CN101979576A - Method for preparing vasonatrin peptide (VNP) by genetic engineering recombination technology - Google Patents
Method for preparing vasonatrin peptide (VNP) by genetic engineering recombination technology Download PDFInfo
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- CN101979576A CN101979576A CN2010105034025A CN201010503402A CN101979576A CN 101979576 A CN101979576 A CN 101979576A CN 2010105034025 A CN2010105034025 A CN 2010105034025A CN 201010503402 A CN201010503402 A CN 201010503402A CN 101979576 A CN101979576 A CN 101979576A
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Abstract
The invention discloses a method for preparing vasonatrin peptide (VNP). In order to overcome the defects such high cost, low biological activity and the like in a conventional VNP chemical synthesis method, in the process of preparing the VNP by the method, a target gene is amplified by using polymerase chain reaction (PCR) technology; after performing enzyme cutting by taking pGEX-4T-1 as a vector plasmid, directionally inserting a VNP target gene to form a recombinant plasmid; Escherichia coli BL21 (DE3) serves as an expression strain; and a target protein is separated by using a GST affinity column. By using the method, a protein at a higher expression level can be obtained, and the protein with higher purity can be obtained by using affinity. The VNP obtained by utilizing the technology of the invention has higher biological activity and lower cost compared with the chemical synthesis method, which lays a foundation for further producing the VNP by a biological method.
Description
Technical field
The present invention relates to biological field, is the method for preparing blood vessel sodium peptide (VNP) by the genetically engineered recombinant technology specifically.
Background information
Cardiovascular and cerebrovascular diseases is " first killer " of universally acknowledged human health, wherein heart failure is almost unavoidable final results such as most organic heart disease people such as myocardium infarct, cor pulmonale, hypertrophic cardiomyopathy, 2 annual death rates of heart failure reach 20%, 5 annual death rate up to 40%.At present, China's patients with heart failure has reached more than 5,000,000 people, along with patients' such as population astogeny and hypertension, hyperlipidemia, diabetes and coronary heart disease number constantly increases, estimates will reach more than 7,000,000 people in the coming five years China patients with heart failure.Treat medicine in heart failure clinically in China several big class medicines such as cardiotonic drug, diuretic(s), vasodilation agent, hypertensin enzymeinhibitor, suprarenin retarding agent are arranged.But there are shortcomings such as dosage instability, toxic side effect be big.Therefore it is very urgent and necessary developing the new drug that a kind of effect is good, dosage stable, toxic side effect is little.In recent years, by to heart failure pathogenic process and the research of neurohormone activated, make people recognize that gradually it is crucial to be only treatment by regulating the neurohormone activation level to change generation, the development process of heart failure.
Natriuretic peptide family is a peptide family of discovered in recent years, has 17 amino acid whose looped domains that formed by disulfide linkage by two halfcystines.Natriuretic peptide family is keeping body water salt balance, blood pressure stabilization, cardiovascular and the kidney and other organs function aspects is significant.In recent years, along with people gradually to natriuretic peptide family member's understanding, natriuretic peptide also becomes the research focus.Human brain natriuretic peptide (human brain natriuretic peptide, hBNP) be one of natriuretic peptide family member, its gene recombination medicine--Nesiritide (nesiritide) in calendar year 2001 by drugs approved by FDA as the treatment acute decompensated heart failure clinical application.China's national biological product I kind new medicine " new live plain " (being rhBNP) that also gone on the market in 2005 is used for the treatment of the acute congestive heart failure equally.
Natural natriuretic peptide mainly comprises ANP, BNP and CNP, they all have ring that disulfide linkage forms, that be made of 17 amino-acid residues, wherein the nitrogen end at people ANP amino-acid residue ring has 6 amino-acid residues, one of carbon tip has 5 amino-acid residues, people CNP amino-acid residue ring nitrogen end has 5 amino-acid residues, and one of carbon tip does not have amino-acid residue.ANP has the sharp sodium of intensive, diuresis and expansion artery effect, but the effect of expansion of veins is very weak, and the activity of the sharp sodium of CNP, diuresis and expansion artery is very weak, but the effect of intensive expansion of veins is arranged.ANP, CNP structure and bioactive difference show, the amino acid residue sequence that natural natriuretic peptide amino acid ring and carboxyl terminal thereof extend may have material impact to its biological activity, 5 amino-acid residues of ANP amino acid ring carboxyl terminal are added to the corresponding site of CNP, may obtain a kind of new active natriuretic peptide that has, based on above-mentioned imagination, Wei equal 1993 at first design and synthetic have a new bioactive natriuretic peptide---blood vessel sodium peptide (Vasonatrin pep tide, VNP).
VNP is the mosaic of people CNP and ANP, and it is that carboxyl terminal at CNP adds 27 peptides that 5 amino-acid residues of ANP carboxyl terminal form, its secondary structure is also had disulfide linkage forms, 17 rings that amino-acid residue is formed, and molecular weight is about 2867Da.Its aminoacid sequence is: Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr.Newcomer with natriuretic peptide family of novel bioactive, existing studies show that, VNP may be in disease treatments such as chronic heart failure, hypertension, and more natural natriuretic peptide has more application prospect.But, have unique bioactive VNP and as if do not attract much attention and pay attention to, trace it to its cause, the one, chemosynthesis can only guarantee the consistence of aminoacid sequence, and can not guarantee the similarity of the pharmacodynamics structure of this compound in compound and the body; They are two years old, the amino acid ring that contains one 17 amino acid tool pharmacodynamics structure folding through spirals, that be connected to form by disulfide linkage in the VNP secondary structure, this is essential for its biological activity, though the VNP of chemosynthesis has guaranteed the consistence of aminoacid sequence and the formation of disulfide linkage, but, thereby be difficult to the correct space conformation of assurance because the chemosynthesis environment is with it is bigger in intracellular folding environment difference.Find that the VNP that obtains with biological process compares behind this research department's process molecular dynamics simulation and the nuclear magnetic resonance spectroscopy, there is very big difference in chemical method synthetic VNP main chain topological framework, and its pharmaceutical activity is difficult to guarantee.
Summary of the invention
Purpose of the present invention can't obtain this bottleneck by biological process in order to break through present blood vessel sodium peptide (VNP) exactly, and a method for preparing blood vessel sodium peptide by gene engineering research is provided.
The e. coli bl21 that we adopted (DE3) prokaryotic expression system makes up pGEX-4T-1 plasmid system, gives expression to soluble fusion protein GST-VNP, through affinity chromatography, can obtain the blood vessel sodium peptide of biologically active after the enteropeptidase enzyme is cut.
The preparation method that the present invention prepares blood vessel sodium peptide (VNP) may further comprise the steps:
(1) adopt round pcr will express the goal gene amplification of VNP, and its product of purifying;
(2) with the PCR product of purifying with the directed pGEX-4T-1 that inserts through same double digestion behind EcoR I and the Not I double digestion, acquisition recombinant plasmid pGEX-4T-1-VNP;
(3) recombinant plasmid pGEX-4T-1-VNP is converted into intestinal bacteria JM 109, through cultivating amplification, extracting and purifying, sequencing analysis, the correct recombinant plasmid of acquisition sequence;
(4) will check order correct recombinant plasmid transformed to BL21 (DE3) bacterium, at 30 ℃, express recombinant protein under the 0.5mM IPTG condition;
(5) the activation bacterium with express recombinant protein is inoculated in the substratum of LB+AMP, carries out shake flat experiment, and engineering bacteria carries out IPTG and induces in fermentation cylinder for fermentation during to OD600nm0.8, put jar after 6 hours, the centrifugal thalline that obtains;
(6) bacterial sediment after centrifugal is carried out cytoclasis, the centrifugal collection supernatant liquor in broken back carries out the GST affinity chromatography, and wash-out obtains fusion rotein GST-VNP;
(7) the fusion rotein GST-VNP that wash-out is obtained is by Sephadex G25 gel-filtration desalination, and the enteropeptidase cutting is adopted in the back.The cutting condition: 25 ℃, pH 7.6,12 hours;
(8) product after enzyme is cut removes the GST label through the GST affinity chromatography again, and adopting molecular weight cut-off is 10, and the sample after the ultra-filtration membrane of 000Da is cut enzyme carries out purifying, removes a spot of remaining enteropeptidase, obtains pure product VNP.
(9) pure product VNP is adopted myocardium vessel ring strain method on rat and remove bladder urine collecting method activity rating.
The above-mentioned recombinant vascular sodium peptide that obtains is tested rat and is shown that VNP possesses the vasodilation of ANP, the dual powerful activity of the diuresis of CNP.
Description of drawings
The structure of Fig. 1 .pGEX-4T-1-VNP.
The structure of Fig. 2 .pGEX-4T-1-VNP.
Fig. 3 .PCR amplification; M.DNA Maker D; 1.VNP
Fig. 4. the SDS-PAGE of fusion rotein analyzes; M. be the low molecular weight protein (LMWP) standard substance; 1.pGEX-4T-1 the empty plasmid abduction delivering, applied sample amount 25 μ L; 2.GST-VNP the fusion rotein abduction delivering, applied sample amount 25 μ L
Fig. 5. the Tricine-SDS-PAGE that fusion rotein is cut with the enteropeptidase enzyme analyzes; M. be ultra-low molecular amount protein standard substance Zadaxin; 1-2. enzyme cuts except that protein sample behind the GST; 3. the centrifugal back of ultrafiltration protein sample
Embodiment
The invention will be further described in conjunction with the embodiments.
The technical application route is as follows:
1, VNP goal gene amplification, construction of recombinant plasmid and Recombinant Protein Expression:
The VNP target gene sequences is consulted according to the NCBI website, synthetic gene pUC57-VNP.To express the goal gene amplification of VNP with round pcr, and its product of purifying.PCR product directed pGEX-4T-I that inserts through same double digestion behind EcoR I and Not I double digestion with purifying obtains recombinant plasmid pGEX-4T-1-VNP.Adopt pGEX-4T-1 plasmid vector multiple clone site place's design universal primer P3, P4 uses as identifying.PGEX-4T-1-VNP is converted into e. coli jm109 with recombinant plasmid, through cultivating amplification, extracting and purifying, sequencing analysis, the correct recombinant plasmid of acquisition sequence.With the correct recombinant plasmid transformed of order-checking to BL21 (DE3) bacterium, at 30 ℃, abduction delivering recombinant protein under the 0.5mM IPTG condition.Behind the centrifugal collection bacterial cell disruption of the fermented liquid cell, centrifuging and taking supernatant liquor, cell debris carry out SDS-PAGE and detect and analyze.
2, the fermentation of expression strain:
The activation bacterium of express recombinant protein is inoculated in the substratum of LB+AMP, carries out shake flat experiment, engineering bacteria carries out high density fermentation in the 5L fermentor tank.Plant daughter bacteria and inoculate in 1% ratio, 37 ℃, 500r/min, ampicillin 100 μ g/mL, high density fermentation is to OD
600nm0.8 in time, carries out IPTG and induces, and puts jar after 6 hours, the centrifugal thalline that obtains.
3, the protein purification of VNP:
3.1 the cracking of cell: the centrifugal bacterial sediment that obtains of fermented liquid adds lysis buffer and carries out cytoclasis, and broken intact back is centrifugal, collects supernatant liquor.Broken damping fluid: Tris 6.09g/L, NaCl 9g/L, HCl transfers pH 8.3.
3.2 affinity chromatography and gel-filtration: the supernatant liquor after fragmentation is centrifugal carries out GST sepharose FF affinity chromatography, collects the fusion rotein GST-VNP that wash-out obtains.Level pad: Tris 6.09g/L, NaCl 9g/L, HCl transfers pH 8.3.Elution buffer: Tris 6.09g/L, NaCl 9g/L, reduced glutathion 3.07g/L, HCl transfers pH 8.3.Fusion rotein GST-VNP is carried out Sephadex G25 gel-filtration desalination, and the Bradford method is carried out quantification of protein, gets sample segment and carries out the SDS-PAGE analysis.
3.3 proteolytic cleavage and GST label are removed: freeze dried GST-VNP fusion rotein carries out the enteropeptidase enzyme and cuts, by 1 μ L enteropeptidase fully the proteic consumption of cleavage of fusion proteins 0.4mg enteropeptidase is added in the fusion rotein liquid of purifying, cut 8h in 25 ℃ of enzymes, enzyme is cut the back sample and is carried out the unnecessary label protein GST of GST sepharose FF affinity chromatography removal again, gets partially digested product and carries out the Tricine-SDS-PAGE analysis.
3.4 ultrafiltration is centrifugal: adopting molecular weight cut-off is 10, and the sample after the ultra-filtration membrane of 000Da is cut enzyme carries out purifying, and enzyme cuts except that the sample behind the label G ST and has a spot of enteropeptidase, 4, and 000r/min, 30min, this step can effectively be removed enteropeptidase.Getting product behind the partial purification carries out Tricine-SDS-PAGE and analyzes.
4, the activity of recombinant vascular sodium peptide
4.1 the test of myocardium vessel ring strain: rat is with vetanarcol (30mg/kg) intraperitoneal anesthesia, cut chest, abdominal cavity after the carotid artery bloodletting open, clip pulmonary artery, aorta abdominalis and abdominal vein are put and are filled cold Krebs liquid (mmol/L:NaCl 118.3, KCl 4.7, MgCl
26H
2O 2.5, CaCl
22.5, KH
2PO
41.2, NaHCO
325.0 Ca-NaEDTA 0.03, Glucose 11.1) plate in, remove the blood vessel surface reticular tissue, be prepared into the long annular vascular specimen of 3mm.In part experiment, this inserts tube chamber with light gage wire to get wherein a segment mark, destroys blood vessel endothelium through rolling.With vascular circle hang on 37 ℃ of Krebs liquid bath in, feed 95%O
2Add 5%CO
2Mixed gas.Connect tension pick-up, change with two road physiographs (LMS-2B type Chengdu) record vascular circle tensile.The basic tension force of pulmonary artery, aorta abdominalis vascular circle is 1g, behind balance 1h, adds 10 μ mol/LNE inspection specimen activity (shrinkage amplitude discards less than 300mg person), and flushing makes antiotasis return to baseline then.Repeat to give NE after stablizing 30min, treat that vasoconstriction reaches stationary value, progressively increase the concentration (10 of VNP or CNP or ANP or GST-VNP
-10～10
-6M), to make cumulative concentration-stretching reaction curve, obtain maximum stretching reaction value (Rmax).With 10 μ mol/L Ach whether vascular circle is produced the diastole effect as the standard of judging whether blood vessel endothelium exists.
4.2 go bladder urine collecting method to detect the diuretic properties of natriuretic peptide: normal healthy Adult SD female rats, Sodital (40mg/kg) intraperitoneal anesthesia, trachea cannula is kept autonomous respiration.Insert from abdomen median incision with homemade polyethylene catheter, by the retrograde intraurethral cannula of bladder, with note urine amount, at bladder root ligation bladder.Urine collecting is in vitro urinating Na in order to detecting
+And K
+After stablizing 30min, write down before the administration respectively and give urine amount in the 15min of natriuretic peptide (50 μ g/kg) back.Each dosing interval is 20min.
In preparation blood vessel sodium peptide (VNP) process, use the round pcr amplifying target genes, orientation forms recombinant plasmid after inserting the VNP goal gene for the vector plasmid enzyme is cut afterwards with pGEX-4T-1, with e. coli bl21 (DE3) as expression strain, with the main purpose of GST affinity column as purifying, this method can obtain the albumen of higher expression amount, can obtain purer albumen by affinity again.
The VNP that uses this method to obtain have better biologic activity compared with chemical synthesis, and cost is low than chemical synthesis, for VNP further lays a good foundation by biological process production.
The specific embodiments of front only is the distance explanation, need not further elaboration, believes that along with the disclosed content in front, those skilled in the art can use the present invention to greatest extent.
Claims (4)
1. the preparation method of blood vessel sodium peptide (VNP) is characterized in that following steps:
Adopt round pcr will express the goal gene amplification of VNP, and its product of purifying;
With the PCR product of purifying with the directed pGEX-4T-1 that inserts through same double digestion behind EcoR I and the Not I double digestion, acquisition recombinant plasmid pGEX-4T-1-VNP;
Recombinant plasmid pGEX-4T-1-VNP is converted into intestinal bacteria JM 109, through cultivating amplification, extracting and purifying, sequencing analysis, the correct recombinant plasmid of acquisition sequence;
With the correct recombinant plasmid transformed of order-checking to BL21 (DE3) bacterium, at 30 ℃, express recombinant protein under the 0.5mM IPTG condition;
The activation bacterium of express recombinant protein is inoculated in the substratum of LB+AMP, carries out shake flat experiment, engineering bacteria is in fermentation cylinder for fermentation, to OD
600nm0.8 in time, carries out IPTG and induces, and puts jar after 6 hours, the centrifugal thalline that obtains;
Bacterial sediment after centrifugal is carried out cytoclasis, and the centrifugal collection supernatant liquor in broken back carries out the GST affinity chromatography, and wash-out obtains fusion rotein GST-VNP;
The fusion rotein GST-VNP that wash-out is obtained passes through Sephadex G25 gel-filtration desalination, and the enteropeptidase cutting is adopted in the back.The cutting condition: 25 ℃, pH 7.6,12 hours;
Product after enzyme is cut removes the GST label through the GST affinity chromatography again, and adopting molecular weight cut-off is that sample after 10000 ultra-filtration centrifuge tube is cut enzyme carries out purifying, removes a spot of remaining enteropeptidase, obtains pure product VNP.
Pure product VNP is adopted myocardium vessel ring strain method and removes bladder urine collecting method activity rating on rat.
2. by the preparation method of the described blood vessel sodium of claim 1 peptide (VNP), it is characterized in that: GST is an affinity tag.
3. by the preparation method of the described blood vessel sodium of claim 1 peptide (VNP), it is characterized in that: the enteropeptidase enzyme is cut.
4. by the preparation method of the described blood vessel sodium of claim 1 peptide (VNP), it is characterized in that: the VNP that biological process obtains has better activity than chemical synthesis.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104059154A (en) * | 2014-05-06 | 2014-09-24 | 中国人民解放军第四军医大学 | Novel natriuretic peptide chimera CNAAC with cardiac failure resistance |
| CN104231088A (en) * | 2014-09-24 | 2014-12-24 | 上海交通大学医学院 | Fusion protein of human vasonatrin peptide and human serum albumin and preparation thereof |
| CN110004170A (en) * | 2019-04-10 | 2019-07-12 | 潍坊医学院 | The recombinant plasmid of the gene of SMIM25 containing someone, genetic engineering bacterium, recombinant protein, more anti-and preparation method and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059154A (en) * | 2014-05-06 | 2014-09-24 | 中国人民解放军第四军医大学 | Novel natriuretic peptide chimera CNAAC with cardiac failure resistance |
| CN104231088A (en) * | 2014-09-24 | 2014-12-24 | 上海交通大学医学院 | Fusion protein of human vasonatrin peptide and human serum albumin and preparation thereof |
| CN110004170A (en) * | 2019-04-10 | 2019-07-12 | 潍坊医学院 | The recombinant plasmid of the gene of SMIM25 containing someone, genetic engineering bacterium, recombinant protein, more anti-and preparation method and application |
| CN110004170B (en) * | 2019-04-10 | 2021-05-07 | 潍坊医学院 | Recombinant plasmid containing human SMIM25 gene, gene engineering bacterium, recombinant protein, polyclonal antibody, preparation method and application |
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Application publication date: 20110223 |