Under stress conditions, molecular chaperones plays the effect of stabilizing protein result and prevents protein condenses, sex change, also has the function of repairing metaprotein, significant to the performance of protein function.
First molecular chaperones---nucleoplasmin (nucleoplasmin) is that the people such as Laskey found in the leach liquor at Xenopus Egg in 1978, they are combined with DNA and are formed in the process of nucleosome by study group's albumen, must rely on a kind of participation of acid nuclear protein just can complete their assembling.
Most of molecular chaperoneses of finding up to now all belong to heat shock protein(HSP) (heat shock protein, HSP) category, roughly can mainly be divided into (HSP 100 family) such as chaperonin family (chaperonin, Cpn), heat shock protein 70 family (HSP 70 family), heat shock protein 90 family (HSP 90 family) and heat shock protein(HSP) 100 families.Molecular chaperones can promote the correct structure of Protein formation, improve protein stability industrial significant because of it.
Summary of the invention
The present invention includes the theme described in the following paragraph of arranging in order:
1. a method for solubility expression human brain natriuretic peptide recombinant protein, comprises the following steps:
1) obtain recombination: obtain the recombination rhBNP that expresses human brain natriuretic peptide's recombinant protein, the sequence of described recombination rhBNP comprises purification tag gene order, molecular chaperone protein gene order, proteolytic enzyme recognition site sequence and the human brain natriuretic peptide's gene order that 5 ' to 3 ' direction is arranged in order;
2) obtain recombinant plasmid: the recombination rhBNP that previous step is obtained inserts plasmid, obtain the recombinant plasmid that contains recombination rhBNP;
3) obtain genetic engineering bacterium: the recombinant plasmid transformed host cell that previous step is obtained obtains genetic engineering bacterium;
4) expression alien gene: the fusion rotein that genetic engineering bacterium fermentation expression contains recombinant human brain natriuretic peptide;
5) purifying target protein: cracking thalline, collect fusion rotein, by the enzyme of fusion rotein cut, purification step obtains recombinant human brain natriuretic peptide recombinant protein.
2. according to the method described in paragraph 1, it is characterized in that described molecular chaperone protein is selected from intestinal bacteria disulfide linkage and forms albumin A (DsbA), intestinal bacteria disulfide linkage formation albumin A mutant (DsbA
mut), CMP-KDO synthetic enzyme (CKS), Trx (Trx) or maltose binding protein (MBP).
3. according to the method described in paragraph 1, it is characterized in that described purification tag albumen is selected from 6 * His(His-Tag), glutathione s-transferase (GST) or FLAG.
4. according to the method described in paragraph 1, it is characterized in that described proteolytic enzyme is selected from zymoplasm or enteropeptidase.
5. according to the method described in paragraph 1, it is characterized in that human brain natriuretic peptide's gene order is the gene order that codon is optimized.
6. according to the method described in paragraph 1, it is characterized in that recombination rhBNP two ends can comprise restriction enzyme site or for carrying out the sequence of homologous recombination with plasmid.
7. according to the method described in paragraph 1, it is characterized in that described plasmid is the prokaryotic expression plasmid that contains T7 strong promoter.
8. according to the method described in paragraph 7, it is characterized in that described plasmid is the prokaryotic expression plasmid that contains kalamycin resistance.
9. according to the method described in paragraph 1, it is characterized in that described host cell is intestinal bacteria.
10. according to the method described in paragraph 1, it is characterized in that the enzyme of described fusion rotein is cut, purification step adopts five step method of purification, this five steps method of purification is followed successively by affinity chromatography, enzyme is cut fusion rotein, ion exchange chromatography, reversed phase chromatography and ion exchange chromatography.
11. according to the method described in paragraph 10, it is characterized in that described five step method of purification concrete steps are:
1) affinity chromatography: Chelating Sepharose F.F. chromatography column initial gross separation fusion rotein;
2) enzyme is cut fusion rotein: with zymoplasm or enteropeptidase enzyme, cut the fusion rotein that step 1) obtains, obtain enzymolysis solution;
3) cation-exchange chromatography: enzymolysis solution cationic exchange coloum purification step 2) obtaining, separation obtains the recombinant human brain natriuretic peptide of preliminary purification;
4) reversed phase chromatography: Source 15 RPC reversed phase chromatography media, obtain the recombinant human brain natriuretic peptide being further purified;
5) cation-exchange chromatography: sample SP Sepharose Fast Flow cationic exchange coloum purification step 4) obtaining, remove impurity, obtain target protein recombinant human brain natriuretic peptide.
12. 1 kinds of solubility expression human brain natriuretic peptides' recombination rhBNP, it is characterized in that, the sequence of described recombination rhBNP comprises purification tag gene order, molecular chaperone protein gene order, proteolytic enzyme recognition site sequence and the human brain natriuretic peptide's gene order that 5 ' to 3 ' direction is arranged in order.
13. according to the recombination rhBNP described in paragraph 12, it is characterized in that, described molecular chaperone protein is selected from intestinal bacteria disulfide linkage and forms albumin A (DsbA), intestinal bacteria disulfide linkage formation albumin A mutant (DsbA
mut), CMP-KDO synthetic enzyme (CKS), Trx (Trx) or maltose binding protein (MBP).
14. according to the recombination rhBNP described in paragraph 12, it is characterized in that, described purification tag albumen is selected from 6 * His(His-Tag), glutathione s-transferase (GST) or FLAG.
15. according to the recombination rhBNP described in paragraph 12, it is characterized in that described proteolytic enzyme is selected from zymoplasm or enteropeptidase.
16. according to the recombination rhBNP described in paragraph 12, it is characterized in that recombination hBNP two ends can comprise restriction enzyme site or for carrying out the sequence of homologous recombination with plasmid.
The recombinant human brain natriuretic peptide albumen of recombinant gene expression described in 17. paragraph 12-16 any one.
The 18. recombinant human brain natriuretic peptide albumen that obtain by method described in paragraph 1-11 any one.
The application of recombinant human brain natriuretic peptide in the medicine of preparation treatment heart failure disease described in 19. paragraph 17-18 any one.
For solve expressing fusion protein easily form inclusion body so that cause renaturation process complexity and cost high, and the problem such as the protein-active obtaining by renaturation process is poor, a kind of method that the invention provides solubility expression human brain natriuretic peptide recombinant protein, the method comprises the steps:
1) obtain recombination: obtain the recombination rhBNP that expresses human brain natriuretic peptide's albumen, the sequence of described recombination rhBNP comprises purification tag gene order, molecular chaperone protein gene order, proteolytic enzyme recognition site sequence and the human brain natriuretic peptide's gene order that 5 ' to 3 ' direction is arranged in order;
2) obtain recombinant plasmid: the recombination rhBNP that previous step is obtained inserts plasmid, obtain the recombinant plasmid that contains recombination rhBNP;
3) obtain genetic engineering bacterium: the recombinant plasmid transformed host cell that previous step is obtained obtains genetic engineering bacterium;
4) expression alien gene: the fusion rotein that genetic engineering bacterium fermentation expression contains recombinant human brain natriuretic peptide;
5) purifying target protein: cracking thalline, collect fusion rotein, by the enzyme of fusion rotein cut, purification step obtains recombinant human brain natriuretic peptide recombinant protein.
In a kind of preferably embodiment, described molecular chaperone protein is selected from intestinal bacteria disulfide linkage and forms albumin A (DsbA), intestinal bacteria disulfide linkage formation albumin A mutant (DsbA
mut), CMP-KDO synthetic enzyme (CKS), Trx (Trx) or maltose binding protein (MBP).
In a kind of preferably embodiment, described purification tag albumen is selected from 6 * His(His-Tag), glutathione s-transferase (GST) or FLAG, be preferably 6 * His(His-Tag).
In a kind of preferably embodiment, described proteolytic enzyme is selected from zymoplasm or enteropeptidase, is preferably zymoplasm.
In a kind of preferably embodiment, described human brain natriuretic peptide's gene order is that the preference on codon uses was carried out codon optimized gene order according to expressive host.
In embodiment preferably, recombination rhBNP two ends can comprise restriction enzyme site or for carrying out the sequence of homologous recombination with carrier.
In a kind of preferably embodiment, described plasmid is the prokaryotic expression plasmid that contains T7 strong promoter, is preferably pET series plasmid.Better, described plasmid is the prokaryotic expression plasmid that contains kalamycin resistance.Preferably, described plasmid is pET28a.
In a kind of preferably embodiment, described host cell is intestinal bacteria, is preferably e. coli bl21 (DE3).
In a kind of preferably embodiment, the enzyme of described fusion rotein is cut, purification step adopts five step method of purification, and this five steps method of purification is followed successively by affinity chromatography, enzyme is cut fusion rotein, ion exchange chromatography, reversed phase chromatography and ion exchange chromatography.Better, described five step method of purification concrete steps are:
1) affinity chromatography: with Chelating Sepharose F.F. chromatography column initial gross separation fusion rotein;
2) enzyme is cut fusion rotein: with zymoplasm or enteropeptidase enzyme, cut the fusion rotein that step 1) obtains, obtain enzymolysis solution;
3) enzymolysis solution cation-exchange chromatography: by SP Sepharose Fast Flow positively charged ion chromatography column purification step 2) obtaining, separation obtains the recombinant human brain natriuretic peptide of preliminary purification;
4) reversed phase chromatography: with Source 15 RPC reversed-phase columns, obtain the recombinant human brain natriuretic peptide being further purified;
5) cation-exchange chromatography: the sample obtaining by SP Sepharose Fast Flow positively charged ion chromatography column purification step 4), remove impurity, obtain target protein recombinant human brain natriuretic peptide.
Further, the invention provides a kind of solubility expression human brain natriuretic peptide's recombination rhBNP, the sequence of described recombination comprises purification tag gene order, molecular chaperone protein gene order, proteolytic enzyme recognition site sequence and the human brain natriuretic peptide's gene order that 5 ' to 3 ' direction is arranged in order.
In a kind of preferably embodiment, described molecular chaperone protein is selected from intestinal bacteria disulfide linkage and forms albumin A (DsbA), intestinal bacteria disulfide linkage formation albumin A mutant (DsbA
mut), CMP-KDO synthetic enzyme (CKS), Trx (Trx) or maltose binding protein (MBP).
In a kind of preferably embodiment, described purification tag albumen is selected from 6 * His(His-Tag), glutathione s-transferase (GST) or FLAG.
In a kind of preferably embodiment, described proteolytic enzyme is selected from zymoplasm or enteropeptidase.
In a kind of preferably embodiment, described human brain natriuretic peptide's gene order is that the preference on codon uses was carried out codon optimized gene order according to expressive host.
In a kind of preferably embodiment, recombination rhBNP two ends can comprise restriction enzyme site or for carrying out the sequence of homologous recombination with plasmid.
Further, the invention provides the recombinant human brain natriuretic peptide albumen of above-mentioned recombination rhBNP expression or the recombinant human brain natriuretic peptide albumen obtaining by aforesaid method.
Further, the invention provides the application of above-mentioned recombinant human brain natriuretic peptide albumen in the medicine of preparation treatment heart failure disease.
Beneficial effect of the present invention: recombination rhBNP provided by the invention, the recombinant protein of its expression does not form inclusion body, and is mainly expressed in colibacillus periplasm space.After broken like this bacterium, can directly from broken bacterium supernatant liquor, obtain target protein, avoid obtaining having from renaturing inclusion bodies the process of bioactive albumen.Expression of recombinant proteins purification process provided by the invention, easy repetition simple to operation, can obtain efficiently and meet the high purity of drug approval standard, highly active recombinant human brain natriuretic peptide albumen.
Accompanying drawing explanation
Fig. 1 pET-28a plasmid physical map
Fig. 2 pET-28a-DsbA
mut-BNP plasmid sequencing result
Fig. 3 His-DsbA
mut-BNP fusion rotein is at engineering bacteria
e.ColibL21(DE3) the SDS-PAGE electrophoretic analysis of expressing in
Fig. 4 affinity chromatography separation and purification His-DsbA
mut-BNP fusion protein S DS-PAGE electrophoretic analysis
Fig. 5 His-DsbA
mut-BNP fusion rotein zymoplasm enzyme is cut front and back SDS-PAGE electrophoretic analysis
Fig. 6 His-DsbA
mut-BNP fusion rotein zymoplasm enzyme is cut rear HPLC Liquid Detection spectrogram
Fig. 7 fusion protease is cut rear cation exchange purification Buffer E wash-out Liquid Detection spectrogram
The anti-phase refined solution phase of Fig. 8 recombinant human brain natriuretic peptide spectrogram
Fig. 9 mass spectroscopy detects recombinant human brain natriuretic peptide molecular weight spectrogram
Figure 10 high performance liquid chromatography detects recombinant human brain natriuretic peptide purity spectrogram
The active spectrogram that detects of Figure 11 recombinant human brain natriuretic peptide
Figure 12 recombinant human brain natriuretic peptide amino acid sequence analysis spectrogram.
Embodiment
The present invention utilizes engineered method, to express the recombination rhBNP of recombinant human brain natriuretic peptide albumen, by this recombination sequence clone to expression vector, transformed host cell, by fermentation, the steps such as separation and purification obtain recombinant human brain natriuretic peptide albumen.
Specifically, recombination rhBNP sequence comprises purification tag gene order, molecular chaperone protein gene order, proteolytic enzyme recognition site sequence and the human brain natriuretic peptide's gene order that 5 ' to 3 ' direction is arranged in order.Recombination two ends also can comprise restriction enzyme site or for carrying out the sequence of homologous recombination with carrier.Method by molecular cloning is inserted into recombination rhBNP in prokaryotic expression carrier, transforms e. coli host cell, expresses the fusion rotein that contains purification tag albumen, molecular chaperone protein, proteolytic enzyme recognition site and human brain natriuretic peptide's albumen.Finally, by step separation and purification fusion roteins such as affinity chromatography, cationic exchange, reversed phase chromatographies, obtain recombinant human brain natriuretic peptide albumen.
The method that obtains recombination comprises artificial gene synthetic method or pcr amplification method etc.
To further describe by specific embodiment below.Yet should be appreciated that specific embodiment is only for explaining the present invention, the protection domain being not intended to limit the present invention.In the application, instrument used, equipment, reagent, method etc., as do not specialized, are the conventional instrument in this area, equipment, reagent and method.
embodiment mono-BNP is codon optimized
Difference synthetic DsbA
mut-wtBNP(SEQ ID No11) and DsbA
mut-optBNP (SEQ ID No3) gene order (Invitrogen is synthetic), and in gene fragment, introduce respectively the histidine-tagged sequence of His-Tag and zymoplasm restriction enzyme site sequence, at the gene fragment 5' of synthetic end, introduce
ncoi restriction enzyme site, 3' holds introducing
hindiII restriction enzyme site.To synthesize fragment and pET28a(+) carrier warp respectively
ncoi with
hindiII double digestion, T4 DNA ligase connects gene fragment and carrier segments, and the conventional DH5 α competent cell that transforms, utilizes kalamycin resistance screening to obtain positive colony, after extraction plasmid, carries out gene sequencing, leaves and takes correct clone and plasmid.
WtBNP gene order is (SEQ ID No 1):
5’-AGC CCC AAG ATG GTG CAA GGG TCT GGC TGC TTT GGG AGG AAG ATG GAC CGG ATC AGC TCC TCC AGT GGC CTG GGC TGC AAA GTG CTG AGG CGG CAT TAA TAA AAG CTT-3’
OptBNP gene order is (SEQ ID No 2):
5’-TCT CCG AAA ATG GTT CAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GAC CGT ATC TCT TCT TCT TCT GGT CTG GGT TGC AAA GTT CTG CGT CGT CAC TAA TAA AAG CTT-3’
Plasmid is converted in e. coli bl21, is cultured to OD
600be that to add final concentration at 0.8 o'clock be that the IPTG induction target protein of 0.5 mM is expressed.After inducing culture 4 h, the centrifugal results thalline of 10000rpm, carries out protein expression component analysis by 12% SDS-PAGE electrophoresis, and result is as shown in table 1.
Wild-type BNP (wtBNP) and codon optimized rear gene order (optBNP) all can expressed fusion proteins, use optimize after the resulting biomass of BNP gene order and expressing fusion protein efficiency all higher.If no special instructions, the application uses the BNP sequence after optimization.
embodiment bis-recombinant vectorss and engineering bacteria build
Entrust the synthetic His-DsbA of Invitrogen company
mut-zymoplasm recognition site-BNP sequence (SEQ ID No 3), and introduce at gene fragment 5' end
ncoi restriction enzyme site, 3' holds introducing
hindiII restriction enzyme site.By synthetic gene fragment and pET28a(+) carrier (Fig. 1) warp respectively
ncoi with
hindiII double digestion, T4 DNA ligase connects gene fragment and carrier segments, and the conventional DH5 α competent cell (TIANGEN Biotech (Beijing) Co., Ltd.) that transforms, according to kalamycin resistance screening positive clone, extracts plasmid.Recombinant plasmid warp
ncoi with
hindiII double digestion and agarose gel electrophoresis are identified, entrust Invitrogen company to carry out sequencing to recombinant plasmid simultaneously, use BioEdit software to analyze sequencing result, and result identical with implementation sequence (as Fig. 2), illustrates that recombinant bacterium successfully constructs.Preservation plasmid is pET-28a-DsbA
mut-BNP fusion rotein carrier.
By the positive colony plasmid pET-28a-DsbA obtaining
mut-BNP transforms after BL21 (DE3) (TIANGEN Biotech (Beijing) Co., Ltd.) competent cell, by blocking that resistance screening positive colony; Positive colony bacterium is seeded to LB liquid nutrient medium (containing 50 μ g/mL kantlex), is cultured to OD
600be that to add final concentration at 0.8 o'clock be that the IPTG induction target protein of 0.5 mM is expressed.After inducing culture 4 h, the centrifugal results thalline of 10000 rpm; Ultrasonication thalline, 12000 rpm are centrifugal, obtain respectively supernatant liquor and precipitation, by 12% SDS-PAGE electrophoresis detection, arrive DsbA
mut-BNP fusion rotein is present in brokenly in bacterium supernatant; Positive colony bacterium carries out DsbA
mut-BNP fusion rotein abduction delivering, the engineering bacteria of screening frozen high expression level.
embodiment tri-engineering bacterium fermentations
1. get pET-28a-DsbA
mut-BNP/BL21 (DE3) plants liquid, and 1:500 is inoculated in (containing 50 μ g/mL kantlex) 37 ℃ in LB liquid nutrient medium, 150 rpm incubated overnight;
2. record incubated overnight bacterium liquid OD
600it is 4 o'clock.According to 1:20 ratio, be seeded in the fermentor tank that contains 30 L TB substratum, it is that 30%, pH control is 7.0,37 ℃ of cultivations, fermentor tank mixing speed and dissolved oxygen auto-associating that dissolved oxygen is controlled.Timing sampling, surveys OD
600value;
3. when cultivating bacterium liquid OD
600reach at 12 o'clock, in fermentor tank, adding the control of IPTG (final concentration 0.5 mM) dissolved oxygen is that 30%, pH control is 7.0,37 ℃ of cultivation inductions, and OD is surveyed in sampling per hour
600value;
4. engineering bacteria stops fermentation culture after inducing 4 h, records OD
600be the centrifugal 15 min results thalline of 28,8500 rpm, obtain thalline weight in wet base 1200 g;
5. Buffer A(20 mM imidazoles, 20 mM Tris-HCl pH=8.0,0.5 M NaCl for the thalline that takes a morsel) damping fluid is resuspended, add albumen loading Loading buffer boiling water bath 10 min to break bacterium, after centrifugal, get supernatant, 12% SDS-PAGE detects rhBNP expressing fusion protein and accounts for whole bacterial protein more than 20% (as Fig. 3).
the separation and purification of embodiment quadruple histone
1. bacterial cell disruption
With Buffer A damping fluid according to 1:10(W/V) resuspended, 8500 rpm, centrifugal 15 min, washing thalline.Thalline is suspended in to Buffer A damping fluid with 10% (W/V) ratio, with tissue refiner, mixes, with the broken bacterium twice of high pressure homogenizer, control pressure is at 600-700 bar, and broken bacterium rate, more than 95%, 4 ℃, after centrifugal 30 min of 8500 rpm, obtains and comprises DsbA
mutthe supernatant liquor of-BNP fusion rotein, a collection of 30 L fermentation thalline weight in wet bases are about 1200 g, and after broken bacterium, thalline supernatant is about 10 L, and protein concentration is about 20 mg/ml.
2. affinity chromatography separation and purification fusion rotein
DsbA
mutthe His-Tag label that contains continuous 6 Histidines in-BNP fusion rotein, broken bacterium supernatant liquor can pass through nickel affinity chromatography purifying; Chelating-Sepharose F.F. post is (purchased from GE company, chromatography column is BPG100/500 chromatography column, gel volume is 1.5L) after Buffer A damping fluid balance, to break bacterium supernatant liquor loading, loading step: 1) three column volumes of Buffer A 100 ml/min flow velocity balances (20 mM Tris-HCl pH=8.0,0.5 M NaCl, 20 mM imidazoles); 2) broken bacterium supernatant loading step 1 being obtained, flow velocity is 100 ml/min; 3) with Buffer A damping fluid, be washed till baseline, flow velocity 100 ml/min; 4) with Buffer B damping fluid (20 mM Tris-HCl pH=8.0,200 mM imidazoles) wash-out and collect elution peak, most DsbA
mut-BNP fusion rotein is adsorbed on filler and can be eluted by Buffer B DsbA
mut-BNP fusion rotein purity reaches 70%(as Fig. 4), protein content is 40 mg/ml, after wash-out, sample volume is about 2000 ml.
3. DsbA
mut-BNP fusion protease is cut
Utilize zymoplasm enzyme to cut fusion rotein, adjusting fusion rotein concentration is 30 mg/ml, at 2000 ml enzymes, cut in system,, use 37 ℃ of enzymes of 1200 U zymoplasm to cut 4 h, can cut entirely by enzyme.Fusion rotein is digested is label protein DsbA
mutwith rhBNP polypeptide, as shown in Figure 5, enzyme is cut rear HPLC Liquid Detection result as shown in Figure 6, more than rhBNP peptide concentration reaches 1.6 mg/ml.
4. cationic exchange, removes enzyme and cuts rear label protein and other impurity
Use SP-Sepharose F.F. gel column (purchased from GE company, BPG100/500 chromatography column, gel volume 800 ml), 1) Buffer C balance (20 mM Tris-HCl pH=8.0) chromatography column, 2) upper step enzyme cuts solution loading, and flow velocity is 100 ml/min; 3) Buffer C balance (20 mM Tris-HCl pH=8.0) pillar, flow velocity is 100 ml/min; 4) with Buffer D(20 mM Tris-HCl pH=8.0,0.1 M NaCl) clean pillar, remove partial impurities; 5. with Buffer E(20 mM Tris-HCl pH=8.0,0.6 M NaCl) wash-out target protein.Liquid phase result shows, most of label protein is removed, rhBNP target protein is adsorbed on filler and can be by Buffer E(20 mM Tris-HCl pH=8.0,0.6 M NaCl) elute, purity reaches more than 60%, concentration is greater than 3 mg/ml, therefore can obtain good purification effect, the sample volume under wash-out is about 1000 ml.As shown in Figure 7.
5. reversed phase chromatography
Use the separated rhBNP polypeptide of Source 15RPC gel of GE company, the FINELINE100LP chromatography column of chromatography column WeiGE company, gel volume is 1.2 L.With Buffer F (5% acetonitrile, 0.1%TFA) balanced gel, the elution peak that step 4 is collected is diluted to 1 ~ 1.5 mg/ml loading, after having gone up sample, use Buffer F(5% acetonitrile, 0.1%TFA) balance, flow velocity is 100 ml/min, with 75%Buffer F+ 25%Buffer G (95% acetonitrile+0.1%TFA) wash-out foreign protein, continuous gradient wash-out with 75%Buffer F+25%Buffer G to 50%Buffer F+50%Buffer G, collect each several part 215 nm ultraviolet absorption peaks, with 11% Buffer F+89% Buffer G buffer solution for cleaning chromatography column; Through HPLC, analyze, the rhBNP target protein peak purity of collecting is as shown in Figure 8 greater than 95%.
6. cationic exchange, removes acetonitrile
Buffer H balance (20 mM Tris-HCl pH=8.0) the SP-Sepharose F.F chromatography column (XK50/60 of GE company chromatography column, gel volume 400 ml), step 5 is collected to liquid loading, Buffer H balance (20 mM Tris-HCl pH=8.0) pillar, flow velocity is 50 ml/min; With Buffer I(20 mM Tris-HCl pH=8.0,0.1 M NaCl) clean chromatography column, remove acetonitrile and trifluoroacetic acid, Buffer J(20 mM Tris-HCl pH=8.0,0.6 M NaCl) wash-out target protein collect elution peak.Through HPLC, analyze, the rhBNP polypeptide purity of collection reaches more than 95%, after 0.22 μ m filter Sterile Filtration, is rhBNP stoste, and rhBNP concentration is greater than 2 mg/ml as calculated, and output is about 2.0 g, and the finished product meet medicine and declare quality standard.
embodiment five result verification
The recombinant human brain natriuretic peptide albumen that above-mentioned steps is obtained, by relevant regulations, protein mass is studied, quality determining method adopts 2010 editions three disclosed methods of < < Chinese Pharmacopoeia > >.BNP Product Activity measuring method is with reference to Rao Chunming etc.; Recombinant human brain natriuretic peptide quality standard and calibration method research, pharmaceutical analysis magazine [J]; 2002,22(5): 346-349 and Dong Peizhi etc.; RhBNP biological activity quality controlling means and improvement [J]; Medical science forum of basic unit; 2007,11 (10): 898-899 relevant references is measured the biologic activity of rhBNP product, the plain product of new work of employing Chengdu Nuodikang company in contrast product carries out correlated results checking work.
1. molecular weight detection
Entrust Shanghai Inst. of Life Science, CAS to carry out the mass spectroscopy molecular weight determination of product.Experimental result shows by the result of mass spectrometric determination consistent with theoretical supposition, and molecular weight is 3464 dalton (Fig. 9);
2. purity check
With reference to 2010 editions three appendix III B high performance liquid chromatography of < < Chinese Pharmacopoeia > >, Liquid Detection condition: use Waters XBridge C18 liquid-phase chromatographic column to detect, rhBNP applied sample amount is not less than 10 μ g; Mobile phase A: containing 5% acetonitrile solution of 0.1% TFA, Mobile phase B: containing 95% acetonitrile solution of 0.1% TFA, gradient: 0-30 min, B:0%-60%; 30-40 min, B:60%-100%, 40-42 min, B:100%-0%; 42-45 min, B:0%; Flow velocity: 1 ml/min, 40 ℃ of column temperatures, 215 nm ultraviolet detection, area normalization method calculation sample purity is 99.02%.(Figure 10).
3. active detection
The rhBNP activity determination method providing with reference to Rao Chunming etc., get rabbit Arterial Rings In Vitro bar and be cut into approximately 1.5 cm length, the wide spiral ring of 2 ~ 3 mm, hang in the Magnus' bath of 37 ℃ of tyrode's solutions that contain the logical oxygen of 10 ml application of load 1 g, stablize 1 h, during every 20 min change 1 tyrode's solution.Connect polygraph, regulate sensitivity, record the tension variation of blood vessel.After tensammetric curve baseline stability, adding 1.25 mg/ml AK-Nefrin 0.05 ml in Magnus' bath, to make its final concentration is 6.25 * 10
-5mg/ml, curve be increased to the highest and stable after, start to add people's human brain profit sodium skin reference substance of recombinating by accumulative total concentration dose regimen, record the tension variation of blood vessel.Complete after above-mentioned administration, wash-out is also stablized 1 h, during every 20 min change tyrode's solution 1 time.After baseline stability, by the method for measuring reference substance, measure testing sample, record measurement result.That calculates reference substance and each laboratory sample partly imitates extension rate, is calculated as follows sample and tires: tire * sample of tire=reference substance of sample is partly imitated extension rate/reference substance and partly imitated extension rate (RU/ml).Carry out 3 laboratory test results and show rhBNP product and the new plain reference substance activity basically identical (Figure 11) of living.
4. amino acid sequence analysis
Entrust Shanghai Inst. of Life Science, CAS by the aminoacid sequence of mass spectrometric determination target protein, measurement result is:
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His(Figure 12).
In sum, recombination His-DsbA provided by the invention
mutthe albumen that-BNP sequence is expressed is present in the broken bacterium supernatant rather than inclusion body of host cell.And then by supernatant liquor is carried out the steps such as chromatography can separation and purification to high purity, highly active target protein, (protein yield is 18 mg/10 g wet thallus; purity is greater than 95%); and albumen is carried out to the complicated processes such as renaturation without also needing obtain albumen from inclusion body after as ordinary method, and this process can cause the reduction of activity or yield conventionally.
the experiment that embodiment six contains other companion/label proteins
Entrust Invitrogen synthetic following gene order: DsbA-BNP (SEQ ID No 4), Trx-BNP(SEQ ID No 5), GST-BNP(SEQ ID No 6), MBP-BNP(SEQ ID No 7), CKS-BNP(SEQ ID No 8), according to the method for above-described embodiment, build rhBNP fusion rotein recombinant plasmid pET28a-DsbA-BNP, pET28a-Trx-BNP, pET28a-GST-BNP, pET28a-MBP-BNP and pET28a-CKS-BNP carrier.After obtaining correct carrier sequence, proceed to e. coli bl21 (DE3) competent cell, by resistance screening, obtain positive colony.After enlarged culturing, carry out fusion rotein abduction delivering, the engineering bacteria of screening high expression level.
By after the engineering bacteria incubated overnight of different chaperones, by 1:100, be inoculated in containing in the LB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃ of shaking culture are to O.D
600be worth 0.8 left and right, adding IPTG is 0.5 mM to final concentration, 37 ℃ of induction 4 h, centrifugal collection thalline.
Different chaperone thalline are respectively taken to 10 g wet thallus, and thalline is suspended in ultrasonic in ice bath in PBS damping fluid (240W, ultrasonic 4 s, gap 8 s) broken thalline with 10% (W/V) ratio, centrifugal 30 min of 12000 rpm, and supernatant is retained to be measured; Precipitation is with after PBS washing 2 times, use again the sample-loading buffer of same volume resuspended, the upper cleer and peaceful precipitation of equivalent (equal-volume suspension) is carried out respectively to 12% SDS-PAGE detection, and SDS-PAGE electrophoresis result is used Quantity One software analysis, qualitatively judges fusion rotein solubility.Fusion rotein solubility expression situation in Table 3 (+represent that fusion protein expression is larger) more.
DsbA, MBP all can realize fusion rotein great amount of soluble as molecular chaperones and express; Trx also can realize the relatively large solubility expression of fusion rotein as molecular chaperones.GST also can realize the solubility expression of fusion rotein in broken bacterium supernatant as purification tag albumen.
DsbA, Trx, MBP are all higher as chaperone gained target protein yield and purity; Use that GST is higher as label protein gained target protein purity, yield is compared with DsbA, DsbA
mut, MBP, GST be low as label protein.
Other molecular chaperone proteins of above-mentioned description of test and label protein also can be used for this invention.
embodiment seven proteolytic enzyme restriction enzyme sites are selected
Respectively at pET28a-DsbA
mutin-BNP sequence, add zymoplasm restriction enzyme site and enteropeptidase restriction enzyme site, by carrying out enzyme after above-described embodiment step purifying, cut, both all can correctly excise label protein and rhBNP albumen.Therefore, zymoplasm and enteropeptidase all can be used for the present invention.
Zymoplasm restriction enzyme site gene order is: CTG GTT CCG CGT (SEQ ID No 9)
Enteropeptidase restriction enzyme site gene order is: GAT GAC GAT GAC AAG (SEQ ID No 10)
Sequence table
Wo Tai bio tech ltd, <110> Shijiazhuang
The methods and applications of a <120> solubility expression human brain natriuretic peptide recombinant protein
<130> 2014HBWT002
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 108
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 1
agccccaaga tggtgcaagg gtctggctgc tttgggagga agatggaccg gatcagctcc 60
tccagtggcc tgggctgcaa agtgctgagg cggcattaat aaaagctt 108
<210> 2
<211> 108
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 2
tctccgaaaa tggttcaggg ttctggttgc ttcggtcgta aaatggaccg tatctcttct 60
tcttctggtc tgggttgcaa agttctgcgt cgtcactaat aaaagctt 108
<210> 3
<211> 771
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 3
accatgggca gctctcatca tcatcatcat cactctagcg gtgctcagta cgaagatggt 60
aaacagtaca ctaccctgga aaaaccggta gctggcgcgc cgcaagtgct ggagtttttc 120
tctttcttca gcccgcattc ctatcagttt gaagaagttc tgcatatttc tgataatgtg 180
aagaaaaaac tgccggaagg cgtgaagatg actaaatacc acgtcaactt catgggtggt 240
gacctgggca aagatctgac tcaggcatgg gctgtggcga tggcgctggg cgtggaagac 300
aaagtgactg ttccgctgtt tgaaggcgta cagaaaaccc agaccattcg ttctgcttct 360
gatatccgcg atgtatttat caacgcaggt attaaaggtg aagagtacga cgcggcgtgg 420
aacagcttcg tggtgaaatc tctggtcgct cagcaggaaa aagctgcagc tgacgtgcaa 480
ttgcgtggcg ttccggcgat gtttgttaac ggtaaatatc agctgaatcc gcagggtatg 540
gataccagca atatggatgt ttttgttcag cagtatgctg atacagtgaa atatctgtcc 600
gagaaaaaag gtgatgaaga tgaagatgaa gatagcggat ccgcagaatt cctggttccg 660
cgttctccga aaatggttca gggttctggt tgcttcggtc gtaaaatgga ccgtatctct 720
tcttcttctg gtctgggttg caaagttctg cgtcgtcact aataaaagct t 771
<210> 4
<211> 774
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 4
accatgggca gctctcatca tcatcatcat cacaaaaaga tttggctggc gctggctggt 60
ttagttttag cgtttagcgc atcggcggcg cagtatgaag atggtaaaca gtacactacc 120
ctggaaaaac cggtagctgg cgcgccgcaa gtgctggagt ttttctcttt cttctgcccg 180
cactgctatc agtttgaaga agttctgcat atttctgata atgtgaagaa aaaactgccg 240
gaaggcgtga agatgactaa ataccacgtc aacttcatgg gtggtgacct gggcaaagat 300
ctgactcagg catgggctgt ggcgatggcg ctgggcgtgg aagacaaagt gactgttccg 360
ctgtttgaag gcgtacagaa aacccagacc attcgttctg cttctgatat ccgcgatgta 420
tttatcaacg caggtattaa aggtgaagag tacgacgcgg cgtggaacag cttcgtggtg 480
aaatctctgg tcgctcagca ggaaaaagct gcagctgacg tgcaattgcg tggcgttccg 540
gcgatgtttg ttaacggtaa atatcagctg aatccgcagg gtatggatac cagcaatatg 600
gatgtttttg ttcagcagta tgctgataca gtgaaatatc tgtccgagaa aaaactggtt 660
ccgcgtagcc ccaagatggt gcaagggtct ggctgctttg ggaggaagat ggaccggatc 720
agctcctcca gtggcctggg ctgcaaagtg ctgaggcggc attaataaaa gctt 774
<210> 5
<211> 504
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 5
accatgggca gcagccatca tcatcatcat cacggttcaa ctagtggttc tggttctgat 60
aaaattattc atctgactga tgattctttt gatactgatg tacttaaggc agatggtgca 120
atcctggttg atttctgggc acactggtgc ggtccgtgca aaatgatcgc tccgattctg 180
gatgaaatcg ctgacgaata tcagggcaaa ctgaccgttg caaaactgaa catcgatcac 240
aacccgggca ctgcgccgaa atatggcatc cgtggtatcc cgactctgct gctgttcaaa 300
aacggtgaag tggcggcaac caaagtgggt gcactgtcta aaggtcagtt gaaagagttc 360
ctcgacgcta acctggctgg ctctctggtt ccgcgttctc cgaaaatggt tcagggttct 420
ggttgcttcg gtcgtaaaat ggaccgtatc tcttcttctt ctggtctggg ttgcaaagtt 480
ctgcgtcgtc actaataaaa gctt 504
<210> 6
<211> 783
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 6
accatgggcc ctatactagg ttattggaaa attaagggcc ttgtgcaacc cactcgactt 60
cttttggaat atcttgaaga aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat 120
aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc ttattatatt 180
gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc tgacaagcac 240
aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga aggagcggtt 300
ttggatatta gatacggtgt ttcgagaatt gcatatagta aagactttga aactctcaaa 360
gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat 420
aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta tgacgctctt 480
gatgttgttt tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt 540
aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata 600
gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg 660
gatctggttc cgcgttctcc gaaaatggtt cagggttctg gttgcttcgg tcgtaaaatg 720
gaccgtatct cttcttcttc tggtctgggt tgcaaagttc tgcgtcgtca ctaataaaag 780
ctt 783
<210> 7
<211> 1236
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 7
accatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60
ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120
catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180
attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240
atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300
tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360
aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420
ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480
ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540
aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600
attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660
aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720
aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780
gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840
ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900
ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960
gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020
ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080
gaagccctga aagacgcgca gactaattcg agctcgctgg ttccgcgtag ccccaagatg 1140
gtgcaagggt ctggctgctt tgggaggaag atggaccgga tcagctcctc cagtggcctg 1200
ggctgcaaag tgctgaggcg gcattaataa aagctt 1236
<210> 8
<211> 891
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 8
accatgggca gctctcatca tcatcatcat cacaaaattg tcggtgtgat ccccgctcgt 60
tttggctcta cacgcttccc cggaaaaccc ctggtgaact tgaaaggccg ccccctgatt 120
cagtggacgg ttgaaggagc caagaagtcg aaacttctga gcgaggtgat tgtcgccacc 180
gatcacgaag gcatcaaagc cgccgccgaa gctgtcggtg ttaaagtcgt catgacggac 240
agcgatcttc ccaccggaag cgaccgaatc aatgctgcca ttaaagatgt tgcctgtgat 300
gtcgttgtca atatccaggg ggatgaaccg ctggtgaccg gcgagttgat tgaccgcctg 360
gcgcaggtgt ttgtcgatga tccgaaaatg gacatggcca cgctggctca tccgatcagt 420
gctgaagagc tgcaatccat gaactcggtg aaagttgttg tgaactgccg tgacgaggct 480
ctttatttca gccgctatcc aatgccttat tcccgtatgt cggcacaaga ggccggcagc 540
atggatggtt gtctgaaaca catcggcatg tatgcctatt ccagaaattt tttaaaacag 600
ttctgcgaag ctcctccggc cctcattgaa aaagccgaga gtctggagca gctgcgcgct 660
ctgtatttag gtgcaaaaat caaagtaatc agagtcaagg aagccagtgt cggggtggat 720
actcccgaag atctggcgcg actcgaaaaa ctcttaagct cacaggggat gctggttccg 780
cgtagcccca agatggtgca agggtctggc tgctttggga ggaagatgga ccggatcagc 840
tcctccagtg gcctgggctg caaagtgctg aggcggcatt aataaaagct t 891
<210> 9
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 9
ctggttccgc gt 12
<210> 10
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 10
gatgacgatg acaag 15
<210> 11
<211> 771
<212> DNA
<213> Artificial Sequence
<220>
<223> artificial seq
<400> 11
accatgggca gctctcatca tcatcatcat cactctagcg gtgctcagta cgaagatggt 60
aaacagtaca ctaccctgga aaaaccggta gctggcgcgc cgcaagtgct ggagtttttc 120
tctttcttca gcccgcattc ctatcagttt gaagaagttc tgcatatttc tgataatgtg 180
aagaaaaaac tgccggaagg cgtgaagatg actaaatacc acgtcaactt catgggtggt 240
gacctgggca aagatctgac tcaggcatgg gctgtggcga tggcgctggg cgtggaagac 300
aaagtgactg ttccgctgtt tgaaggcgta cagaaaaccc agaccattcg ttctgcttct 360
gatatccgcg atgtatttat caacgcaggt attaaaggtg aagagtacga cgcggcgtgg 420
aacagcttcg tggtgaaatc tctggtcgct cagcaggaaa aagctgcagc tgacgtgcaa 480
ttgcgtggcg ttccggcgat gtttgttaac ggtaaatatc agctgaatcc gcagggtatg 540
gataccagca atatggatgt ttttgttcag cagtatgctg atacagtgaa atatctgtcc 600
gagaaaaaag gtgatgaaga tgaagatgaa gatagcggat ccgcagaatt cctggttccg 660
cgtagcccca agatggtgca agggtctggc tgctttggga ggaagatgga ccggatcagc 720
tcctccagtg gcctgggctg caaagtgctg aggcggcatt aataaaagct t 771。
Claims (10)
1. a method for solubility expression human brain natriuretic peptide recombinant protein, comprises the following steps:
1) obtain recombination: obtain the recombination rhBNP that expresses human brain natriuretic peptide's recombinant protein, the sequence of described recombination rhBNP comprises purification tag gene order, molecular chaperone protein gene order, proteolytic enzyme recognition site sequence and the human brain natriuretic peptide's gene order that 5 ' to 3 ' direction is arranged in order;
2) obtain recombinant plasmid: the recombination rhBNP that previous step is obtained inserts plasmid, obtain the recombinant plasmid that contains recombination rhBNP;
3) obtain genetic engineering bacterium: the recombinant plasmid transformed host cell that previous step is obtained obtains genetic engineering bacterium;
4) expression alien gene: the fusion rotein that genetic engineering bacterium fermentation expression contains recombinant human brain natriuretic peptide;
5) purifying target protein: cracking thalline, collect fusion rotein, by the enzyme of fusion rotein cut, purification step obtains recombinant human brain natriuretic peptide recombinant protein.
2. method according to claim 1, is characterized in that described molecular chaperone protein is selected from intestinal bacteria disulfide linkage and forms albumin A (DsbA), intestinal bacteria disulfide linkage formation albumin A mutant (DsbA
mut), CMP-KDO synthetic enzyme (CKS), Trx (Trx) or maltose binding protein (MBP).
3. method according to claim 1, is characterized in that described purification tag albumen is selected from 6 * His(His-Tag), glutathione s-transferase (GST) or FLAG.
4. method according to claim 1, is characterized in that described proteolytic enzyme is selected from zymoplasm or enteropeptidase.
5. method according to claim 1, is characterized in that human brain natriuretic peptide's gene order is the gene order that codon is optimized.
6. method according to claim 1, is characterized in that recombination rhBNP two ends can comprise restriction enzyme site or for carrying out the sequence of homologous recombination with plasmid.
7. method according to claim 1, is characterized in that described plasmid is the prokaryotic expression plasmid that contains T7 strong promoter.
8. method according to claim 7, is characterized in that described plasmid is the prokaryotic expression plasmid that contains kalamycin resistance.
9. method according to claim 1, is characterized in that described host cell is intestinal bacteria.
10. method according to claim 1, the enzyme that it is characterized in that described fusion rotein is cut, purification step adopts five step method of purification, and this five steps method of purification is followed successively by affinity chromatography, enzyme is cut fusion rotein, ion exchange chromatography, reversed phase chromatography and ion exchange chromatography.
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| CN107177649A (en) * | 2017-06-22 | 2017-09-19 | 西藏诺迪康药业股份有限公司 | A kind of zymotechnique for improving recombinant human brain natriuretic peptide fusion protein expression |
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| CN110257347A (en) * | 2019-06-25 | 2019-09-20 | 成都英普博集生物科技有限公司 | Thioredoxin mutant, preparation method and its application in recombination fusion protein production |
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