CN103159847A - 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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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 the snake class, and the gene of this polypeptide of encoding.
Background technology
Natriuretic peptide (Natriuretic Peptide, NP) refers to that a class has than 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 that obtains from mammal comprises: atrial natriuretic peptide, brain natriuretic peptide and c-type natriuretic peptide.Atrial natriuretic peptide is mainly by the atrial muscle cell secretion, and brain natriuretic peptide is mainly by the ventricular muscle cell secretion, and c-type natriuretic peptide is mainly by the vascular endothelial cell secretion 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 comprised of 28 amino-acid residues.The Main Function of ANP is to make to relax the VSM and promote kidney row sodium, draining.When being subjected to tractive, atrial walls can stimulate atrial muscle cell to discharge ANP.ANP mainly contains following several respects to the effect of kidney: 1, on the impact of glomerular filtration rate(GFR.ANP descends vascular smooth muscle cytoplasmic calcium ionic concn by second messenger cGMP, makes the 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, on the impact of other hormones.ANP also suppresses the secretion of feritin, aldosterone and beta-hypophamine.
Japanese scientist Sudoh in 1988 etc. isolate second member---brain natriuretic peptide natriuretic peptide family from the 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 the patient blood of 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 the pig brain, because it has stronger inhibition vascular remodeling and certain vasodilative effect is paid close attention to widely.
Studies show that, 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.
Studies show that, 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 the 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; Load before and after reducing heart; 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 the glomerular capillary resistance, increase glomerular filtration rate(GFR, promote the transhipment of uriniferous tubules sodium and water, and sodium, diuretic properties are arranged in performance.The characteristics of natriuretic peptide are: there is no positive inotropic action; Forcibly do not strengthen the contractility of cardiac muscle; Do not increase myocardial consumption of oxygen; There is no to expand at present blood vessel medicine and diuretic for the side effect of blood pressure and alteration in heart rate.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 the 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.
U.S. Scios company (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.
China also has two enterprise's production and selling research and development recombinant human brain natriuretic peptide medicines at present, one is that (Listed Company is called for short: the Tibet medicine company) in Tibet Nuodikang limited-liability company, trade(brand)name " the new element of living ", another family is Suzhou Su Lan biological medicine scientific and technological development company limited, trade(brand)name " Bu Luonatai " is lyophilized injectable powder.These two kinds of medicines all granted list marketing in 2005, are the cardiovascular diseases first class national new drugs, dyspneic acute heart failure patient when 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 every year is no less than 2,000,000 people, and every year is with 10% ratio increase, 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 the patient more than 90% 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 to obtain D type natriuretic peptide (Dendroaspis Natriuretic Peptide, DNP); Ho equal 1997 from the South America coral snake (
Micrurus corallinus) snake venom in separate and to obtain M type natriuretic peptide (Micrurus Natriuretic Peptide, MNP), be separated to V-type natriuretic peptide (Ventricle Natriuretic Peptide, VNP) (Takei etc., 1991) from the eel heart.Than ANP, BNP and CNP, the research of other natriuretic peptides report is less.
The green mamba snake in East Africa (
Dendroaspis angusticeps) be Elapidae mamba snake belong to a kind of, be considered to one of the fastest snake of present creep speed, be also one of the most august poisonous snake of African grassland, 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 jump between branch flexibly.The too late black mamba snake of the toxin of green mamba snake (
Dendroaspis polylepis), but they have inherited the innate advantage of rapid movement equally, become a kind of snake that wins victory with speed and venom.
Therefore, the present invention is devoted to seek new natriuretic peptide from the snake venom glandular tissue of the green mamba snake in East Africa.
Summary of the invention
The purpose 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) the snake venom glandular tissue in the basis of natural polypeptides on the polypeptide that obtains.
Natriuretic peptide of the present invention be a kind of 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 are comprising 25 amino acid whose signal peptides and 38 amino acid whose mature peptides.
The present invention is separated the ripe natriuretic peptide that obtains and compare with the DNP mature peptide of having reported in same source, they are 38 amino acid, but homology between the two is only 36.80%.Although natriuretic peptide of the present invention is different from other known natriuretic peptides on aminoacid sequence, but has equally the core texture of natriuretic peptide namely 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 will this brand-new natriuretic peptide be called GNP(G from Green Mamba), G type natriuretic peptide namely.Be simplified illustration, below represent natriuretic peptide of the present invention with GNP.
According to prior art, can derive the gene order of coding natriuretic peptide of the present invention from aminoacid sequence disclosed in this invention, therefore, all natriuretic peptides of the present invention of can encoding are within the gene of GNP all falls into protection scope of the present invention.The contriver also obtains the gene order of coding GNP from the snake venom glandular tissue cDNA library that builds.
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 that obtains 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, express and be purified into restructuring GNP polypeptide in escherichia expression system.In this embodiment, adopt to contain the GST(glutathione S-transferase) the pGEX-6P-3 carrier, give expression to the GNP amyloid protein precursor, and cracking obtains the GNP mature peptide.Those skilled in the art can according to disclosed principle and method, adopt known arbitrarily protokaryon or eukaryotic expression system to give expression to restructuring GNP polypeptide.Therefore; the recombinant expression vector that comprises the gene of coding GNP of the present invention; and the transformant that adopts this recombinant expression vector transformed host cell gained, and the restructuring natriuretic peptide that obtains 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, transformant for the recombinant expression vector transformed host cell gained that adopts arbitrarily the gene that comprises coding GNP of the present invention, can select suitable culture condition to cultivate described transformant, and reclaim the expressed restructuring natriuretic peptide GNP that namely recombinates by suitable extracting method.Within the method for these preparations restructuring of the present invention GNP all falls 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), studies the effect that this polypeptide stimulates the cGMP secretion.PC12 and HCD cell are all mainly expressed NPRA(natriuretic peptide receptor A), and the RASMC cells 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, but GNP all effective stimulus cGMP secretions in above-mentioned three kinds of cells, 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 than 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 prepare the medicine that patient after 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 the 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.
Description of drawings
Fig. 1 is that natriuretic peptide of the present invention is the analysis chart of the aminoacid sequence of the full length cDNA sequence of GNP and derivation.
Fig. 2 A is ANP(1 * 10
-6M) impel the column diagram of cGMP secretion in the PC12 cell.
Fig. 2 B is GNP(1 * 10
-6M) impel the column diagram of cGMP secretion in the PC12 cell.
Fig. 3 is five kinds of natriuretic peptides impel the cGMP secretion in the PC12 cell graphic representation.
Fig. 4 shows GNP(1 * 10
-6M) impel the column diagram of cGMP secretion in the RASMC cell.
Fig. 5 has shown five kinds of graphic representations that natriuretic peptide impels cGMP to secrete in the RASMC cell.
Fig. 6 A is ANP(1 * 10
-6M) impel the column diagram of cGMP secretion in the HCD cell.
Fig. 6 B is GNP(1 * 10
-6M) impel the column diagram of cGMP secretion in the HCD cell.
Fig. 7 is five kinds of natriuretic peptides impel the cGMP secretion in the HCD cell graphic representation.
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
Embodiment one: cDNA clone and the genetic analysis of GNP
The fast m RNA extraction test kit of employing Invitrogen company extracts from the snake venom glandular tissue of the fresh separated of the green mamba snake in East Africa and obtains mRNA, then adopts the SMART of Clontech company
TMCDNA library builds the cDNA library that test kit builds the snake venom glandular tissue.This test kit adopts 5 ' and 3 ' RACE technology to carry 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 the RACE product, the full length cDNA sequence that to obtain a kind of new natriuretic peptide be 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 is 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 check order after the PCR product cloning, the full-length cDNA of results verification GNP is present in the snake venom glandular tissue really.
The following part that merits attention is arranged in the genetic analysis of GNP:
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 was considered to be designed for physiological response is made rapid reaction.
2. GenBank BLAST retrieval shows, similarity is quite significantly arranged between the signal peptide of GNP and MNP, have in 25 amino acid 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 the GNP gene is:
KSTPDGCFGHKLDPIGSHSGLGCPGAGPHPKPTPGAGR（38aa）。
The following part that merits attention is arranged in the analysis of protein of GNP:
1. although GNP and DNP are 38 amino acid, and all from the green mamba snake in East Africa, homology between the two is only 36.80%.GNP and other difference of known natriuretic peptide on sequence are larger.
2. between signal peptide and mature peptide, there are a closely similar aminoacid sequence in GNP and DNP, have in 26 amino acid 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 resulting GNP of the present invention is a brand-new natural natriuretic peptide, is the newcomer of natriuretic peptide family.
Embodiment two: the synthetic of GNP albumen and analysis
The contriver carries out the synthetic of GNP albumen according to the analytical results of embodiment one, and whether the synthetic polypeptide sample of entrusting the biochemical institute in Shanghai that the contriver is provided 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 the disulfide linkage positioning analysis.
1. molecular weight analyse
Sample state: solid.
Detection method: mass spectroscopy.
Key instrument used: mass spectrograph.
The 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).
The 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 holds the 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）；
The 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 the 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 correct higher structure, keeps 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, the 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: to the detection of disulfide linkage, MALDI-TOF/TOF and LCMSMS technology are combined in this experiment, from molecular weight, the aspects such as firsts and seconds ion are proved conclusively disulfide linkage accurately and effectively.On the one hand, use AB SCIEX 4800 MALDI-TOF/ TOF that the trial-product relative molecular mass is tested, obtain accurately and reliably protein relative molecular mass information.On the other hand, use the LCMSMS mass-spectrometric technique that the peptide molecule of disulfide linkage pairing is resolved, 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 the 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 specimen: select linear method specimen molecular weight under positive ion mode.
4) MALDI-TOF/TOF mass-spectrometric data and collection of illustrative plates are processed
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 is separated by the Ettan MDLC of GE company, then tests by Thermo LTQ-Velos.
6) LCMSMS Mass Spectrometric Identification.
7) the LCMSMS mass-spectrometric data is processed
Peptide section mass-spectrogram is first mated by the computer software checking storehouse, then by artificial coupling checking peptide section second order ms, determines at last the on-link mode (OLM) of disulfide linkage.
Experimental result and analysis
At first this experiment is determined the relative molecular mass of peptide molecule and the MS2 collection of illustrative plates is carried out the sequence conclusive evidence 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 the reduction conforms to the theoretical molecular of this polypeptide.
Whether consistent with theoretical sequence for the aminoacid sequence of further determining this peptide molecule, by gathering second order spectrum, and MS2 collection of illustrative plates and theoretical sequence are mated to verify, 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 to exist in trial-product by coupling the 1 pair of disulfide linkage that conforms to theoretical matching method, for: Cys7 ~ Cys23.
Because the sensitivity of modern biological mass spectrometry is very high, micro-mispairing in the disulfide linkage analysis also may obtain false-positive result in the MS2 appraising datum, the confirmation work of the effect of the chromatogram mass spectrum (XICs) that the disulfide linkage of therefore needs being determined matches just seems very important.Before and after this experiment has carried out analyzing and calculating reduction to the XICs of disulfide linkage matching form, signal divides peak area, further the disulfide linkage of theory pairing is proved conclusively.
Can get from the XICs atlas analysis, be descended before reduction after reduction by the preliminary disulfide linkage Cys7 that confirms of MS2 collection of illustrative plates ~ Cys23 place peptide segment molecule strength of signal remarkable, further illustrate disulfide linkage Cys7 ~ Cys23 and really exist, rather than false-positive result.
Conclusion
In sum, trial-product GNP polypeptide (lot number: have 1 pair of disulfide linkage that conforms to theory GNP20120817), be Cys7 ~ Cys23.
Embodiment three: the cGMP secretion experiment on the PC12 cell
Experiment material: PC12 cell (a kind of adult rat adrenal tissue cell), business is bought.
According to having been reported, in the 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 5 kinds of natriuretic peptide family members that comprise GNP effect of stimulation to cGMP secretion on the PC12 cell.Wherein, GNP is the sample from two synthetic of embodiment, and other 4 kinds of natriuretic peptides all derive from business purchase or scholar's present.
Experiment one: cultivate the 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) impel the dynamic process of cGMP secretion in the PC12 cell.Through as seen, after administration 2-4 hour, cGMP concentration all reached peak value, although the peak value in the peakedness ratio ANP experiment in the GNP experiment is low, after reaching peak value, the drug effect of GNP experiment is subdued than ANP experiment slower (still keeping high peaks after 8 hours).
Experiment two: cultivate the 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 cultivating in 2 hours, the nutrient solution that takes out in the hole carries out cGMP concentration analysis (employing immunoassay).
Fig. 3 has shown five kinds of curves that natriuretic peptide impels cGMP to secrete in the PC12 cell.As shown in the figure, when 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 was above, short cGMP secretion effect separately began differentiation, and the stimulation ability of GNP is swum between two parties, though lower than ANP, BNP and DNP, be significantly higher than CNP.
But the present embodiment above-mentioned experiment showed, the GNP useful effect in NPRA to stimulate the cGMP secretion.
Embodiment four: the cGMP secretion experiment on the RASMC cell
Experiment material: RASMC cell (a kind of rat aorta smooth muscle cell), business is bought.
According to having been reported, in the RASMC cell, the ratio of NPRA and NPRB is 1:3, namely, and take NPRB as main.
This experimental study by 5 kinds of natriuretic peptide family members that comprise GNP effect of stimulation to cGMP secretion on the RASMC cell.
Experiment one: cultivate the 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) impel the dynamic process of cGMP secretion in the RASMC cell.As seen from the figure, after administration 2-4 hour, cGMP concentration reached peak value, still keeps high peaks after 8 hours, and drug effect is subdued slower.Because ANP only acts on NPRA, thus in the RASMC cell experiment to no effect.
Experiment two: cultivate the 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 cultivating in 2 hours, the nutrient solution that takes out in the hole carries out cGMP concentration analysis (employing immunoassay).
Fig. 5 has shown five kinds of curves that natriuretic peptide impels cGMP to secrete in the RASMC cell.As shown in the figure, when 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 was above, short cGMP secretion effect separately began differentiation, and the stimulation ability of GNP is swum between two parties, though lower than CNP, be significantly higher than ANP, BNP and DNP.
But the present embodiment above-mentioned experiment showed, the GNP useful effect in NPRB to stimulate the cGMP secretion.
Embodiment five: the cGMP secretion experiment on the HCD cell
Experiment material: HCD cell (a kind of people's renal cortex duct cells), business is bought.
According to having been reported, in the 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 5 kinds of natriuretic peptide family members that comprise GNP effect of stimulation to cGMP secretion on the PC12 cell.
Experiment one: cultivate the 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) impel the dynamic process of cGMP secretion in the HCD cell.Through as seen, after administration 2-4 hour, cGMP concentration all reached peak value, although the peak value in the peakedness ratio ANP experiment in the GNP experiment is low, after reaching peak value, the drug effect of GNP experiment is subdued than ANP experiment slower (still keeping high peaks after 8 hours).
Experiment two: cultivate the 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 cultivating in 2 hours, the nutrient solution that takes out in the hole carries out cGMP concentration analysis (employing immunoassay).
Fig. 7 has shown five kinds of curves that natriuretic peptide impels cGMP to secrete in the HCD cell.As shown in the figure, when 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 was above, short cGMP secretion effect separately began 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).
But the present embodiment above-mentioned experiment showed, the GNP useful effect in NPRA to stimulate the cGMP secretion.
The interpretation of result of comprehensive embodiment two 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 the known natriuretic peptide family member of this explanation biases toward a certain acceptor.Can find pleasantly surprisedly, GNP of the present invention 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 that can be simultaneously plays a role in conjunction with two kinds of acceptor NPRA and NPRB.
Embodiment six: the preparation of restructuring GNP
The present embodiment utilizes engineered method, gives expression to restructuring GNP polypeptide in escherichia expression system.
Employing intestinal bacteria (
E. coli) in the plasmid of efficient expressed fusion protein, namely contain the GST(glutathione S-transferase) the 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 comprised 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, is used 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 the pGEX-6P-3 carrier.With GNP amyloid protein precursor gene and GST after pGEX-6P-3 carrier endomixis, high-level abduction delivering in the e. coli bl21 cell.Inductor is IPTG, also can induce by salt, can realize great expression cheaply like this.
Then, utilize glutathione agarose (Glutathione Sepharose) filler to be purified into the fusion rotein of GST-GNP by affinity chromatography from the Bacillus coli cells lysate, its molecular weight is 37.80 kDa, carry out single step digestion in post by front scinderin enzyme again, wash-out obtains the 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 the 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 the Poly-His label, and they are widely used in the recombinant protein that obtains purifying; Also optionally reach carrier with following table: pET, pUCH33 etc., perhaps 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, perhaps select following excretion vector: PPIC9K, PPICZ α, PGAPZ α, perhaps 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 embodiment six is prepared restructuring GNP as activeconstituents, perhaps the synthetic GNP that embodiment two is prepared is as activeconstituents, allocate in pharmaceutically acceptable vehicle, carrier or thinner, be adjusted to can concentration, filtration sterilization, depyrogenation, can, 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 the 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 preparing the medicine that patient after 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 the systolic heart failure patient.
Sequence table (SEQUENCE LISTING)
＜110〉Ye Liang; Chen Guangming; Wang Hongmin
＜120〉a kind of natriuretic peptide and gene thereof and purposes
<130>
<160> 6
<170> PatentIn version 3.5
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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 encode gene of 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. the recombinant expression vector that comprises gene as claimed in claim 3.
- 5. with the transformant of recombinant expression vector transformed host cell gained as claimed in claim 4.
- 6. the restructuring natriuretic peptide that obtains from transformant as claimed in claim 5.
- 7. prepare the method for restructuring natriuretic peptide as claimed in claim 6, it is characterized in that, comprise the following steps: cultivation comprises transformant as claimed in claim 5, reclaims 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. 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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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9018168B2 (en) | 2010-08-12 | 2015-04-28 | 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 |
| CN112710852A (en) * | 2021-03-26 | 2021-04-27 | 上海美迪西生物医药股份有限公司 | GNP polypeptide detection kit and detection method |
| WO2024222771A1 (en) * | 2023-04-28 | 2024-10-31 | 成都奥达生物科技有限公司 | Long-acting natriuretic peptide compound |
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 variant |
-
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 variant |
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》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9018168B2 (en) | 2010-08-12 | 2015-04-28 | 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 |
| CN112710852A (en) * | 2021-03-26 | 2021-04-27 | 上海美迪西生物医药股份有限公司 | GNP polypeptide detection kit and detection method |
| 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 |
| WO2024222771A1 (en) * | 2023-04-28 | 2024-10-31 | 成都奥达生物科技有限公司 | Long-acting natriuretic peptide compound |
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|---|---|
| CN103159847B (en) | 2014-05-28 |
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