CN103159847B - Natriuretic peptide, as well as gene and use thereof - Google Patents
Natriuretic peptide, as well as gene and use thereof Download PDFInfo
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- CN103159847B CN103159847B CN201310127277.6A CN201310127277A CN103159847B CN 103159847 B CN103159847 B CN 103159847B CN 201310127277 A CN201310127277 A CN 201310127277A CN 103159847 B CN103159847 B CN 103159847B
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- natriuretic peptide
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Abstract
The invention relates to a natriuretic peptide from a snake and a gene encoding the polypeptide. The natriuretic peptide disclosed by the invention is the natriuretic peptide from dendroaspis angusticeps. The natriuretic peptide disclosed by the invention is the brand new natriuretic peptide and a new member in a natriuretic peptide family, and is further called as GNP, namely G type natriuretic peptide. The invention further provides a preparation method of the recombinant natriuretic peptide. The natriuretic peptide provided by the invention comprises the recombinant natriuretic peptide and an artificially synthesized natriuretic peptide, which can be applied to preparation of medicaments for treating patients with acute heart failure, acute decompensated heart failure, acute myocardial infarction after coronary intervention, chronic heart failure, and acute anterior wall myocardial infarction with systolic heart failure of old people.
Description
Technical field
The present invention relates to a kind of natriuretic peptide, especially relate to a kind of natriuretic peptide from snake class, and the gene of this polypeptide of encoding.
Background technology
Natriuretic peptide (Natriuretic Peptide, NP) refers to that a class has compared with bioactive polypeptide such as forced-ventilated sodium, diuresis, expansion blood vessels.These polypeptide have homology in heredity and identical receptor system.
At present, the natriuretic peptide obtaining from mammal comprises: atrial natriuretic peptide, brain natriuretic peptide and c-type natriuretic peptide.Atrial natriuretic peptide is mainly secreted by atrial muscle cell, and brain natriuretic peptide is mainly secreted by ventricular muscle cell, and c-type natriuretic peptide is mainly secreted by vascular endothelial cell and at part performance vasodilation and antiproliferative effect.
The heart extracting solution of the discovery rats such as Canadian scientist de Bold in 1981 has very strong sharp sodium, diuretic properties.After 1 year, they confirm that its active principle is polypeptide.In 2-3 subsequently, the structure of first member's atrial natriuretic peptide in natriuretic peptide family is identified.Atrial natriuretic peptide (atrial natriuretic peptide, ANP) is called again " atrial natriuretic peptide " or " atrial natriuretic peptide ", is the peptide class of being synthesized and being discharged by atrial muscle cell, is called again " A type natriuretic peptide " according to its english abbreviation.ANP in human body is made up of 28 amino-acid residues.The Main Function of ANP is to make to relax the VSM and promote kidney row sodium, draining.In the time that being subject to tractive, atrial walls can stimulate atrial muscle cell to discharge ANP.ANP mainly contains following several respects to the effect of kidney: 1, the impact on glomerular filtration rate(GFR.ANP declines vascular smooth muscle cytoplasmic calcium ionic concn by second messenger cGMP, makes afferent glomerular arteriole diastole, and glomerular filtration rate(GFR increases; 2, the impact of pair set pipe.ANP closes the sodium channel on collecting tubule epithelial cell luminal membrane by cGMP, suppresses the heavily absorption of NaCl; 3, the impact on other hormones.ANP also suppresses the secretion of feritin, aldosterone and beta-hypophamine.
Japanese scientist Sudoh in 1988 etc. isolate second member---the brain natriuretic peptide natriuretic peptide family from pig brain.Brain natriuretic peptide (Brain Natriuretic Peptide, BNP) claims again " B-Type natriuretic peptide ", is called again " B-typeNatriuretic Peptide " according to its english abbreviation.In patient blood in heart failure, brain natriuretic peptide, atrial natriuretic peptide all obviously raise.In fact brain natriuretic peptide is mainly derived from ventricle.BNP has important physiopathology meaning, it can promote to arrange sodium, urinate, the vasodilator effect that tool is stronger, can resist the contracting blood vessel function of renin-angiotensin-aldosterone system (RAAS), be equally that human body is resisted the overweight and hypertensive main endocrine system of volume load with ANP.Cardiac dysfunction can greatly activate Natriuretic Peptide System Played, and the increase of ventricle load causes BNP to discharge.
C-type natriuretic peptide (C-type Natriuretic Peptide, CNP) is that Sudoh equals the 3rd the natriuretic peptide family member that nineteen ninety extracts from pig brain, is paid close attention to widely because it has compared with strong inhibition vascular remodeling and certain vasodilative effect.
Research shows, the core texture of natriuretic peptide be one by 17 amino acids formed ring texturees that connect by a pair of halfcystine disulfide linkage, this core texture for the combination of natriuretic peptide and acceptor with and biological activity be very important.
Research shows, the physiological and pharmacological mechanism of natriuretic peptide is: act on the cardiovascular system urinary system of unifying; Be combined with the guanylate cyclase receptor of vascular smooth muscle and endotheliocyte, in irritation cell, second messenger cGMP concentration raises; Promote smooth muscle cell diastole; Expansion artery and vein; Reduce systemic vascular resistance; Increase the permeability of blood vessel; Increase VC; Improve haemodynamics; Reduce SAP, right atrial pressure, pulmonary capillary wedge pressure; Before and after reducing heart, load; It is the natural agonist of renin-angiotensin-aldosterone system; The activity of antagonism endothelin, norepinephrine and aldosterone; The afferent glomerular arteriole of nephrectasia bead, contraction efferent glomerular arteriole, increase glomerular capillary resistance, increases glomerular filtration rate(GFR, promotes the transhipment of uriniferous tubules sodium and water, and sodium, diuretic properties are arranged in performance.The feature of natriuretic peptide is: there is no positive inotropic action; Forcibly do not strengthen myocardium contractility; Do not increase myocardial consumption of oxygen; There is no to expand at present the side effect for blood pressure and alteration in heart rate of blood vessel medicine and diuretic.Therefore, the potential clinical indication of natriuretic peptide comprises: after acute heart failure, acute decompensated heart failure, acute myocardial infarction intervention of coronary artery, patient, chronic heart failure, older patients with acute Anterior wall myocardial infarction merge systolic heart failure patient etc.
At present, developed rhBNP medicine both at home and abroad.These medicines are products of synthetic, have 32 identical aminoacid sequences with the natural brain natriuretic peptide of ventricular muscles secretion.
Scios company of the U.S. (now belonging to Johson & Johnson) is in list marketing in September calendar year 2001 Nesiritide (Nesiritide), trade name " Natrecor ", and it is the recombinant human brain natriuretic peptide (rhBNP) of first listing.Nesiritide has clear and definite expansion blood vessel and natriuretic diuretic effect, gets permission to be used for the treatment of acute decompensated heart failure in the U.S..Clinical study shows, Nesiritide can improve the haemodynamics of heart failure patient, improves the clinical symptom of heart failure patient.
Current Ye Youliangjia enterprise of China production and selling research and development recombinant human brain natriuretic peptide medicine, one is Tibet Nuodikang limited-liability company (Listed Company is called for short: Tibet medicine company), trade(brand)name " the new element of living ", another family is Suzhou Su Lan biological medicine Science and Technology Development Co., Ltd., trade(brand)name " Bu Luonatai ", is lyophilized injectable powder.These two kinds of medicines, all granted list marketing in 2005, are cardiovascular diseases first class national new drugs, dyspneic acute heart failure patient while being used for the treatment of rest or light activity.It is reported, " the new element of living " completes fourth phase clinical trial in April, 2010, enters by the end of May national stem " acute heart failure diagnosis and treatment guide ".
It is reported, the sickness rate of the annual heart failure of the U.S. is 0.23-0.27%, and patient's number that China is in hospital because of acute heart failure is every year no less than 2,000,000 people, and increases with 10% ratio every year, along with standard of living continues to rise and aging population, number of patients will continue to rise.It is the rescue medication of acute heart failure that restructuring human brain profit is received peptide, and wherein more than 90% patient can use this medicine.
In non-mammal, also found the natriuretic peptide of similar structures and function, for example, Schweitz equal 1992 from East Africa green mamba snake (
dendroaspis angusticeps, claim again Green Mamba) snake venom in separate and obtain D type natriuretic peptide (Dendroaspis Natriuretic Peptide, DNP); Ho equal 1997 from South America coral snake (
micrurus corallinus) snake venom in separate obtain M type natriuretic peptide (Micrurus Natriuretic Peptide, MNP), from eel heart, be separated to V-type natriuretic peptide (Ventricle Natriuretic Peptide, VNP) (Takei etc., 1991).Than ANP, BNP and CNP, the research report of other natriuretic peptides is less.
The green mamba snake in East Africa (
dendroaspis angusticeps) be the one that Elapidae mamba snake belongs to, be considered to one of the fastest snake of current creep speed, be also one of the most august poisonous snake of Veld, be called as " East Africa Death ".Green mamba snake is green from head to foot must look like green bamboo, and head and body are generally thin, can between branch, jump flexibly.The toxin of green mamba snake not as good as black mamba snake (
dendroaspis polylepis), but they have inherited the innate advantage of rapid movement equally, become a kind of snake winning victory with speed and venom.
Therefore, the present invention is devoted to find new natriuretic peptide from the snake venom glandular tissue of the green mamba snake in East Africa.
Summary of the invention
The object of this invention is to provide a kind of new natriuretic peptide, it be from East Africa green mamba snake (
dendroaspis angusticeps, claim again Green Mamba) snake venom glandular tissue in the basis of natural polypeptides on the polypeptide that obtains.
Natriuretic peptide of the present invention be one derive from the green mamba snake in East Africa (
dendroaspis angusticeps) natriuretic peptide, its aminoacid sequence is as shown in SEQ ID NO:1,125 amino acid of total length, comprising 25 amino acid whose signal peptides and 38 amino acid whose mature peptides.
The present invention is separated to the ripe natriuretic peptide obtaining and compare with the DNP mature peptide of having reported in same source, they are 38 amino acid, but homology is between the two only 36.80%.Although natriuretic peptide of the present invention is different from other known natriuretic peptides on aminoacid sequence, but there is equally the core texture of natriuretic peptide by 17 amino acids formed ring texturees that connect by a pair of halfcystine disulfide linkage, therefore, natriuretic peptide of the present invention is a brand-new natural natriuretic peptide, is the newcomer of natriuretic peptide family.The present invention is called GNP(G from Green Mamba by this brand-new natriuretic peptide), namely G type natriuretic peptide.For simplified illustration, below represent natriuretic peptide of the present invention with GNP.
According to prior art, can from aminoacid sequence disclosed in this invention, derive the gene order of coding natriuretic peptide of the present invention, therefore, within the gene that all natriuretic peptides of the present invention of can encoding are GNP all falls into protection scope of the present invention.Contriver also obtains the gene order of coding GNP from the snake venom glandular tissue cDNA library building.
Preferably, the natriuretic peptide of the present invention of encoding is the gene of GNP, and its nucleotide sequence is as shown in SEQ ID NO:2.SEQ ID NO:2 has shown the full length cDNA sequence that the coding GNP obtaining is cloned in the mRNA construction cDNA library of the snake venom glandular tissue fresh separated of contriver green mamba snake from East Africa then, wherein, 5 '-UTR(5 ' non-translational region) be 150bp, 3 '-UTR(3 ' non-translational region) be 160bp, open reading frame 125 amino acid of coding and 1 terminator, comprise 25 amino acid whose signal peptides and 38 amino acid whose mature peptides.
According to a specific embodiment of the present invention, utilize engineered method, in escherichia expression system, express and be purified into restructuring GNP polypeptide.In this embodiment, adopt containing GST(glutathione S-transferase) pGEX-6P-3 carrier, give expression to GNP amyloid protein precursor, and cracking obtains GNP mature peptide.Those skilled in the art can be according to disclosed principle and method, adopts known arbitrarily protokaryon or eukaryotic expression system to give expression to restructuring GNP polypeptide.Therefore; the recombinant expression vector of the gene that comprises coding GNP of the present invention; and adopt the transformant of this recombinant expression vector transformed host cell gained, and the restructuring natriuretic peptide obtaining from described transformant, within all falling into protection scope of the present invention.
Those skilled in the art can be according to disclosed principle and method, for the transformant that adopts arbitrarily the recombinant expression vector transformed host cell gained of the gene that comprises the GNP of coding of the present invention, can select suitable culture condition to cultivate described transformant, and reclaim the expressed restructuring natriuretic peptide GNP that recombinates by suitable extracting method.Within these methods of preparing restructuring GNP of the present invention all fall into protection scope of the present invention.
According to a particular embodiment of the invention, GNP of the present invention is applied to PC12 cell (adult rat adrenal tissue cell), RASMC cell (rat aorta smooth muscle cell) and HCD cell (people's renal cortex duct cells), and studying this polypeptide stimulates the effect of cGMP secretion.PC12 and HCD cell are all mainly expressed NPRA(natriuretic peptide receptor A), RASMC cell is mainly expressed NPRB(natriuretic peptide receptor B).According to the experimental result of cell levels provided by the present invention, can find pleasantly surprisedly, GNP all can secrete by effective stimulus cGMP in above-mentioned three kinds of cells, and namely, it is NPRA and NPRB that GNP can activate two kinds of natriuretic peptide receptors, although effect is slightly lower than ANP and CNP.Therefore, GNP of the present invention has compared with biological activitys such as forced-ventilated sodium, diuresis, expansion blood vessels.Be similar to aforesaid " Nesiritide " and " the new element of living ", natriuretic peptide provided by the invention, comprise restructuring natriuretic peptide and synthetic natriuretic peptide, all can be applicable to the medicine that patient after preparation treatment acute heart failure, acute decompensated heart failure, acute myocardial infarction intervention of coronary artery, chronic heart failure, older patients with acute Anterior wall myocardial infarction merge systolic heart failure patient.
The present invention also provides a kind of pharmaceutical composition, and it comprises natriuretic peptide of the present invention, comprises restructuring natriuretic peptide and synthetic natriuretic peptide, and pharmaceutically acceptable vehicle, carrier or thinner.
According to the further feature of pharmaceutical composition of the present invention, described pharmaceutical composition can be injection, can be also freeze-dried preparation.
The present invention for the physiological and pharmacological research of described natriuretic peptide GNP with and Application and Development established good basis.
Accompanying drawing explanation
Fig. 1 is that natriuretic peptide of the present invention is the analysis chart of the full length cDNA sequence of GNP and the aminoacid sequence of derivation.
Fig. 2 A is ANP(1 × 10
-6m) column diagram that impels cGMP to secrete in PC12 cell.
Fig. 2 B is GNP(1 × 10
-6m) column diagram that impels cGMP to secrete in PC12 cell.
Fig. 3 is the graphic representation that five kinds of natriuretic peptides impel cGMP secretion in PC12 cell.
Fig. 4 shows GNP(1 × 10
-6m) column diagram that impels cGMP to secrete in RASMC cell.
Fig. 5 has shown five kinds of graphic representations that natriuretic peptide impels cGMP to secrete in RASMC cell.
Fig. 6 A is ANP(1 × 10
-6m) column diagram that impels cGMP to secrete in HCD cell.
Fig. 6 B is GNP(1 × 10
-6m) column diagram that impels cGMP to secrete in HCD cell.
Fig. 7 is the graphic representation that five kinds of natriuretic peptides impel cGMP secretion in HCD cell.
Fig. 8 is the collection of illustrative plates of pGEX-6P-3 carrier.In figure, arrow has shown the cleavage site of front scinderin enzyme (Prescission Protease).
Fig. 9 is the SDS-PAGE gel electrophoresis figure of GNP amyloid protein precursor.In the figure, swimming lane 1 is protein molecular weight standard; Swimming lane 2 is first protein fragments of wash-out in the past scinderin enzymic digestion post; Swimming lane 3 is second protein fragments; Swimming lane 3 is the 3rd protein fragments.
Embodiment
cDNA clone and the genetic analysis of embodiment mono-: GNP
Adopt the fast m RNA extraction test kit of Invitrogen company to extract and obtain mRNA from the snake venom glandular tissue of the fresh separated of the green mamba snake in East Africa, then adopt the SMART of Clontech company
tMcDNA library builds the cDNA library of test kit structure snake venom glandular tissue.This test kit employing 5 ' and 3 ' RACE technology are carried out cDNA end quick clone.
The primer of 5 ' RACE: SEQ ID NO:3
5’-TGGTCGATCTTGTGGCCGAAGCAGCCAT-3’ 。
The primer of 3 ' RACE: SEQ ID NO:4
5’-TAGACTCGCGTCGTATGAAGGGGCTGGA-3’ 。
Cut glue and reclaim RACE product, obtain the full length cDNA sequence that a kind of new natriuretic peptide is GNP of the present invention.
The full length cDNA sequence of GNP and the aminoacid sequence of derivation can be referring to Fig. 1 and sequence tables.This cDNA total length 718bp, its nucleotide sequence is as shown in SEQ ID NO:2.Wherein, 5 '-UTR(5 ' non-translational region) be 150bp, 3 '-UTR(3 ' non-translational region) be 160bp, open reading frame 125 amino acid of coding and 1 terminator, comprise 25 amino acid whose signal peptides and 38 amino acid whose mature peptides.Natriuretic peptide of the present invention is that the aminoacid sequence (being ripe peptide sequence) of GNP is SEQ ID NO:1; its modified outcome; the product that for example partial amino-acid is removed, replaced, produce after modification or addition, within being also included within protection domain of the present invention.
For confirm to exist the cDNA of above-mentioned total length in the first chain cDNA, adopt two primers based on 5 ' end and 3 ' end design, carry out PCR take the first chain cDNA as template.
Forward primer: SEQ ID NO:5
5’-CTATAGGGCAAGCAAGCAGTGGTAAC-3’
Reverse primer: SEQ ID NO:6
5’-CTGCGGATGGGGTGTGGGGTGTCC-3’
To after PCR product cloning, check order, the full-length cDNA of results verification GNP is present in snake venom glandular tissue really.
In the genetic analysis of GNP, there is the following part that merits attention:
1. have two ATTTA structural domains at 3 '-UTR, this sequence was once found (but not existing in ANP) in the cDNA of BNP and CNP, and its function is to make mRNA unstable, and is considered to be designed for physiological response is made to rapid reaction.
2. GenBank BLAST retrieval shows, has similarity quite significantly between GNP and the signal peptide of MNP, in 25 amino acid, have 23 identical.This chances are because they from the green mamba snake in East Africa and South America coral snake all belong to poisonous snake.
The theoretical aminoacid sequence (primary structure) of deriving according to GNP gene is:
KSTPDGCFGHKLDPIGSHSGLGCPGAGPHPKPTPGAGR（38aa）。
In the analysis of protein of GNP, there is the following part that merits attention:
Although 1. GNP and DNP are 38 amino acid, and all from the green mamba snake in East Africa, homology is between the two only 36.80%.GNP and other difference of known natriuretic peptide in sequence are larger.
2. between signal peptide and mature peptide, there is a closely similar aminoacid sequence in GNP and DNP, in 26 amino acid, have 24 identical, this chances are because they from the poison gland of the green mamba snake in East Africa.
3. similar with the report of other natriuretic peptides, the core texture of GNP is also by 17 amino acids formed ring texturees that connect by a pair of halfcystine disulfide linkage.
In sum, the GNP that the present invention obtains is a brand-new natural natriuretic peptide, is the newcomer of natriuretic peptide family.
embodiment bis-: the synthetic of GNP albumen and analysis
Contriver carries out the synthetic of GNP albumen according to the analytical results of embodiment mono-, and whether the synthetic polypeptide sample of entrusting the biochemical institute in Shanghai to provide contriver carries out molecular weight analyse (mass spectroscopy), purity check (HPLC method), N end order-checking test analysis, the interior disulfide linkage of peptide molecule and exist and disulfide linkage positioning analysis.
1. molecular weight analyse
Sample state: solid.
Detection method: mass spectroscopy.
Key instrument used: mass spectrograph.
Analytical test result:
Mass spectrum molecular weight 3690.5146 3690.5146
Theoretical molecular 3689.7900 3689.79.
2. purity check
Sample state: solid.
Detect foundation: 2010 editions three appendix III B high performance liquid chromatography of Chinese Pharmacopoeia.
Key instrument used: HPLC(Agilent, 1200 types).
Analytical test result: sample is analyzed through HPLC, take peak area normalization method calculation sample main peak purity as: 97.34%(is anti-phase).
3. N end order-checking test analysis
Actual measurement sequence and theoretical sequence alignment are as follows:
Theoretical sequence:
K-S-T-P-D-G-C-F-G-H-K-L-D-P-I-G-S-H-S-G-L-G-C-P-G-A-G-P-H-P-K-P-T-P-G-A-G-R （38aa）；
Actual measurement sequence:
NH2-K-S-T-P-D-G-C-F-G-H-K-L-D-P-I-G-S-H-S-G-L-G-C-P-G-A-G-P-H-P-K-P-T-P-G-A-G-R （38aa）。
4. in peptide molecule, whether disulfide linkage exists and disulfide linkage positioning analysis
Disulfide linkage is important protein post-translational modification form, is also a kind of more special modification.Protein analysis links together by the halfcystine in various interchains and chain, and protein molecule is kept to correct higher structure, maintains necessary biological activity most important.Therefore in the biochemical analysis of protein drug, disulfide linkage is the emphasis of being concerned about always.In the present invention, Binding peptide molecule is enzymolysis and LCMSMS in solution, disulfide linkage matching method to trial-product is analyzed, the clear and definite various intrachain disulfide bond matching methods of trial-product, and the experimental evidence of definite disulfide linkage matching method is provided, comprise firsts and seconds mass-spectrometric data etc.
Trial-product state: solid.
Theoretical matching method is: Cys7 and Cys23.
Laboratory apparatus:
1) LTQ-velos，Thermofinnigan
2) MDLC，GE Healthcare
3) 4800 MALDI-TOF/TOF, AB SCIEX 。
Experimental principle: the detection to disulfide linkage in this experiment, MALDI-TOF/TOF and LCMSMS technology are combined, from molecular weight, the aspects such as firsts and seconds ion are confirmed disulfide linkage accurately and effectively.On the one hand, use AB SCIEX 4800 MALDI-TOF/ TOF to test trial-product relative molecular mass, obtain accurately and reliably protein relative molecular mass information.On the other hand, use LCMSMS mass-spectrometric technique to resolve the peptide molecule of disulfide linkage pairing, in conjunction with software and additional artificial parsing, and carried out the chromatogram mass spectrogram analysis of target disulfide linkage peptide molecule before and after reduction, pin-point accuracy has obtained the information of disulfide linkage pairing reliably.
Experimental technique
1) chymotrypsin enzymolysis and reduction.
2) MALDI-TOF/TOF point sample.
3) MALDI-TOF/TOF test sample: select linear method test molecular weight analyte under positive ion mode.
4) MALDI-TOF/TOF mass-spectrometric data and collection of illustrative plates processing
Raw data and collection of illustrative plates that 4800 MALDI-TOF/TOF produce are derived by 4000 Series Explorer V3.5 softwares.
5) capillary high performance liquid chromatography
Peptide section after enzymolysis separates by the Ettan MDLC of GE company, then tests by Thermo LTQ-Velos.
6) LCMSMS Mass Spectrometric Identification.
7) LCMSMS mass-spectrometric data processing
Peptide section mass-spectrogram is first mated by computer software checking storehouse, then by artificial coupling checking peptide section second order ms, finally determines the on-link mode (OLM) of disulfide linkage.
Experimental result and analysis
First this experiment is determined the relative molecular mass of peptide molecule and MS2 collection of illustrative plates is carried out to sequence confirmation by MALDI TOF/TOF technology.
The detected result of molecular weight is: before reduction, molecular weight is about 3688.75Da; After IAA reduction and alkylation, molecular weight is about 3804.98Da; More than the molecule measuring test result before and after reduction conforms to the theoretical molecular of this polypeptide.
Whether consistent with theoretical sequence for further determining the aminoacid sequence of this peptide molecule, by gathering second order spectrum, and MS2 collection of illustrative plates is mated to verify with theoretical sequence, matching result finds that this second order spectrum is identical with trial-product polypeptide theory sequence.
Further, by artificial coupling MS2 collection of illustrative plates, determine the on-link mode (OLM) of disulfide linkage.Find by coupling 1 pair of disulfide linkage that in trial-product, existence conforms to theoretical matching method, for: Cys7 ~ Cys23.
Because the sensitivity of modern biological mass spectrometry is very high, micro-mispairing in disulfide linkage analysis also may obtain false-positive result in MS2 appraising datum, and therefore the confirmation work of the effect of the chromatogram mass spectrum (XICs) to the definite disulfide linkage pairing of needs just seems very important.This experiment has been carried out analyzing and calculating reduction front and back signal to the XICs of disulfide linkage matching form and has been divided peak area, further the disulfide linkage of theory pairing is confirmed.
Can obtain from XICs atlas analysis, before reduction, be declined significantly after reduction by the preliminary disulfide linkage Cys7 ~ Cys23 place peptide segment molecule strength of signal confirmed of MS2 collection of illustrative plates, further illustrate disulfide linkage Cys7 ~ Cys23 and really exist, rather than false-positive result.
Conclusion
In sum, having 1 pair of disulfide linkage conforming to theory in trial-product GNP polypeptide (lot number: GNP20120817), is Cys7 ~ Cys23.
embodiment tri-: the cGMP secretion experiment on PC12 cell
Experiment material: PC12 cell (a kind of adult rat adrenal tissue cell), business is bought.
According to having been reported, in PC12 cell, NPRA(natriuretic peptide receptor A) with NPRB(natriuretic peptide receptor B) ratio be 3:1, namely, take NPRA as main.
This experimental study by the effect of stimulation to cGMP secretion on PC12 cell of 5 kinds of natriuretic peptide family members including GNP.Wherein, GNP is the sample from bis-synthetic of embodiment, and other 4 kinds of natriuretic peptides all derive from business purchase or scholar's present.
Experiment one: cultivate PC12 cell to every hole 1 × 10 on 24 well culture plates
6cell, adds respectively 1 × 10
-6the ANP of M and GNP.The nutrient solution that takes out 6 different holes at each time point carries out cGMP concentration analysis (employing immunoassay).
Fig. 2 A and Fig. 2 B show respectively ANP(1 × 10
-6m) with GNP(1 × 10
-6m) dynamic process that impels cGMP to secrete in PC12 cell.Through more visible, 2-4 hour after administration, cGMP concentration all reaches peak value, although the peak value in the peakedness ratio ANP experiment in GNP experiment is low, is reaching after peak value, and the drug effect of GNP experiment is subdued than slower (still maintaining high peaks after 8 hours) of ANP experiment.
Experiment two: cultivate PC12 cell to every hole 1 × 10 on 24 well culture plates
6cell, adds respectively ANP, BNP, CNP, DNP and the GNP of each concentration, and after within 2 hours, cultivating, the nutrient solution taking out in hole carries out cGMP concentration analysis (employing immunoassay).
Fig. 3 has shown five kinds of curves that natriuretic peptide impels cGMP to secrete in PC12 cell.As shown in the figure, in the time of lower concentration, the effect of stimulation between various natriuretic peptides is more or less the same; Reach 1 × 10 in natriuretic peptide concentration
-9when M is above, short cGMP secretion effect separately starts differentiation, and the stimulation ability of GNP is swum between two parties, though lower than ANP, BNP and DNP, be significantly higher than CNP.
The above-mentioned of the present embodiment experiment showed, that GNP can secrete with stimulation cGMP in NPRA in useful effect.
embodiment tetra-: the cGMP secretion experiment on RASMC cell
Experiment material: RASMC cell (a kind of rat aorta smooth muscle cell), business is bought.
According to having been reported, in RASMC cell, the ratio of NPRA and NPRB is 1:3, namely, and take NPRB as main.
This experimental study by the effect of stimulation to cGMP secretion on RASMC cell of 5 kinds of natriuretic peptide family members including GNP.
Experiment one: cultivate RASMC cell to every hole 1 × 10 on 24 well culture plates
6cell, adds respectively 1 × 10
-6the ANP of M and GNP.The nutrient solution that takes out 6 different holes at each time point carries out cGMP concentration analysis (employing immunoassay).
Fig. 4 shows GNP(1 × 10
-6m) dynamic process that impels cGMP to secrete in RASMC cell.As seen from the figure, 2-4 hour after administration, cGMP concentration reaches peak value, still maintains high peaks after 8 hours, and drug effect is subdued slower.Because ANP only acts on NPRA, thus in RASMC cell experiment to no effect.
Experiment two: cultivate RASMC cell to every hole 1 × 10 on 24 well culture plates
6cell, adds respectively ANP, BNP, CNP, DNP and the GNP of each concentration, and after within 2 hours, cultivating, the nutrient solution taking out in hole carries out cGMP concentration analysis (employing immunoassay).
Fig. 5 has shown five kinds of curves that natriuretic peptide impels cGMP to secrete in RASMC cell.As shown in the figure, in the time of lower concentration, the effect of stimulation between various natriuretic peptides is more or less the same; Reach 1 × 10 in natriuretic peptide concentration
-8when M is above, short cGMP secretion effect separately starts differentiation, and the stimulation ability of GNP is swum between two parties, though lower than CNP, be significantly higher than ANP, BNP and DNP.
The above-mentioned of the present embodiment experiment showed, that GNP can secrete with stimulation cGMP in NPRB in useful effect.
embodiment five: the cGMP secretion experiment on HCD cell
Experiment material: HCD cell (a kind of people's renal cortex duct cells), business is bought.
According to having been reported, in HCD cell, NPRA(natriuretic peptide receptor A) with NPRB(natriuretic peptide receptor B) ratio be 3:1, namely, take NPRA as main.
This experimental study by the effect of stimulation to cGMP secretion on PC12 cell of 5 kinds of natriuretic peptide family members including GNP.
Experiment one: cultivate HCD cell to every hole 1 × 10 on 24 well culture plates
6cell, adds respectively 1 × 10
-6the ANP of M and GNP.The nutrient solution that takes out 6 different holes at each time point carries out cGMP concentration analysis (employing immunoassay).
Fig. 6 A and Fig. 6 B show respectively ANP(1 × 10
-6m) with GNP(1 × 10
-6m) dynamic process that impels cGMP to secrete in HCD cell.Through more visible, 2-4 hour after administration, cGMP concentration all reaches peak value, although the peak value in the peakedness ratio ANP experiment in GNP experiment is low, is reaching after peak value, and the drug effect of GNP experiment is subdued than slower (still maintaining high peaks after 8 hours) of ANP experiment.
Experiment two: cultivate HCD cell to every hole 1 × 10 on 24 well culture plates
6cell, adds respectively ANP, BNP, CNP, DNP and the GNP of each concentration, and after within 2 hours, cultivating, the nutrient solution taking out in hole carries out cGMP concentration analysis (employing immunoassay).
Fig. 7 has shown five kinds of curves that natriuretic peptide impels cGMP to secrete in HCD cell.As shown in the figure, in the time of lower concentration, the effect of stimulation between various natriuretic peptides is more or less the same; Reach 1 × 10 in natriuretic peptide concentration
-9when M is above, short cGMP secretion effect separately starts differentiation, and the stimulation ability of GNP is swum between two parties, though lower than ANP, BNP and DNP, be significantly higher than CNP(and almost do not have effect).
The above-mentioned of the present embodiment experiment showed, that GNP can secrete with stimulation cGMP in NPRA in useful effect.
The interpretation of result of comprehensive embodiment bis-to four, in the dominant cell of NPRA, the effect of stimulation of ANP, BNP, DNP is more obvious, and in the dominant cell of NPRB, the effect of stimulation of CNP is more obvious, and this illustrates that known natriuretic peptide family member biases toward a certain acceptor.Can find, to the action effect of two kinds of acceptor NPRA and NPRB all clearly, this shows that new GNP is a kind of natriuretic peptide can be simultaneously playing a role in conjunction with two kinds of acceptor NPRA and NPRB to GNP of the present invention pleasantly surprisedly.
embodiment six: the preparation of restructuring GNP
The present embodiment utilizes engineered method, gives expression to restructuring GNP polypeptide in escherichia expression system.
Adopt intestinal bacteria (
e. coli) in the plasmid of efficient expressed fusion protein, containing GST(glutathione S-transferase) pGEX-6P-3 carrier, this carrier is commercial plasmid, its collection of illustrative plates is referring to Fig. 8.The fusion rotein that the albumen of this vector expression is made up of 26 kDa GST and target protein.Encoded between GST structural domain and the multiple clone site recognition site of PreScission proteolytic enzyme of this carrier, has a front scinderin enzyme (Prescission Protease) recognition site, for from fusion rotein specificity cutting target protein.
Utilize PCR method to add BamH I and Not I restriction enzyme site at the two ends of GNP amyloid protein precursor (preproGNP) gene, therefore will insert pGEX-6P-3 carrier.By GNP amyloid protein precursor gene and GST after pGEX-6P-3 carrier endomixis, high-level abduction delivering in e. coli bl21 cell.Inductor is IPTG, also can induce by salt, can realize like this great expression cheaply.
Then, utilize glutathione agarose (Glutathione Sepharose) filler from Bacillus coli cells lysate, to be purified into the fusion rotein of GST-GNP by affinity chromatography, its molecular weight is 37.80 kDa, carry out single step digestion in post by front scinderin enzyme again, wash-out obtains GNP amyloid protein precursor, and its molecular weight is 11.80 kDa.Fig. 9 has shown the SDS-PAGE gel electrophoresis result (gel adopts SimplyBlue SafeStain dyeing) of GNP amyloid protein precursor.By the effect of bacteria protease, can make GNP amyloid protein precursor generation protein cleavage, further obtain GNP mature peptide.
The present invention also can adopt other prokaryotic expression systems to express and purifying, for example, can adopt the expression plasmid with Poly-His label, and they are widely used in the recombinant protein that obtains purifying; Also optionally reach carrier by following table: pET, pUCH33 etc., or other carriers of selling of commercialization; Select following prokaryotic expression bacterial strain: e. coli bl21, e. coli jm109 etc., or other host cells of conduct or commercialization sale.
The present invention also can adopt other eukaryotic expression systems to express and purifying, for example, select with type carrier: PAO815, PPIC3K, PPICZ, PHWO10, PGAPZ in lower eyelid, or select following excretion vector: PPIC9K, PPICZ α, PGAPZ α, or other carriers of commercialization sale; Select following eukaryotic expression bacterial strain or cell: Pichia yeast KM71, MC100-3, SMD1168, SMD1165, SMD1163 etc., or other host cells of conduct or commercialization sale.
embodiment seven: pharmaceutical preparation
The present invention can be using restructuring GNP prepared embodiment six as activeconstituents, or using synthetic GNP prepared embodiment bis-as activeconstituents, allocate in pharmaceutically acceptable vehicle, carrier or thinner, be adjusted to filling concentration, filtration sterilization, depyrogenation, filling, make injection, further can be made into lyophilized injectable powder.Relevant technological can be with reference to the pharmacy common process of this area.
With reference to domestic and international research and application for recombinant human brain natriuretic peptide medicine at present, the restructuring GNP medicine that the present invention is prepared and synthetic GNP medicine, after clinical trial, also can be used for the medicine that patient after preparation treatment acute heart failure, acute decompensated heart failure, acute myocardial infarction intervention of coronary artery, chronic heart failure, older patients with acute Anterior wall myocardial infarction merge systolic heart failure patient.
Sequence table (SEQUENCE LISTING)
<110> leaf is bright; Chen Guangming; Wang Hongmin
<120> natriuretic peptide and gene and purposes
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 125
<212> PRT
<213> Dendroaspis angusticeps
<400> 2
Met Val Gly Leu Ser Arg Leu Ser Gly Val Gly Leu Leu Leu Val Leu
1 5 10 15
Ala Leu Leu Pro Leu Ala Leu Asp Gly Lys Ser Pro Pro Gln Ala Leu
20 25 30
His Lys Pro Pro Pro Ala Leu Ser Ala Pro Ser Arg Leu Met Gly Ala
35 40 45
Leu Arg Pro Asp Ser Lys Gln Ser Arg Ala Ser Trp Asp Arg Met Val
50 55 60
His Pro Glu Pro His Val Gly Gly Gly Gly Thr Gly Val Asp Ser Arg
65 70 75 80
Arg Met Lys Gly Leu Asp Lys Lys Ser Thr Pro Asp Gly Cys Phe Gly
85 90 95
His Lys Leu Asp Pro Ile Gly Ser His Ser Gly Leu Gly Cys Pro Gly
100 105 110
Ala Gly Pro His Pro Lys Pro Thr Pro Gly Ala Gly Arg
115 120 125
<210> 2
<211> 718
<212> DNA
<213> Dendroaspis angusticeps
<400> 1
gaggcaaacg agcgtagctc gacgagagac gctcctgcag cgacagcacc cggctcggct 60
ctcttcggct cagctggtcc gcaccctcga ggattcatct ctctctctct ctctctctct 120
tccacctgca cttccgcagc ccggggaag atg gtc ggc ctc tcc cgt ctg tcg 173
Met Val Gly Leu Ser Arg Leu Ser
1 5
ggc gtc ggg ctg ctg ctg gtg ctg gcc ctg ctg cct ctc gcc ctc gat 221
Gly Val Gly Leu Leu Leu Val Leu Ala Leu Leu Pro Leu Ala Leu Asp
10 15 20
ggg aag tcg ccg cct cag gcg ctg cac aag cct ccg ccg gct ctc tca 269
Gly Lys Ser Pro Pro Gln Ala Leu His Lys Pro Pro Pro Ala Leu Ser
25 30 35 40
gcg ccg tca cgg ctc atg ggg gct ttg cgc ccc gac agc aag cag tca 317
Ala Pro Ser Arg Leu Met Gly Ala Leu Arg Pro Asp Ser Lys Gln Ser
45 50 55
cgg gcc tcc tgg gac cgg atg gtg cac cct gag ccc cat gta gga ggc 365
Arg Ala Ser Trp Asp Arg Met Val His Pro Glu Pro His Val Gly Gly
60 65 70
ggc ggc act ggg gta gac tcg cgt cgt atg aag ggg ctg gac aag aaa 413
Gly Gly Thr Gly Val Asp Ser Arg Arg Met Lys Gly Leu Asp Lys Lys
75 80 85
agc acg ccc gat ggc tgc ttc ggc cac aag ctc gac ccc atc ggc agt 461
Ser Thr Pro Asp Gly Cys Phe Gly His Lys Leu Asp Pro Ile Gly Ser
90 95 100
cat agc ggc ttg ggc tgc ccg ggc gcc ggg ccc cac ccg aag cca aca 509
His Ser Gly Leu Gly Cys Pro Gly Ala Gly Pro His Pro Lys Pro Thr
105 110 115 120
cct ggt gca ggt cgt tga agggacac cccacacccc atccgcagac atttctggac 565
Pro Gly Ala Gly Arg *
125
atcccctgca gtcacccggg gcccccactg gcctgacccc caagggccac ccacagctgt 625
tggaacgagg gtatatattg tttataaaga gatatttata aaaaatacta ataattttat 685
ttatggaaac gaaaaaaaaa aaaaaaaaaa aaa 718
<210> 3
<211> 28
<212> DNA
<213> artificial sequence
<400> 3
tggtcgatct tgtggccgaa gcagccat 28
<210> 4
<211> 28
<212> DNA
<213> artificial sequence
<400> 4
tagactcgcg tcgtatgaag gggctgga 28
<210> 5
<211> 26
<212> DNA
<213> synthetic
<400> 5
ctatagggca agcaagcagt ggtaac 26
<210> 6
<211> 24
<212> DNA
<213> synthetic
<400> 6
ctgcggatgg ggtgtggggt gtcc 24
Claims (10)
- One kind derive from the green mamba snake in East Africa ( dendroaspis angusticeps) natriuretic peptide, its aminoacid sequence is as shown in SEQ ID NO:1.
- 2. the gene of coding natriuretic peptide as claimed in claim 1.
- 3. gene according to claim 2, its nucleotide sequence is as shown in SEQ ID NO:2.
- 4. comprise the recombinant expression vector of gene as claimed in claim 3.
- 5. by the transformant of recombinant expression vector transformed host cell gained as claimed in claim 4.
- 6. the restructuring natriuretic peptide obtaining from transformant as claimed in claim 5.
- 7. the method for preparation restructuring natriuretic peptide as claimed in claim 6, is characterized in that, comprises the following steps: cultivate and comprise transformant as claimed in claim 5, reclaim expressed restructuring natriuretic peptide.
- 8. natriuretic peptide as claimed in claim 1 patient, chronic heart failure, older patients with acute Anterior wall myocardial infarction after preparation treatment acute heart failure, acute decompensated heart failure, acute myocardial infarction intervention of coronary artery merges the purposes of systolic heart failure patient's medicine.
- 9. purposes according to claim 8, is characterized in that: described natriuretic peptide is restructuring natriuretic peptide or synthetic natriuretic peptide.
- 10. a pharmaceutical composition, is characterized in that: comprise natriuretic peptide according to claim 1 or restructuring natriuretic peptide according to claim 6, and pharmaceutically acceptable vehicle, carrier or thinner; Described pharmaceutical composition is injection or freeze-dried preparation.
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| CN201310127277.6A CN103159847B (en) | 2013-04-12 | 2013-04-12 | Natriuretic peptide, as well as gene and use thereof |
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| CN103159847B true CN103159847B (en) | 2014-05-28 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012019237A1 (en) | 2010-08-12 | 2012-02-16 | Madeleine Pharmaceuticals Pty Ltd | Therapeutic method for treating congestive heart failure |
| CN109172808A (en) * | 2018-11-02 | 2019-01-11 | 广州雷恩康亚生物医药科技有限公司 | Natriuretic peptide GNP is used to prepare the purposes of the drug for the treatment of pulmonary hypertension correlation indication or heart failure merging pulmonary hypertension |
| CN112710852B (en) * | 2021-03-26 | 2021-08-03 | 上海美迪西生物医药股份有限公司 | GNP polypeptide detection kit and detection method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020082219A1 (en) * | 1999-12-17 | 2002-06-27 | John C. Burnett, Jr. | Chimeric natriuretic peptides |
| CN102481330A (en) * | 2009-05-20 | 2012-05-30 | 生物马林药物股份有限公司 | C-type natriuretic peptide variants |
-
2013
- 2013-04-12 CN CN201310127277.6A patent/CN103159847B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020082219A1 (en) * | 1999-12-17 | 2002-06-27 | John C. Burnett, Jr. | Chimeric natriuretic peptides |
| CN102481330A (en) * | 2009-05-20 | 2012-05-30 | 生物马林药物股份有限公司 | C-type natriuretic peptide variants |
Non-Patent Citations (1)
| Title |
|---|
| Hugues Schweitz.A New Member of the Natriuretic Peptide Family Is Present in the Venom of the Green Mamba (Dendroaspis angusticeps）.《THE JOURNAL BIOLOGICAL CHEMISTRY》.1992,第267卷(第20期),13928-13932. * |
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