CN100386341C - Polypeptide of extracellular domain of sodium pump and use in preparation of drugs for adjusting the activity of sodium pump - Google Patents
Polypeptide of extracellular domain of sodium pump and use in preparation of drugs for adjusting the activity of sodium pump Download PDFInfo
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- CN100386341C CN100386341C CNB2006100427411A CN200610042741A CN100386341C CN 100386341 C CN100386341 C CN 100386341C CN B2006100427411 A CNB2006100427411 A CN B2006100427411A CN 200610042741 A CN200610042741 A CN 200610042741A CN 100386341 C CN100386341 C CN 100386341C
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Abstract
The present invention discloses a polypeptide in a sodium pump alpha-subunit film outer region, and applications of the polypeptide to preparing medicine for regulating the activity of a sodium pump. The polypeptide in the sodium pump alpha-subunit film outer region is a kind of polypeptide containing amino acid sequences of the sodium pump alpha-subunit film outer region, and belongs to the applications of polypeptide or proteins. The polypeptide in the sodium pump alpha-subunit film outer region can influence the biological activity of the sodium pump of cell membranes, and has the potential for treating cardiovascular diseases. Moreover, the functions of the polypeptide for blocking or antagonizing the restriction of sodium pumps by Ouabain are tested in in-vitro experiments. Animal pharmacological experiments indicate that the polypeptide can reduce the blood pressure of animal models with high blood pressure, and therefore, the present invention can be used for treating cardiovascular diseases, and is particularly used for treating high blood pressure. Meanwhile, the present invention has the characteristics of conspicuous therapeutic effect, small side effect, etc.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to the application of polypeptide fragment, relate in particular to a kind of active sodium pump film of sodium pump outskirt polypeptide and this sodium pump film outskirt polypeptide thereof of can influencing and regulate application in the sodium pump active medicine in preparation.
Background technology
Sodium pump (Na
+, K
+-ATPase) be a kind of cell transmembrane albumen, keeping Na inside and outside the cell
+, K
+Ion transport, the electrochemical gradient that keeps the inside and outside sodium of cell, potassium ion, and this ion gradient is that the activity of body basic function is necessary, such as nutritive substance by the extracellular to intracellular transhipment, the balance of the inside and outside osmotic pressure of cell and the adjusting of cell-volume, and the keeping etc. of excitable cell film resting potential.In a word, sodium pump is being kept cylinder electrolyte balance, stabilized cell membrane potential, is being kept aspects such as cell-volume, signal transduction all to have vital role.
One, the structure of sodium pump:
Sodium pump is made up of α, β, three subunits of γ.The minimum working energy unit of sodium pump is a dimer, and promptly α β often exists with tetramer form, i.e. 2 α, 2 β.Studies show that in recent years, alpha subunit are its catalytic subunits, and its configuration decision sodium pump activity is the functional unit of sodium pump.Alpha subunit is to promoting that synthetic β subunit leaves endoplasmic reticulum and its glycosylation is played an important role.β subunit is a kind of glycosyl albumen, closely is connected with alpha subunit, plays an important role to keeping the sodium pump activity.It assembles in endoplasmic reticulum, can promote alpha subunit fixing in film, forms stable and activated α β dimer, and it can influence the transhipment of sophisticated alpha subunit.Newfound in recent years γ subunit, its function is still indeterminate at present.
The sodium pump alpha subunit has and many sodium pump regulatory factors (multiple aglucon such as cardiac stimulant glucoside material) bonded site, can with Na
+, K
+, Ca
2+, combination such as ATP, interrelate with phosphorylation and energy delivery system, be the main attemperator of sodium pump function, in the active adjusting of sodium pump, have vital role.The earliest, people only know that purple foxglove is Na
+, K
+Unique antagonist of-ATPase recently, is found to exist endogenous sodium pump inhibition (SPIs, EDLS, EO etc.) in the mammalian body, and is found that they may participate in keeping of active adjusting of sodium pump and electrolyte balance.Evidence suggests endogenous ouabain (EO) and Na
+, K
+The cardiotonic glycoside combining site of-ATPase catalytic subunit (the Butt AN that reversibly combines, Tennant BP, Gillingwater SD, et al.Binding of ouabain and humanouabainlike substance to different Na+, K+-ATPase isoforms.HypertensRes, 2000; 23:S45-S50).
According to sodium pump catalytic subunit and Na
+, K
+, Ca
2+The difference that reaches multiple aglucon avidity such as cardiac stimulant glucoside material is divided into α
1, α
2, α
3And α
4Hypotype (α
4Hypotype is known little about it).In fact, the equal express alpha of all cells
1Subunit, and the avidity of α 1-subunit and ouabain is starkly lower than α
2And α
3Subunit, α
2And α
3In all vertebratess with the higher (IC of the avidity of ouabain
50=10-100nM).Simultaneously sodium pump subunit in tissue distribution and also have certain species variation with the binding ability of sodium pump inhibitor (as ouabain).As, the α of rodent
1The avidity of subunit and ouabain (ki=1-5 * 10
-5Mol/L) than α
2, α
3Subunit (ki=1-5 * 10
-7Mol/L) height.And the catalytic subunit of different subtype has different dynamicss.Use Na
+, K
+-ATPase suppresses the α that active method records dog respectively
1The α of subunit and primates
3Subunit and the similar (IC of the bonding force of ouabain
50=10nM).Ferrandi recorded dog kidney α respectively with similar methods in 1992
1The IC50 of subunit is 14nM, rat brain α
3The IC of subunit
50Be 21nM, in addition, rat kidney α
1The IC of subunit
50Be 105nM, the prompting rat is than dog kidney α
1Subunit is low to the susceptibility of ouabain.Studies show that in recent years, rabbit kidney α
1Subunit is reactionless fully to ouabain, when the concentration of ouabain reaches 500nM, does not also see significant reaction even.1998, people such as Anner recorded rabbit kidney α
1The IC of subunit
50Be 600nM.When the concentration of ouabain reaches 500nM, only can be observed 5% inhibited reaction.The rat brain α that records with Ferrandi in 1992
3The IC of subunit
50Record primates pallium α with Butt in 2000
3The IC of subunit
50Compare, the latter obviously increases.
However, the different subtype basic structure of sodium pump α catalytic subunit is high conservative still, Na
+, K
+-ATPase alpha subunit is made up of 1016 amino acid, and 10 transmembrane domains (the sodium pump structural representation is referring to shown in Figure 7) are arranged.Its N-terminal, H
2-H
3, H
4-H
5, H
6-H
7, H
8-H
9Between sequence and carboxyl terminal all be positioned at the endochylema face of cytolemma, be called the film inner region, its sequence high conservative comprises ATP-binding site and phosphorylation site, can with Na
+, K
+, the ATP combination, interrelate with phosphorylation and energy delivery system, in the active performance of sodium pump, have vital role.H
1-H
2, H
3-H
4, H
5-H
6, H
7-H
8, H
9-H
10Between sequence be positioned at outside the cytolemma, be called the film outskirt, be the binding site of sodium pump part, as the cardiotonic glycoside binding site, all be positioned at cytolemma film outskirt such as digoxin and ouabain binding site.Sodium pump activation/supressor is by the function influence sodium pump activity to these film outskirts.Discover sodium pump H at present
1-H
2, H
3-H
4The film outskirt is the main binding site of parts such as sodium pump regulatory factor, and H
5-H
6, H
7-H
8, H
9-H
10It is relevant to interact between other subunits of film outskirt and sodium pump.
People such as Shull thought that the ouabain binding site may be to be positioned at H in 1985
3-H
4Between the outer region sequence of film.But also there is research to think carboxyl terminal, H
3-H
4And H
7-H
8Also may there be the binding site of ouabain in the film outskirt, prove that simultaneously these zones not merely are (the Shull GE that combines with the purple foxglove material, Schwartz A, Lingrel JB.Amino-acid sequence of the catalytic subunit of the (Na+-K+) ATPase deduced from a complementary DNA.Nature 1985; 316 (22): 691-695).
Discoveries such as Shull in 1986, ouabain not only with H
3-H
4Between amino acid combine and H
1-H
2Between amino acid also participated in this combination, (Shull GE, GreebJ, Lingrel JB.Molecular cloning of three distingct forms of the Na also may combine with lactonic ring
+, K
+-ATPase α-subunit from rat brain.Biochemistry 1986; 25 (16): 8125-8132).Price in 1988 and Lingral find, rat sodium pump film outskirt H
1-H
2Boundary's point Arg-113 and Asp-124 be the major cause that produces the ouabain opposing.Therefore 6 kinds of different amino acid of human such as nineteen ninety Price are replaced and are positioned at M
1-M
2The amino acid on film outskirt border, research sodium pump structure and function relationship (Price EM, Rice DA, Linger JB.Structure-function studies ofNa
+, K
+-ATPase.Site-directed mutagenesis of the border residues from theH1-H2extracellular domain of the α subunit.J Bio Chem 1990; 265 (12): 6638-6641).No matter replace 111 or 122, or both are replaced simultaneously, and variation has all taken place its avidity to ouabain.111 and 122 amino acids are replaced with Lys-111-Lys-122, Glu-111-Glu-122, Lys-111-Glu-122 and Asp-111-Arg-122 respectively, no matter the sheep Na of transfected any sudden change
+, K
+The Hela cell of-ATPase alpha subunit all can be grown in the toxigenous ouabain of wild-type Hela cell to untransfected, has produced the ouabain opposing.Through the sodium pump of rite-directed mutagenesis, its ouabain IC
50Further improve (4 * 10
-3M-1 * 10
-6M).Wherein produce ouabain opposing the strongest be two sudden changes (Asp-111-Arg-122) of sodium pump film outskirt; The most weak is one-sided sudden change.Therefore think ouabain not only with sodium pump H
3-H
4Between amino acid, but also and M
1-M
2Between amino acid combine, and think that recently the latter is even more important.
As seen sodium pump H
1-H
2, H
3-H
4The film outskirt is the main target spot of sodium pump regulatory factor effect.Studies confirm that sodium pump α catalytic subunit H at present
1-H
2, H
3-H
4The film outskirt plays a significant role in the active adjusting of sodium pump, and many sodium pump parts (sodium pump regulatory factor) in the blood are just by playing significant role with being combined in the active adjusting of sodium pump of above-mentioned 2 film outskirts.
The pathophysiological change of the sodium pump function caused by abnormal that the sodium pump regulatory factor causes, (Hamlyn J plays an important role in the generation of multiple disease, Blaustin M, Bova S, et al.Identificationand characterization of oubain-like compound from human plasma.Proc NatlAcad Sci 1991; 88:625; Ferrandi M, Minotti E, Salardi S, et al.Characteristicof a ouabain-like factor from the Milan hypertensive rats.J CardiovascPharmacol 1993; 22 (suppl 2): S75-78) sodium pump inhibitor horizontal abnormality in comprising multiple disease patient's body of hypertension, and closely related, and play a significant role just because of unusually the sodium pump activity being suppressed in the generation of relative disease and the development of this regulatory factor with the research degree of sufferer.For example:
1) the heart failure patient blood plasma EO level of discovering of heart failure: Gottlieb etc. increases, and increasing degree and cardiac index and mean arterial pressure are negative correlation, its reason may be because be secondary to that the incomplete blood pressure of left chamber function reduces and during heart failure the blood perfusion of tissue such as suprarenal gland reduce, stimulate the secretion of EO to increase, thereby the blood pressure of keeping patients with heart failure and the balance of Q volume of blood are played an important role.The change of EO level when other clinical and experimentation on animals result also confirms heart failure.It is significant that patients with heart failure is carried out blood plasma EO monitoring, because the increase of blood plasma EO level may make body that the susceptibility of digitaloid drugs is reduced, increased the possibility of digitalism.
2) coronary heart disease: discover, first day blood plasma EO content of Acute Myocardial Infarction Patients morbidity raises, and concurrent ventricular arrhythmia person do not have the not normal person's height of the rhythm of the heart, but concurrent person in heart failure is lower, EO promptly reduced to normally in second day, and no considerable change in later two weeks.
3) irregular pulse: EO content increases in the ventricular arrhythmia rat plasma that discoveries such as Bagrov are brought out by myocardial ischemia, but give that EO descends behind the DigiTAb (may be) because DigiTAb and ouabain exist due to cross coupled reacts, simultaneously ARR incidence reduces, and the time length shortens, and the change of prompting EO may participate in ARR generation.
4) other: the Q volume of blood expansion of when gestation, the secretion that has stimulated EO, and concurrent hyperpietic EO content is higher, no matter whether blood pressure normal, divides puerperium EO to descend rapidly.Simultaneously, the higher EO of parent content has passed to the newborn infant by blood and milk etc.Studies show that newborn infant's blood plasma EO content obviously increases, but after birth, recover normal usually in several days.
Essential hypertension relate to the unusual and cytolemma ion transport obstacle of a series of internal secretions relevant and biochemical metabolism with blood pressure regulation, wherein sodium pump activity change, cytolemma ion transport obstacle and kidney row sodium defective are in key link.Particularly sodium pump active transport barrier plays a significant role in hypertensive generation and development unusually with signal transduction, has been acknowledged as one of pathogenesis of essential hypertension (especially salt-sensitive hypertension).
At present, for the endogenous sodium pump inhibitor---the study on mechanism of ouabain comparatively clear, this also may represent the binding mode (Ming-juanZhang of other sodium pump inhibitor to a certain extent, Jun Yang, Zhuo-ren Lu.A New Ouabain Conjugated Peptide Was FoundFrom Phage Displayed Peptide Library.Am J Hypert.2004,17:619-623; HamlynJM, Hamilton BP, Manunta P.Endogenous ouabain, sodium balance and bloodpressure:a review and a hypothesis.J Hypertens 1996; 14 (1): 151-167; Goto A, Yamnada K.Putative roles of ouabainlike compound in hypertension:Revisited.Hypertens Res 2000; 23:S7-S13).
High-caliber ouabain can excessively suppress cytolemma sodium pump activity, influences the resistance vessel function, regulates sympathetic activity and renal function, causes the body elevation of blood pressure.It may be machine-processed as follows: sodium pump is excessively suppressed to bring Na in the cell
+Raise, behind the Na/Ca interchanger, make Ca in the cell
2+Ionic concn raises, and this has just brought sarcoplasmic reticulum/endoplasmic reticulum picked-up Ca
2+Ion strengthens, and causes sarcoplasmic reticulum/endoplasmic reticulum to store Ca
2+Increase, strengthen whole cell Ca
2+The conduction of ionic signal is so that cause biological effect; Simultaneously, 1., suppress blood vessel Na-K-ATPase the restraining effect of sodium pump inhibitor cell membrane sodium pump also can cause hypertensive generation by the signal transduction pathway approach:, the E-C coupling process is strengthened, caused that vascular smooth muscle shrinks, cause antiotasis to increase; 2., ouabain is by suppressing the encephalic sodium pump, increases the release of neurotransmitter, increases periphery sympathetic nervous system activity, causes increased blood pressure; 3., activate renin-angiotensin-aldosterone system (RAS) in the brain, and then make sympathetic activation; 4., suppress the blood vessel sodium pump and cause that also blood vessel increases adrenergic reactivity; 5., find also that recently ouabain is Na not just
+, K
+The supressor of-ATPase is also by to being distributed in II type and the III type H on the uriniferous tubules
+, K
+-ATPase suppresses, and participates in hypertensive generation.
As seen, numerous factors all can be by active inhibition causes Na in the cell to sodium pump
+The change of temporary transient or lasting rising, intracellular ph value and ionic equilibrium, and then cause: 1., Peripheral resistance more; 2., receptor of blood pressure is resetted; 3., sympathetic activity increases; 4., genetic change of table or interaction aggravation; 5., increase vessel wall/chamber ratio.Single or the combined action of above-mentioned factor finally causes the persistence of blood pressure to increase.So think, the endogenous sodium pump inhibitor causes cytolemma ion transport obstacle to the active inhibition of sodium pump, may be the key link that hypertension takes place.
Based on above-mentioned analysis, by of the inhibition of blocking-up sodium pump inhibitor, make sodium pump recover to arrange the sodium function again for sodium pump, will open up a kind of new method and thinking for the medical treatment of cardiovascular disorder.Have and studies confirm that this thinking is feasible.Ferrari in 1999 finds, give behind the Dahl rat high salt diet intracerebral ventricle injection a kind of can with ouabain bonded new drug PST2238, blood pressure drops is to normal level after 18 hours; Give gestational hypertension patient infusion PST2238, also obvious step-down (QuadriL, Bianchi G, Cerri A, et al.17beta-(3-furyl)-5beta-androstane-3beta, 14beta, 17beta-triol (PST2238): A very potent antihypertensive agent with a novelmechanism of action.J Med Chem 1997; 40 (11): 1561-1564).In addition, can block the inhibition of the interior EO of patients with sevious craniocerebral injury body with DigiTAb, and bring high blood pressure down blood vessel Na-K-ATPase.This study group adopts anti-ouabain antibody of specificity and F (ab) thereof
2Fragment equally also can significantly reduce the blood pressure of laboratory animal.
Above-mentioned research illustrates that all selectivity reduces the restraining effect of sodium pump inhibitor to sodium pump of dissociating in the blood circulation, or it is competitive in conjunction with sodium pump, or the quantity of the sodium pump subunit of reduction high-affinity ouabain, all can recover the sodium pump activity, reach and reduce the particularly purpose of the responsive patient's blood pressure of salt of hyperpietic.Therefore, by the inhibition of blocking-up sodium pump inhibitor for sodium pump, make sodium pump recover again to arrange the sodium function, it is in full force and effect and feasible reaching the hypertensive strategy of treatment.
But, because the polytype endogenous factors that exists in the body all can suppress the activity of sodium pump, and adopt specific antibody or antibody fragment is can only block the single type sodium pump inhibitor by the antigen antibody reaction of classics, can not block or effectively alleviate the restraining effect of number of different types endogenous sodium pump inhibitor to sodium pump simultaneously.
Therefore, design can be blocked the restraining effect of polytype sodium pump inhibition to sodium pump simultaneously, and influence as few as possible the cell normal physiological function more reasonable, the effectively preventing strategy is significant for the control of cardiovascular disorder.
Above-mentioned analysis confirms at sodium pump alpha subunit H
1～H
2And H
3～H
4The film outskirt is the binding site of sodium pump inhibitor, plays a significant role in the active adjusting of sodium pump.Sodium pump inhibitor for the restraining effect of sodium pump just by with sodium pump α-H of subunit
1～H
2And H
3～H
4Between film outskirt amino-acid residue in conjunction with realizing, and only comprise sodium pump α-H of subunit
1～H
2And H
3～H
4Blocking property sodium pump α-subunit's fragment of film outskirt just possesses the activity in conjunction with sodium pump inhibitor.
That therefore, utilizes artificial preparation comprises sodium pump α-H of subunit
1～H
2And H
3～H
4The blocking property sodium pump fragment of film outskirt, competitive in conjunction with free sodium pump inhibitor, influence the activity of cytolemma sodium pump, regulate the inside and outside ionic concn of cell, thereby, can reach and block the restraining effect of polytype sodium pump inhibitor simultaneously, reach the purpose of treatment, and reduce the influence of pair cell normal physiological function to greatest extent sodium pump.
Summary of the invention
The object of the present invention is to provide α-H of subunit that sodium pump α-subunit's film outskirt polypeptide comprises
1～H
2And H
3～H
4Blocking property of film outskirt polypeptide fragment.
Another object of the present invention is to provide α-H of subunit that above-mentioned sodium pump α-subunit's film outskirt polypeptide comprises
1～H
2And H
3～H
4The medicine of cardiovascular disorder and the application in the hypertensive medicine of preparation treatment are especially treated in the application of blocking property of film outskirt polypeptide fragment in the medicine of the unusual relative disease of preparation treatment sodium pump function.
For achieving the above object, the present invention takes following technical scheme:
The polypeptide of sodium pump α-subunit's film outskirt is characterized in that, comprises following 6 sections α-H of subunit
1～H
2And H
3～H
4Blocking property of film outskirt polypeptide fragment, concrete aminoacid sequence is as follows:
H
1～H
2Blocking property of film outskirt polypeptide fragment:
Gln?Ala?Ala?Thr?Glu?Glu?Glu?Pro?Gln?Asn?Asp?Asn
Gln?Ala?Ala?Met?Glu?Asp?Glu?Pro?Ser?Asn?Asp?Asn
Gln?Ala?Gly?Thr?Glu?Asp?Asp?Pro?Ser?Asn?Asp?Asn
H
3～H
4Blocking property of film outskirt polypeptide fragment:
Glu?Tyr?Thr?Trp?Leu?Glu
Gly?Tyr?Ser?Trp?Leu?Glu
Gly?Tyr?Thr?Trp?Leu?Glu。
The preparation method of the polypeptide of above-mentioned sodium pump α-subunit's film outskirt adopts external artificial synthesis method to synthesize α-H of subunit
1～H
2(or) H
3～H
4Blocking property of film outskirt polypeptide fragment is through high performance liquid chromatography purifying synthetic sodium pump α-subunit's film outskirt polypeptide.Concrete grammar is as follows:
At first an amino acid of alpha-amino group being protected with the Fmoc group is attached on the insoluble vector resin by a support arm, subsequently with the alpha-amino group deprotection, with solution washing amino acid-support arm-resin; The amino acid of second activatory alpha-amino group protection is in advance connected up by coupled reaction; Perhaps the peptide segment with α-N end and side chain protected replaces one amino acid to carry out coupled reaction, after condensation reaction is finished, uses solution washing, repeats deprotection, coupling connection, up to obtaining α-H of subunit
1～H
2(or) H
3～H
4Blocking property of film outskirt polypeptide fragment gets final product peptide and support arm and resin cracking at last.
Perhaps adopt the insect baculovirus expression system preparation to comprise the sodium pump α 3-H of subunit
1～H
2And H
3～H
4The polypeptide fragment of film outskirt specifically comprises the following steps:
1) extraction of the mRNA of rat artery vascular tissue
Get after fresh rat artery vascular tissue weighs, shred, directly add homogenate in the homogenate, adopt the centrifugal post sharp separation of oligomerization-Mierocrystalline cellulose mRNA;
2) reverse transcription of mRNA
In aseptic 0.5ml centrifuge tube, add 5-10 μ gmRNA, 10 RNasin of unit, 10pmol 3 ' end primer, the various dNTP of 1mmol/L, its pH7.0 and 50 unit left and right sides M-Mulv ThermoScript II, or 5 unit left and right sides Taq archaeal dna polymerases, reaction buffer adds water to 20 μ l, 37 ℃ of insulation 1h;
3) pcr amplification target gene fragment
According to rat sodium pump alpha subunit gene order (Genbank, accession number: M14513.1) design primer, primer sequence: primer 1:5`-c tca cta gtc gtc aag ttc tgc cgg cagctg-3`Primer2:5`-gac aag ctt tta gca gtt ctt gcg agc cat gcg-3; CDNA with reverse transcription is a template, carries out PCR reaction, in the amplification sodium pump alpha subunit gene order between 260～1200 length be the nucleotide sequence of 760bp, as the gene order of sodium pump α-subunit's film outskirt;
Perhaps directly (Genbank, accession number: M14513.1) design comprises the α 3-H of subunit according to rat sodium pump alpha subunit gene order
1～H
2(or) H
3～H
4The upstream and downstream primer of the gene fragment of film outskirt through after the renaturation, carries out pcr amplification or directly cuts the acquisition target gene fragment through Xbal, HindIII restriction endonuclease enzyme.
4) structure of reorganization pFB-sp3 transfer vector
Target DNA fragment and pFB transfer vector all after Xbal, HindIII restriction endonuclease enzyme are cut, T
4Under the effect of-dna ligase, external connection transforms recon DH5 α host bacterium then, and alkaline lysis extracts plasmid, and identifies through PCR, gene sequencing and digestion with restriction enzyme, obtains reorganization pFB-sp3 transfer vector;
5) preparation of reorganization Bacmid DNA
With reorganization pFB-sp3 transfer vector transfection DH10Bac competent cell, screen positive bacterial plaque through Luria Agar selective medium, the alkaline hydrolysis method is extracted reorganization BacmidDNA, identifies positive recombinant chou with PCR;
6) results recombinant baculovirus
27 ℃ of Grace` perfect mediums are cultivated Sf-9 cell 1h at least in the 35mm culture dish; Be configured to down solution:
A liquid: the Bacmid DNA of 5 μ l reorganization adds 95 μ l sterilization deionized water;
B liquid: 6 μ l Cellfectin add 94 μ l sterilization deionized water;
A liquid and B liquid is mixing gently, and room temperature is placed 30min; Behind 3 Sf-9 cells of TNM-FH washing with serum-free, antibiotic-free, dropwise add the AB mixed solution, cultivate 72h altogether and collect culture supernatant, the centrifugal 10min of 500g, supernatant is divided into aliquot as the former seed culture of viruses of recombinant baculovirus, called after reorganization Ac-sp3,4 ℃ keep in Dark Place;
7) sodium pump α-subunit's film outskirt polypeptide expression and evaluation
During 27 ℃ of cultivation Sf-9 cell to 80% cytogamy, add recombinant baculovirus seed culture of viruses 400ul/ bottle, continue to cultivate 72h, collect virus infected cell, after the thermally denature, get the capable SDS-PAGE electrophoresis of 10ul supernatant, i.e. 12% separation gel, 5% spacer gel detects the proteic expression of blocking property sodium pump alpha subunit;
8) purifying of non-blocking property of sex change sodium pump α-subunit's film outskirt polypeptide
Use ProBond
TMThe Column resin absorption infects the Sf-9 cell conditioned medium of recombinant virus through the multigelation cracked, non-sex change washings washing, 50mM, 200mM, the non-sex change of 350mM, 500mM-imidazoles elutriant wash-out, the centrifugal 2min of 800 * g, collect supernatant, promptly obtain 6 sections α-H of subunit
1～H
2And H
3～H
4Blocking property of film outskirt polypeptide fragment.
Sodium pump α-subunit's film outskirt polypeptide of purifying is used for the active preparation of regulating medicine of sodium pump, directly inject mammals (comprising the mankind) or non-mammal by the intravenous injection approach, reach the restraining effect of sodium pump inhibitor of dissociating in the competitive blocking-up body to sodium pump, recover cytolemma sodium pump activity, reduce the influence of pair cell normal physiological function to greatest extent.Thereby use it for the medication preparation of sodium pump active unusual relative disease, especially cardiovascular disorder and the application in the hypertensive medicine of preparation treatment.
Description of drawings
Fig. 1 is 6 energy of the present invention and sodium pump α-subunit's film outskirt amino acid sequence of polypeptide of sodium pump specific combination.
Fig. 2 is that reorganization pFB-sp3 transfer vector PCR identifies.The 1:pFB-sp3 transfer vector; 2: negative control.
Fig. 3 is that reorganization Ac-sp3 baculovirus PCR identifies.1: negative control; The 2:Ac-sp3 baculovirus.
Fig. 4 is the expression that the polypeptide SDS-PAGE of sodium pump α-subunit's film outskirt identifies recombinant baculovirus.The Sf-9 cell pyrolysis liquid of 1:Ac-sp3 baculovirus infection; 2: negative control.
Fig. 5 is that sodium pump α-subunit's film outskirt polypeptide suppresses the red corpuscle picked-up to ouabain
86The extracorporeal biology activity test of the blocking effect of Rb is figure as a result.Wherein, X-coordinate is represented the concentration of sodium pump α-subunit's film outskirt polypeptide; Ordinate zou is represented red corpuscle
86The Rb uptake ratio.
Fig. 6 is sodium pump α-subunit's film outskirt polypeptide hypotensive effect synoptic diagram.X-coordinate is represented the concentration of sodium pump α-subunit's film outskirt polypeptide; Ordinate zou is represented blood pressure level.
Fig. 7 be sodium pump by α and β subunit structure synoptic diagram (Blanco, Am.J.Physiol.1998).
For a more clear understanding of the present invention, provide below in conjunction with accompanying drawing and contriver that the present invention is described in further detail.
Embodiment
1. sodium pump α-subunit's film outskirt polypeptid acid sequence is definite:
By the gene order and the protein sequence of Genebank acquisition sodium pump α-subunit,, determine α-H of subunit according to the structure and the film outskirt peptide sequence of α-subunit
1～H
2(or) H
3～H
4Film outskirt polypeptide.The present invention has obtained following 6 sections sodium pump α-subunit's film outskirt polypeptide (Fig. 1) through aforesaid method, and its aminoacid sequence is:
Gln?Ala?Ala?Thr?Glu?Glu?Glu?Pro?Gln?Asn?Asp?Asn
Gln?Ala?Ala?Met?Glu?Asp?Glu?Pro?Ser?Asn?Asp?Asn
Gln?Ala?Gly?Thr?Glu?Asp?Asp?Pro?Ser?Asn?Asp?Asn
Glu?Tyr?Thr?Trp?Leu?Glu
Gly?Tyr?Ser?Trp?Leu?Glu
Gly?Tyr?Thr?Trp?Leu?Glu。
2. preparing above-mentioned sodium pump α-subunit's film outskirt polypeptide can distinguish by the following technical solutions:
1) external artificial synthesis method:
The Fmoc method is synthesized α-H of subunit
1～H
2(or) H
3～H
4Blocking property of film outskirt polypeptide fragment is through blocking property of high performance liquid chromatography purifying synthetic sodium pump fragment.Concrete grammar is as follows:
At first an amino acid of alpha-amino group being protected with the Fmoc group is attached on the insoluble carrier by a support arm, subsequently with the alpha-amino group deprotection, with solution washing amino acid-support arm-resin.The amino acid of second activatory alpha-amino group protection is in advance connected up by coupled reaction.In addition, also can replace one amino acid to carry out coupled reaction, after condensation reaction is finished, use solution washing, repeat deprotection, coupling connection, up to obtaining the purpose peptide, at last with peptide-support arm-resin cracking with the peptide segment of α-N end and side chain protected.The solid-phase synthesis of this prolongation peptide chain both can adopt the method for interruption, also can use the method for continuous flow.
2) adopt prokaryotic expression system, eukaryotic expression system, insect baculovirus expression system or other eukaryotic expression systems or non-eukaryotic expression system to prepare sodium pump α-subunit's film outskirt.Concrete grammar is as follows:
Adopt methods such as RT-PCR and molecule clone technology to obtain to comprise above-mentioned sodium pump α-subunit's film outskirt gene order, by molecule clone technology it is cloned into carrier for expression of eukaryon or non-carrier for expression of eukaryon, and through the corresponding host cell of corresponding transfection, express sodium pump α-subunit's film outskirt polypeptide, non-sex change condition purifying (method sees molecular cloning for details).
3. sodium pump α-subunit's film outskirt polypeptide extracorporeal biology is active detects
Adopt red corpuscle
86Sodium pump α-subunit's film outskirt polypeptide extracorporeal biology activity is identified in the test of Rb uptake ratio.Influence the cytolemma sodium pump function, regulate the inside and outside ionic equilibrium of cell, thereby point out it to have the latent effect of treatment cardiovascular disorder.At last, the employing animal experiment confirms the effect of its treatment cardiovascular disorder, and the emergency toxicology effect.See embodiment 2 than concrete grammar.
4. in vivo test
Adopt hypertension animal model experimental study sodium pump α-subunit's film outskirt polypeptide pharmacodynamics and toxicology in animal body.Concrete grammar is seen embodiment 3,4.
5. the active preparation of regulating medicine of sodium pump
In order to confirm that sodium pump α-subunit's film outskirt polypeptide can influence the cytolemma sodium pump function, and can be used for treating cardiovascular disorder, especially hypertension, the embodiment of the following stated is used to describe the present invention, rather than restriction the present invention.
Experiment material:
1. the acquisition of the polypeptide fragment of sodium pump α-subunit's film outskirt:
Embodiment 1:
Use the synthetic automatically sodium pump alpha subunit film outskirt polypeptide of Peptide synthesizer, and by the HPLC purifying.Present embodiment synthesizes the sodium pump α 3-H of subunit
1～H
2Film outskirt polypeptide fragment, its aminoacid sequence is as follows: Gln Ala Gly Thr Glu Asp Asp Pro Ser Asn Asp Asn.
The Fmoc solid phase method of peptide synthesis:
At first an amino acid of alpha-amino group being protected with the Fmoc group is attached on the insoluble carrier by a support arm; subsequently with the alpha-amino group deprotection; with solution washing amino acid-support arm-resin. the amino acid of second activatory alpha-amino group protection is in advance connected up by coupled reaction. in addition; also can replace one amino acid to carry out coupled reaction with the peptide segment of α-N end and side chain protected; after condensation reaction is finished; use solution washing; repeat deprotection; the coupling connection; up to obtaining the purpose peptide. at last with peptide-support arm-resin cracking. the solid-phase synthesis of this prolongation peptide chain both can adopt the method for interruption, also can use the method for continuous flow.That is:
(1) C-terminal amino acid and resin-bonded;
(2) Na-Fmoc's removes, washs;
(3) coupling connection, washing;
(4) repeating step (2)～step (3);
(5) deprotection base.
Can obtain other several α-H of subunit involved in the present invention with method
1～H
2(or) H
3～H
4Blocking property of film outskirt polypeptide fragment.
Embodiment 2:
Adopt the insect baculovirus expression system preparation to comprise the sodium pump α 3-H of subunit
1～H
2(or) H
3～H
4The polypeptide fragment of film outskirt, its aminoacid sequence comprises following aminoacid sequence:
Gln?Ala?Gly?Thr?Glu?Asp?Asp?Pro?Ser?Asn?Asp?Asn。
Concrete preparation method is as follows:
(1) extraction of the mRNA of rat artery vascular tissue: get after fresh rat artery vascular tissue weighs, shred, directly add homogenate in the homogenate, adopt oligomerization (the dT)-centrifugal post sharp separation of Mierocrystalline cellulose mRNA.
(2) reverse transcription of mRNA: in aseptic 0.5ml centrifuge tube, add 5-10 μ gmRNA, 10 RNasin of unit, 10pmol 3 ' end primer, various dNTP of 1mmol/L (pH7.0) and 50 unit left and right sides M-Mulv ThermoScript II (or 5 unit left and right sides Taq archaeal dna polymerases), reaction buffer adds water to 20 μ l, 37 ℃ of insulation 1h.
(3) pcr amplification target gene fragment: according to rat sodium pump alpha subunit gene order (Genbank accession number: M14513.1) design primer, primer sequence: primer 1:5`-c tca cta gtcgtc aag ttc tgc cgg cag ctg-3`Primer2:5`-gac aag ctt tta gca gttctt gcg agc cat gcg-3`.CDNA with reverse transcription is a template, carries out PCR reaction, in the amplification sodium pump alpha subunit gene order between 260～1200 length be the nucleotide sequence of 760bp, as the polypeptide (Fig. 2) of sodium pump α-subunit's film outskirt.
(4) structure of reorganization pFB-sp3 transfer vector:
Target DNA fragment and pFB transfer vector all after Xbal, HindIII restriction endonuclease enzyme are cut, T
4Under the effect of-dna ligase, external connection.Then recon is transformed DH5 α host bacterium, alkaline lysis extracts plasmid, and identifies through PCR, gene sequencing and digestion with restriction enzyme, obtains reorganization pFB-sp3 transfer vector.
(5) preparation of reorganization Bacmid DNA:
With reorganization pFB-sp3 transfer vector transfection DH10Bac competent cell, screen positive bacterial plaque (concrete grammar is with reference to the Bac-to-Bac of GIBCO company baculovirus expression system operational manual) through Luria Agar selective medium.The alkaline hydrolysis method is extracted reorganization BacmidDNA, identifies positive recombinant chou (Fig. 3) with PCR.
(6) results recombinant baculovirus:
27 ℃ of Grace` perfect mediums are cultivated Sf-9 cell 1h at least in the 35mm culture dish; Be configured to down solution: A liquid (the Bacmid DNA of 5 μ l reorganization adds 95 μ l sterilization deionized water) and B liquid (6 μ l Cellfectin add 94 μ l sterilization deionized water) is mixing gently, and room temperature is placed 30min; Behind 3 Sf-9 cells of TNM-FH washing with serum-free, antibiotic-free, dropwise add the AB mixed solution, cultivate 72h altogether and collect culture supernatant, the centrifugal 10min of 500g, supernatant is divided into aliquot as the former seed culture of viruses of recombinant baculovirus, called after reorganization Ac-sp3,4 ℃ keep in Dark Place.(concrete grammar is with reference to the Bac-to-Bac of GIBCO company baculovirus expression system operational manual).
(7) sodium pump α-subunit's film outskirt polypeptide expression and evaluation:
During 27 ℃ of cultivation Sf-9 cell to 80% cytogamy, add recombinant baculovirus seed culture of viruses 400ul/ bottle, continue to cultivate 72h, collect virus infected cell, after the thermally denature, get the capable SDS-PAGE electrophoresis of 10ul supernatant (12% separation gel, 5% spacer gel) and detect the blocking property proteic expression of sodium pump alpha subunit (Fig. 4).
(8) purifying of non-blocking property of sex change sodium pump α-subunit's film outskirt polypeptide: use ProBond
TMThe Sf-9 cell conditioned medium of recombinant virus is infected in Column resin (Invitrogen company) absorption through the multigelation cracked, non-sex change washings washing, 50mM, 200mM, the non-sex change of 350mM, 500mM-imidazoles elutriant wash-out, the centrifugal 2min of 800 * g, collect supernatant, obtain the purpose fragment.
Can obtain other several the α-H of subunit that contains involved in the present invention with method
1～H
2(or) H
3～H
4The segmental expression of blocking property of film outskirt polypeptide.
Embodiment 3:
Adopt Protocols in Molecular Biology, blocking property of prokaryotic expression sodium pump α-subunit's film outskirt polypeptide fragments comprises the sodium pump α 3-H of subunit
1～H
2(or) H
3～H
4The polypeptide fragment of film outskirt, its aminoacid sequence comprises following aminoacid sequence:
Gln?Ala?Gly?Thr?Glu?Asp?Asp?Pro?Ser?Asn?Asp?Asn
Specific embodiment is as follows:
(1) extraction of people's renal tissue mRNA: after getting fresh renal tissue samples weighing, shred into about 1cm
3Tissue block, directly add the extraction of being mRNA in the homogenate.Adopt the centrifugal post sharp separation of oligomerization (dT)-Mierocrystalline cellulose.Principle: earlier with guanidinium isothiocyanate and sarcosyl, direct cracking homogenate, cracking renal tissue cell also suppress the RNase activity, cell pyrolysis liquid is directly gone up oligomerization (dT)-cellulose column, with different salt concn damping fluid washing upper prop things, poly (A under the last wash-out
+) RNA, and do not need to use ethanol sedimentation, and reclaim micro-RNA with the glycogen precipitation.Relevant operation steps is with reference to fast m RNA purification test kit product description.
(2) reverse transcription of mRNA: latex 5-10 μ g cell mRNA in aseptic 0.5ml centrifuge tube, 10 RNasin of unit, 10pmol 3 ' end primer, various dNTP of 1mmol/L (pH7.0) and 50 unit left and right sides M-Mulv ThermoScript II (or 5 unit left and right sides Taq archaeal dna polymerases), reaction buffer adds water to 20 μ l, 37 ℃ of insulation 1h.
(3) pcr amplification target gene fragment: designing 4 pairs of primers, is template with mRNA, amplifying target genes fragment under suitable condition.
(4) cloning vector is connected and conversion with the external of target gene fragment: target DNA fragment and expression vector pGEM-Zf are all after suitable restriction endonuclease enzyme is cut, through an amount of T
4The effect of-dna ligase reaches under the suitable condition, carries out external connection.Recon is transformed into corresponding host bacterium.
(5) screening of positive recombinant and evaluation: transform bacteria directly is coated on contains on 100 μ g/ml Amp, 0.5mmol/L IPTG and the 40 μ g/ml X-gal agar plates, after the incubated overnight, can see the white colony of millimeter size.The amplification of picking white colony, a small amount of are extracted DNA, make the restriction map spectrum, determined dna sequence.
(6) prepare goal gene in a large number: the positive recombinant after will screening, identifying increases in a large number, extracts DNA in a large number.
(7) external connection and conversion: target DNA fragment and expression vector pGEX-4t-1 all after suitable restriction endonuclease enzyme is cut, carry out external connection under suitable condition.Recon is transformed into corresponding host bacterium.
(8) screening of positive recombinant: cell is changed in the LB nutrient solution that contains penbritin, filter out and contain recon and transform bacterial strain, extract plasmid DNA and make the restriction map spectrum, determined dna sequence is determined to express after errorless.
(9) expression, evaluation and the purifying of blocking property sodium pump α-subunit's film outskirt polypeptide fragment: recon transform bacterial strain 37 ℃ cultivate a few hours reach the logarithmic growth after date add IPTG to final concentration be 1mmol/L, continue to cultivate the expression of inducing blocking property sodium pump catalytic subunit 3-5 hour.SDS-PAGE identifies, adopts Glutathione Sepharose 4B purifying expressing protein.
Can obtain other several the α-H of subunit that contains involved in the present invention with method
1～H
2(or) H
3～H
4The segmental expression of blocking property of film outskirt polypeptide.
More than the following test that all can be used for embodiment of the polypeptide fragment of the three kinds of sodium pump that embodiment obtained α-subunit's film outskirts detect.The present invention be to be described, the synthetic sodium pump α 3-H of subunit that all adopts embodiment 1 to be obtained in the following content of present embodiment
1～H
2The polypeptide fragment of film outskirt carries out.
2, the evaluation of sodium pump α-subunit's film outskirt polypeptide extracorporeal biology activity (influencing the cytolemma sodium pump function)
Sodium pump α-subunit's film outskirt polypeptide is to ouabain and sodium pump bonded blocking effect: in freshly prepared red cell suspension, behind the ouabain solution of adding fixed concentration, the sodium pump α-subunit's film outskirt polypeptide solution and RingerShi damping fluid of different concns, mix, incubation adds again
86RbCl solution, incubation, termination reaction, centrifugal then, inhale remove supernatant liquor after, insert in the gamma counter, record that red corpuscle takes in
86Rb.Remaining experimental implementation is the same, and the result shows that sodium pump α-subunit's film outskirt polypeptide has blocking-up to suppress red corpuscle
86The effect that Rb takes in, within the specific limits, the concentration of sodium pump α-subunit's film outskirt polypeptide is high more, and it suppresses red corpuscle to ouabain
86The inhibition of Rb picked-up is just big more.
3, sodium pump α-subunit's film outskirt polypeptide is at the body pharmacological experiment
(1) experimental technique and result:
Hypertension model experiment: experiment is divided into 3 groups, i.e. placebo, Sodium Nitroprusside group, blocking property sodium pump segment group, and every group of 8-10 is only.
Each organizes rat all through 25% urethane anesthesia, and carotid artery intubate monitoring blood pressure is treated tongue intravenously administrable behind the blood pressure stabilization, and positive control drug is Sodium Nitroprusside (quiet injecting amount is the 0.3mg/kg body weight), and negative control is physiological saline (placebo).
(2) the animal pharmacology experiment shows: the hypotensive effect of sodium pump α-subunit's film outskirt polypeptide has following characteristics: pressure reduction effect is relevant with dosage, and step-down amplitude and time length and dosage are proportionate; Antihypertensive effect is than antihypertensive drugs Sodium Nitroprusside step-down longer duration commonly used; Step-down is rapid-action, onset in 2-5 minute after the medication.
4, sodium pump α-subunit's film outskirt polypeptide acute toxicity test
Select 20 of the healthy mices of 18-22 gram body weight, male and female half and half are once injected sodium pump α-subunit's film outskirt polypeptide of peak concentration (230mg/Kg body weight) through two kinds of approach of tail vein and abdominal cavity.Observations:
(1) test reaction of animals situation at once: animal activity obviously reduces, but does not have generations such as twitching and is short of breath.
(2) continue to observe 10 days, test first day animal diet followed and movablely obviously reduce, took a turn for the better in second day, recovered normally to observe 10 days no animal deads in the 3rd day.
Conclusion: maximum tolerance test shows that the acute toxicity effect of sodium pump α-subunit's film outskirt polypeptide is little.
From Fig. 5, sodium pump α-subunit's film outskirt polypeptide is used for the result of extracorporeal biology activity test as can be seen: sodium pump α-subunit's film outskirt polypeptide has influenced the biologic activity of cytolemma sodium pump; Its strength of solution heals when high, and ouabain is suppressed red corpuscle
86The blocking-up of Rb picked-up is just stronger, otherwise more.
The animal pharmacology experiment shows (shown in Figure 6): the pressure reduction effect of sodium pump α-subunit's film outskirt polypeptide is relevant with dosage, and step-down amplitude and time length and dosage are proportionate; Step-down is rapid-action, onset in 2～5 minutes after the medication.
In a word, above content has proved absolutely that sodium pump α-subunit's film outskirt polypeptide has influenced the biologic activity of cytolemma sodium pump, and can be applied to the treatment of cardiovascular disorder, especially treats essential hypertension.
2. technique effect
The present invention adopts the polypeptide of sodium pump α-subunit's film outskirt of such scheme acquisition, has inside and outside competitive blocking-up sodium pump inhibitor to the active restraining effect of sodium pump, reach and regulate the active purpose of sodium pump, can combine the sodium pump regulatory factor with the competition of cytolemma sodium pump, reduce the blood pressure of experimental animal.Can directly it be used for the pharmacological agent of active relative disease patient of sodium pump or experimental animal as medicine with injection system, reach the restraining effect of sodium pump inhibitor of dissociating in the competitive blocking-up body to sodium pump, recover cytolemma sodium pump activity, reduce the purpose of the influence of pair cell normal physiological function to greatest extent, thereby, can use it for the preparation of sodium pump active unusual relative disease, especially cardiovascular disorder and antihypertensive drugs.
3. preferred forms
3.1 the acquisition of the polypeptide of sodium pump α-subunit's film outskirt
3.1.1
Directly use the polypeptide of the synthetic sodium pump α of Peptide synthesizer-subunit's film outskirt, and by HPLC purifying (referring to embodiment 1).
3.1.2
Adopt the insect baculovirus expression system preparation to comprise the polypeptide fragment (referring to embodiment 2) of sodium pump α-subunit's film outskirt.
3.1.3
Adopt the prokaryotic expression system preparation to comprise the polypeptide fragment (referring to embodiment 3) of sodium pump α-subunit's film outskirt.
3.2
Has inside and outside competitive blocking-up sodium pump inhibitor to the active restraining effect of sodium pump, reach and regulate the active purpose of sodium pump, the polypeptide of sodium pump α-subunit's film outskirt that can directly the present invention be obtained is used for the pharmacological agent of active relative disease patient of sodium pump or experimental animal with injection system with it as medicine, reach the restraining effect of sodium pump inhibitor of dissociating in the competitive blocking-up body to sodium pump, recover cytolemma sodium pump activity, reduce the purpose of the influence of pair cell normal physiological function to greatest extent, thereby, can use it for the preparation of active unusual relative disease, especially cardiovascular disease medicine of sodium pump.
The polypeptide of sodium pump α-subunit's film outskirt contains
α-the H of subunit
1～H
2And/or H
3～H
4Film outskirt polypeptid acid sequence
Gln?Ala?Ala?Thr?Glu?Glu?Glu?Pro?Gln?Asn?Asp?Asn
Gln?Ala?Ala?Met?Glu?Asp?Glu?Pro?Ser?Asn?Asp?Asn
Gln?Ala?Gly?Thr?Glu?Asp?Asp?Pro?Ser?Asn?Asp?Asn
Glu?Tyr?Thr?Trp?Leu?Glu
Gly?Tyr?Ser?Trp?Leu?Glu
Gly?Tyr?Thr?Trp?Leu?Glu
Claims (4)
1. sodium pump α-subunit's film outskirt polypeptide is characterized in that, by following α-H of subunit
1～H
2And H
3～H
4The repetition peptide sequence fragments of segmental single or two the above peptide sections of blocking property of film outskirt polypeptide are formed, and length is less than 300 amino acid whose small peptides or albumen, and its aminoacid sequence is:
H
1～H
2Blocking property of film outskirt polypeptide fragment:
Gln?Ala?Ala?Thr?Glu?Glu?Glu?Pro?Gln?Asn?Asp?Asn
Gln?Ala?Ala?Met?Glu?Asp?Glu?Pro?Ser?Asn?Asp?Asn
Gln?Ala?Gl?y?Thr?Glu?Asp?Asp?Pro?Ser?Asn?Asp?Asn
H
3～H
4Blocking property of film outskirt polypeptide fragment:
Glu?Tyr?Thr?Trp?Leu?Glu
Gly?Tyr?Ser?Trp?Leu?Glu
Gly?Tyr?Thr?Trp?Leu?Glu
Above-mentioned blocking property of film outskirt polypeptide fragment can be used in the medicine of preparation treatment hypertension or cardiovascular disorder.
2. the described sodium pump α of claim 1-subunit's film outskirt polypeptide is used to prepare the medicinal application for the treatment of cardiovascular or high blood pressure disease.
3. sodium pump α as claimed in claim 1-subunit's film outskirt polypeptide is characterized in that the medicine of described treatment hypertension or cardiovascular disorder is the medicine of injection system.
4. application as claimed in claim 2 is characterized in that, the medicine of described treatment hypertension or cardiovascular disorder is the medicine of injection system.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6846646B1 (en) * | 1998-12-24 | 2005-01-25 | The General Hospital Corporation | Methods for screening HIF like ouabain-resistant Na+-K+-ATPase agents |
-
2006
- 2006-04-27 CN CNB2006100427411A patent/CN100386341C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6846646B1 (en) * | 1998-12-24 | 2005-01-25 | The General Hospital Corporation | Methods for screening HIF like ouabain-resistant Na+-K+-ATPase agents |
Non-Patent Citations (6)
| Title |
|---|
| Molecular Cloning of Three Distinct Forms ofthe Na+,K+-ATPase α-Subunit from Rat Brain. Gary E Shull et al.Biochemistry,Vol.25 No.25. 1986 |
| Molecular Cloning of Three Distinct Forms ofthe Na+,K+-ATPase α-Subunit from Rat Brain. Gary E Shull et al.Biochemistry,Vol.25 No.25. 1986 * |
| 哇巴因结合肽拮抗哇巴因抑制钠泵作用的研究. 张明娟等.西安交通大学学报（医学版），第25卷第3期. 2004 |
| 哇巴因结合肽拮抗哇巴因抑制钠泵作用的研究. 张明娟等.西安交通大学学报（医学版），第25卷第3期. 2004 * |
| 钠泵α_1亚单位基因突变与哈萨克族人原发性高血压的关系. 周玲等.科学技术与工程，第5卷第11期. 2005 |
| 钠泵α_1亚单位基因突变与哈萨克族人原发性高血压的关系. 周玲等.科学技术与工程，第5卷第11期. 2005 * |
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