CN101602796A - A kind of selenium-containing active polypeptide and preparation method thereof and application - Google Patents
A kind of selenium-containing active polypeptide and preparation method thereof and application Download PDFInfo
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Abstract
The present invention relates to a kind of active polypeptide, its synthetic method and purposes in preparing analgesic drugs thereof that contains seleno-cysteine.Described polypeptide has the aminoacid sequence of SEQ ID NO.1～8, adopt the preparation of polypeptide solid state chemistry synthetic method, steady quality, cost are lower, and described peptide has extremely strong analgesic activities and structural stability, have the excellent development prospect as the analgesic of no habituation type.
Description
Technical field
The present invention relates to class selenium-containing active polypeptide and preparation method thereof, and the application of described polypeptide in medication preparation.
Background technology
Cone shell is the general name that a class belongs to Mollusca (Mollusca) Conidae (Conidae) marine animal, and as ocean " snail ", it is slow in action, and hunting food mainly is to lean on the paralysis of venom to be used for conquering prey.Cone shell has the good poisonous organ of evolving, and the liquid gland at radula sac rear can be secreted venom.Have venom when needing and penetrate, cause prey and lose the resistance function rapidly from rhynchodaenm.
The research of cone shell venom starts from the 1950's, carried out the toxicity research of 37 kinds of cone shell venom the beginning of the sixties to the people, find that ground-tint cone shell and the deadly composition of knitting continuous cone shell venom are protein the seventies, and from the venom of ground-tint cone shell, isolated 3 pure polypeptide toxins in 1977.It is said that every kind of cone shell all contains 50～200 kinds of toxin compositions, the someone infers should have 50,000 various active peptide compositions to be present in the cone shell venom in theory.At present, visible hundreds of separation or synthetic conotoxin from various documents and Patent data.The eighties, the cone shell venom has been carried out the systematic research results suggest, the different components in the cone shell venom has specific target spot in neural system: or the different ionic channel of control Nerve impulse in the blocking-up cytolemma; Or blocked the acceptor that transmits the neurotransmitter of signal at iuntercellular.
Cone shell peptide toxin is made up of the individual amino-acid residue in 10～30 (8～42) mostly, cysteine content is very high, and contain 2 or 3 pairs of conservative disulfide linkage, and it is said that conotoxin is the animal nerve toxin peptide of the nucleic acid encoding of the minimum found so far, also be the highest biological peptide of disulfide linkage density.The conotoxin biologic activity is extremely strong, and the pharmacotoxicological effect target spot type that has been found that mainly contains the voltage-dependent ionic channel, as Ca
2+Passage, Na
+Passage etc.; Acceptor dependent form ionic channel is as nAch acceptor, nmda receptor; And the acceptor of other types, as NE acceptor etc.
According to the characteristic framework pattern of rows and columns of the disulfide bond key bridge between conotoxin mature peptide cysteine residues and the signal peptide sequence of the amino acid whose high conservative of conotoxin precursor N end regions, conotoxin can be divided into a plurality of superfamilies such as A, M, O, P, S, T.In each superfamily, can mark off different families again according to its target site and pharmacologically active again, mechanism of action and target position as according to cone shell peptide toxin can be divided into pharmacologist same clan types such as α, ω, μ and δ.
Anodyne is one of main direction of domestic and international conotoxin research and development.It is reported have 1/6 population bearing the torment of pain sufferer approximately in the west, as spinal injury, migraine and with sacroiliitis, diabetes, pain that cancer is relevant.Existing pain therapy medicine mainly is limited to opioid drug (morphine and related drugs) and non-steroidal anti-inflammatory drugs (NSAIDs), there is serious habituation side effect in the former, the latter is then invalid to moderate, severe pain, and therefore the exploitation of new type analgesic thing safely and effectively has very important significance.Ionic channel has vital role at pain sensation physiological mechanism, and these ionic channels relevant with pain sensation conduction all might become the target spot of the new pain therapy medicine of research and development.
Conotoxin ω-MVIIA be exactly a class relevant with pain, have a highly active selective N type Ca
2+Channel blocker, Elan company has developed synthetic analogues ziconotide (Ziconotide, trade(brand)name Prialt), respectively at the listing permission that obtains U.S. FDA and European Union (EC) at the beginning of in December, 04 and 2005, be used for the treatment of severe pain (severe chronic inflammation and the neuralgia relevant with cancer or acquired immune deficiency syndrome (AIDS)), this is that first also is unique so far conotoxin class medicine of being ratified by FDA.The advantage of MVIIA is that analgesic effect is extremely strong, it is said, its analgesia is tired strong 1000 times than morphine, and does not produce tolerance and habituation.As first N-type VSCC blocker medicine, the pain patients that the appearance of MVIIA is uncontrollable for those even morphine is also ineffective is a Gospel, has very important significance for the quality of life that improves these patients.
Conotoxin ω-MVIIA is a small peptide of being made up of 25 amino acid, contains 6 cysteine residues, is connected to form by 3 three pairs of disulfide linkage to have active tertiary structure.Aminoacid sequence is
Cys-Lys-Gly-Lys-Gly-Ala-Lys-
Cys-Ser-Arg-Leu-Met-Tyr-Asp-
Cys-
Cys-Thr-Gly-Ser-
Cys-Arg-Ser-Gly-Lys-
Cys-NH
2, being connected to of three pairs of disulfide linkage: Cys1-Cys16, Cys8-Cys20, Cys15-Cys25.The ω that the U.S. has gone on the market-MVIIA adopts the chemiluminescent polypeptide synthetic method to produce, because three pairs of halfcystines need correct pairing just can be formed with active end product, synthesis step is many, and difficulty in process is with high costs.
In addition, owing to have 3 pairs of disulfide linkage, ω-MVIIA runs in body will be extremely unstable under the reductive conditions such as gsh, Trx and protein disulfide isomerase, disulfide bonds or mispairing may take place, and cause higher structure to change, active descend even lose fully, and the peptide chain that loses higher structure is more prone to by intravital other proteasome degradation of machine.ω-MVIIA eliminates the transformation period and lacks (t in human body
1/2) be (4.6 ± 0.9) h, can be cut by endopeptidase and exopeptidase, advance the peptase/proteolytic enzyme proteolytic cleavage that can be present in after the human circulation in the multiple organ, become peptide class fragment or total free aminoacids.Improving polypeptide drugs in the intravital stability of machine, for very important of the clinical application of medicine, also is an emphasis direction of modern polypeptide drugs research and development.
(Selenocysteine is a kind of naturally occurring amino acid Sec) to seleno-cysteine, and the amino acid of the 21st kind of biosynthetic constitutive protein matter of being known as is present in the active centre of seleno-protein or selenoenzyme (particularly antioxidase) more.With halfcystine (Cysteine, Cys) similar, also can form " the two selenium keys " that be similar to " disulfide linkage ", and have the redox property that is similar to Cys, become the active centre of multiple in the organism " oxygen-go back enzyme ".
Because the bond energy of two selenium keys and disulfide linkage there are differences, the redox electromotive force of selenium hydrogen base and sulfydryl is different, that is to say, the formation condition of two selenium keys and one-tenth key stability are different with disulfide linkage.Utilize this principle,, can guarantee that contained Sec in the synthetic polypeptide and/or Cys are paired into the accuracy of key, guarantee the correct folding of polypeptide, optimization production technology by the controlled oxidation reduction reaction conditions.Adopt Sec to substitute Cys simultaneously and also may improve the stability of polypeptide under reductive condition, thereby obtain more stabilizing effective polypeptide drugs in body.
ω-MVIIA compares with prototype, and the inventor finds that in surprise seleno class ω-MVIIA peptide has the longer transformation period, therefore can correspondingly reduce the dosage of keeping drug effect.
Summary of the invention
An object of the present invention is to provide a kind of active polypeptide that contains seleno-cysteine, described active polypeptide is the synthetic analogues of a class conotoxin ω-MVIIA, promptly, seleno is intended ω-MVIIA peptide (Sec-MVIIA, abbreviate as and intend ω-MVIIA peptide), described plan ω-MVIIA peptide has the aminoacid sequence shown in the SEQ ID NO.1:
SEQ?ID?NO.1：XKGKGAK?X?SRLMYDX?XTGS?X?RSGKX
*
Wherein, amino acid shown in the X is C (Cysteine) and/or U (Selenocysteine), and in described sequence,
(1) the 1st, 16, the 8th, 20, the 15th, 25 X is simultaneously C in couples or be U in couples;
(2) the 1st, 16, the 8th, 20, among the 15th, the 25 amino acids X, has a pair of U that is simultaneously at least;
(3) in the tertiary structure of peptide, the 1st and 16, the 8 and 20, the 15 and 25 amino acids U and/or C form two selenium key and/or disulfide linkage respectively in couples.
*Expression terminal amideization or be the free carboxy end.
Obviously, the active polypeptide that contains seleno-cysteine of the present invention is the synthetic analogues of ω-MVIIA, be equivalent to seleno-cysteine (Selenocysteine, Sec) halfcystine (Cysteine in replacement ω-MVIIA peptide chain, Cys), described replacement comprises that at least one pair of halfcystine in conotoxin ω-MVIIA peptide chain is replaced by seleno-cysteine, and thus, it is strong to form at least one pair of two selenium in proteinic tertiary structure.
The present invention's plan ω-MVIIA peptide is generally in the C-terminal amidation, but also can have the free carboxy end, perhaps connects other group at C-terminal.
More preferably, plan ω-MVIIA peptide provided by the invention comprises and is selected from the plan ω shown in following SEQ ID NO.2～8 sequences-MVIIA peptide A～G:
NO.2：U?KGKGAK?C?SRLMYD?CU?TGS?C?R?SGK?C
*；
(intend ω-MVIIA peptide A, [Sec
1,16] MVIIA, in ω-MVIIA aminoacid sequence the 1st and 16 halfcystines are replaced by seleno-cysteine respectively)
NO.3：C?KGKGAK?U?SRLMYD?CC?TGS?U?R?SGK?C
*；
(intend ω-MVIIA peptide B, [Sec
8,20] MVIIA, in ω-MVIIA peptide ammino acid sequence the 8th and 20 halfcystines are replaced by seleno-cysteine respectively)
NO.4：C?KGKGAK?C?SRLMYD?UC?TGS?C?R?SGK?U
*；
(intend ω-MVIIA peptide C, [Sec
15,25] MVIIA, in ω-MVIIA aminoacid sequence the 15th and 25 halfcystines are replaced by seleno-cysteine respectively)
NO.5：U?KGKGAKU?SRLMYD?CU?TGS?U?R?SGK?C
*；
(intend ω-MVIIA peptide D, [Sec
1,16,8,20] MVIIA, in ω-MVIIA aminoacid sequence the 1st and 16, the 15th and 25 halfcystines are replaced by seleno-cysteine respectively)
NO.6：C?KGKGAK?U?SRLMYD?UC?TGS?U?R?SGK?U
*；
(intend ω-MVIIA peptide E, [Sec
8,20,15,25] MVIIA, in ω-MVIIA aminoacid sequence the 8th and 20, the 15th and 25 halfcystines are replaced by seleno-cysteine respectively)
NO.7：U?KGKGAK?C?SRLMYD?UU?TGS?C?R?SGK?U
*；
(intend ω-MVIIA peptide F, [Sec
1,16,15,25] MVIIA, the 1st and 16, the 15th and 25 halfcystines are replaced by seleno-cysteine respectively in ω-MVIIA aminoacid sequence)
NO.8：U?KGKGAK?U?SRLMYDUUTGS?U?R?SGK?U
*。
(intend ω-MVIIA peptide G, [Sec
1,16,8,20,15,25] MVIIA, the 1st and 16, the 8th and 20, the 15th and 25 halfcystines are replaced by seleno-cysteine respectively in ω-MVIIA aminoacid sequence)
In the above peptide sequence, form three pairs of disulfide linkage and/or two selenium keys between the 1st and 16,8 and 20,15 and 25 the amino-acid residue respectively.
More preferred sequence is with the formed sequence of a pair of halfcystine in the aminoacid sequence of seleno-cysteine replacement ω-MVIIA peptide, the sequence shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.
Another object of the present invention is to provide the pharmaceutical composition that contains bioactive peptide of the present invention, it contains plan ω of the present invention-MVIIA peptide and pharmaceutically acceptable carrier and/or thinner, and pharmaceutical composition of the present invention can be prepared into suitable formulation as required.
The preparation method who intends ω-MVIIA peptide that also provides of the present invention.It will be understood by those skilled in the art that on the basis of the above-mentioned aminoacid sequence of the present invention, can adopt various methods known in the art to prepare peptide of the present invention, for example: expression of synthetic aminoacid sequence, genetically engineered recombinant DNA carrier or the like.Preferred manufacturing procedure is a solid phase synthesis process, comprises the folding and purification step of synthetic, the linear peptides of linear peptides.
The synthetic employing Boc in-situ chemical solid-phase synthesis of linear peptides based on the chemiluminescent polypeptide composition principle,, is begun to synthesize with Boc amino acid as condensing agent by HBTU, and synthesis step is with reference to the solid phase synthesis handbook.Carry out in the 0.1M ammonium formate solution of the folding pH4.0 of being reflected at～8.5 of linear peptides, preferred pH value of solution value is 4.5 and 8.3.
Solid phase synthesis seleno conotoxin adopts the HPLC Analysis and Identification more.
The circular dichroism spectroscopic identification shows that disulfide linkage matching method and the beta sheet of intending ω-MVIIA peptide are similar to conotoxin ω-MVIIA, shows that intending the ω-tertiary structure of MVIIA peptide has kept being similar to the active structure of conotoxin ω-MVIIA.
Proteinic oxidizing and refolding is the process of a complexity, might form the different polytype molecular configurations of disulfide linkage paired mode.Polypeptide just has its biological activity when only being correct folded conformation form.The oxidizing and refolding process of therefore synthetic polypeptide is to influence the active committed step of polypeptide product.The present invention utilizes the difference of two selenium keys and disulfide linkage bond energy, designed employing selenium replace halfcystine (Selenocysteine, Sec) replace thiocysteine (Cysteine, Cys) and the plan ω-MVIIA peptide that forms.Thereby by the controlled oxidation reduction reaction conditions, contained Sec and/or Cys are paired into the accuracy of key in the synthetic polypeptide of assurance.
A further object of the present invention is to provide the purposes of aforementioned polypeptides, and the seleno that the present invention obtains is intended ω-MVIIA derivative biologically active, has analgesic activity.The plan ω of solid phase synthesis-MVIIA peptide acts on the HEK293 cell of transient expression L, N, P/Q, four kinds of hypotype voltage-dependent ca channels of R, optionally suppress N type Calcium Current, show that intending ω-MVIIA peptide can be used as a kind of potential N type voltage-dependent ca channel blocker performance analgesic activity.The test of rat internal metabolism shows, intends ω-MVIIA peptide and obviously prolongs than conotoxin ω-MVIIA in the intravital transformation period of rat.Therefore plan ω-MVIIA peptide that the present invention obtained can further be applied to the no addicted analgesics thing of preparation.
ω-MVIIA compares with conotoxin, and the folding accuracy of the present invention's plan ω-MVIIA peptide improves, and polypeptide stability is better, inhibition to N type calcium road electric current, its effect is more obvious, and has the longer transformation period, can correspondingly reduce the dosage of keeping drug effect.Compare with the chemiluminescent polypeptide synthetic method that conotoxin ω-MVIIA adopts, the present invention's the ω of planing-MVII peptide adopts polypeptide solid state chemistry synthetic method to prepare, and steady quality, cost are lower.
Description of drawings
Fig. 1 is conotoxin ω-MVIIA and intends ω-MVIIA peptide circular dichroism spectroscopic identification CD collection of illustrative plates;
Fig. 2 intends the impact analysis figure of ω-MVIIA peptide to the N type passage activated calcium current of expression;
Fig. 3 intends the impact analysis figure of ω-MVIIA peptide to the non-N type passage of expression.
Embodiment
Further set forth the present invention below in conjunction with example, will help those of ordinary skill in the art further to understand content of the present invention, but should be appreciated that listed examples only plays illustration, and do not limit the present invention in any form.
[embodiment 1] [Sec
1,16] synthetic (the intending ω-MVIIA peptide A) of MVIIA
(1) solid phase synthesis: according to intending ω-MVIIA peptide [Sec
1,16] the MVIIA aminoacid sequence, use Boc solid state chemistry synthesis method, adopt HBTU as coupling reagent and Boc-amino acid (Sigma), MBHA (4-methylbenzhy-drylamine) resin (Advanced Technology ﹠amp; Industrial company) carries out synthetic.Boc-Sec (MeBzl)-OH's is synthetic: 5g seleno-cysteine (Sigma) adds in the 20ml 1M NaOH solution, and 0 ℃ of stirring adds the sodium borohydride solution of 20ml 0.8M, about 40 minutes simultaneously.Regulate pH to 7.0 with Glacial acetic acid.1.5g in the step reaction solution, 0 ℃ was reacted 2.5 hours before alpha-brominated xylene soluble in 20ml ethanol, was added dropwise to.6M hydrochloric acid is regulated pH to 2, obtains white precipitate and promptly gets 4-phenmethyl-L-seleno-cysteine.4-phenmethyl-L-seleno-cysteine 4g and K
2CO
3Be dissolved in the 30ml water 4.5g mix, added 3.2g tertbutyloxycarbonyl-sodium bicarbonate stirring at room 1.5 hours, after add water 150ml.After diethyl ether was washed twice, it was 4.0 that water layer is transferred ph with citric acid.Equal-volume ethyl acetate extraction three times, 10% citric acid solution are given a baby a bath on the third day after its birth inferior, filter, and vacuum-drying gets Boc-Sec (MeBzl)-OH.The cracking of peptide chain resin, to first (benzene) phenol/toluene-(18: 1: 1, volume ratio) solution effects polypeptide, 0 ℃ was reacted about 2.5 hours with HF/, and vacuum is removed HF, and the cold ethyl acetate washing precipitation promptly gets crude product.
(2) two selenium keys/disulfide linkage forms and the peptide chain folding: the gained crude product is dissolved in 400ml 0.1M ammonium formiate, in 50% aqueous isopropanol, and 4.3～4.8 oxidations of pH value, two selenium keys form.After this use 0.01M MH
4OH solution is regulated pH value to 8.0～8.5, and further oxidation forms disulfide linkage.Regularly take a sample from reaction solution, the HPLC method is analyzed.Oxidation finishes to regulate pH value 2～3.Vacuum is removed Virahol, by RP-HPLC method purified polypeptide, can obtain the correct folding plan ω-MVIIA peptide A of purity 98%, than ω-efficient raising of MVIIA renaturation more than 10%.
[embodiment 2] synthesize [Sec
8,20], [Sec
15,25], [Sec
1,16,8,20], [Sec
1,16,15,25], [Sec
8,20,15,25], [Sec
1,16,8,20,15,25] MVIIA (intending ω-MVIIA peptide B, C, D, E, F, G)
According to intending ω-MVIIA peptide [Sec
8,20], [Sec
15,25], [Sec
1,16,8,20], [Sec
1,16,15,25], [Sec
8,20,15,25], [Sec
1,16,8,20,15,25] the MVIIA sequence, prepare described plan ω-MVIIA peptide according to described synthetic, the Cheng Jian of embodiment 1, folding step.
[embodiment 3] are intended ω-MVIIA peptide molecular weight and are measured
Intend ω-MVIIA peptide purification freeze-drying, the BIFLEX of Bruker company type MALDI-TOF-MS carries out molecular weight determination.The result shows, replaces plan ω-MVIIA peptide ([Sec of a pair of halfcystine (Cys) with seleno-cysteine (Sec)
1,16] MVIIA, [Sec
8,20] MVIIA, [Sec
15,25] MVIIA) molecular weight is 2732.61Da, 2733.02Da conforms to theoretical molecular.There are two halfcystines to be replaced in 6 halfcystines of prompting formation ω-MVIIA by seleno-cysteine.
[embodiment 4] intend the mensuration of ω-MVIIA peptide ammino acid sequence
Adopt ABI Procise 491 type protein sequencers, with the order-checking of standard Edman degradation method.The present invention's plan ω-MVIIA peptide ammino acid sequence is:
(A)SecLysGlyLysGlyAlaLys?CysSerArgLeuMetTyrAsp?Cys?SecThrGlySerCys?ArgSerGlyLys?Cys
*
(B)CysLysGlyLysGlyAlaLys?SecSerArgLeuMetTyrAsp?Cys?Cys?ThrGlySerSec?ArgSerGlyLys?Cys
*
(C)CysLysGlyLysGlyAlaLys?CysSerArgLeuMetTyrAsp?Sec?CysThrGlySerCys?ArgSerGlyLys?Sec
*
(D)SecLysGlyLysGlyAlaLys?SecSerArgLeuMetTyrAsp?Cys?SecThrGlySer?SecArgSerGlyLys?Cys
*
(E)CysLysGlyLysGlyAlaLys?SecSerArgLeuMetTyrAsp?Cys?Sec?ThrGlySerSec?ArgSerGlyLys?Sec
*
(F)SecLysGlyLysGlyAlaLys?CysSerArgLeuMetTyrAsp?SecSecThrGlySer?Cys?ArgSerGlyLys?Sec
*
(G)SecLysGlyLysGlyAlaLys?SecSerArgLeuMetTyrAsp?Sec?SecThrGlySer?SecArgSerGlyLys?Sec
*
In the above sequence, Sec represents seleno-cysteine,
*Expression peptide chain terminal amideization.
[embodiment 5] intend the circular dichroism spectroscopic identification of ω-MVIIA peptide
Under 25 ℃, respectively conotoxin ω-MVIIA, plan ω-MVIIA peptide aqueous solution are placed the 0.1cm quartz colorimetric utensil, Japanese JASCO J-810 circular dichroism spectrometer 190～250nm scanning.Fig. 1 is ω-MVIIA, the CD collection of illustrative plates of intending ω-MVIIA peptide A.As seen it is similar to ω-MVIIA to intend ω-MVIIA peptide A disulfide linkage matching method and beta sheet, and the tertiary structure of intending ω-MVIIA peptide A does not change.
[embodiment 6] intend ω-MVIIA peptide
1H-NMR detects
U.S. Varian INOVA
1H-NMR (500MHz, D
2O) result is as shown in table 1, and halfcystine among ω-MVIIA (Cys) is ([Sec after seleno-cysteine (Sec) replaces
1,16] MVIIA), link to each other-CH with-SeH
2Middle hydrogenation displacement study changes.
Table 1. is intended ω-MVIIA's
1The H-NMR spectral data
| ??ω-MVIIA | Intend ω-MVIIA peptide A |
| ??3.05(m，2H，C-H) | ??1.7(m，1H，C-H)??1.9(m，1H，C-H) |
[embodiment 7] intend the analgesia pharmacodynamic action of ω-MVIIA peptide
(1) mouse acetic acid twisting method analgesic test
Mouse is divided into blank group and various dose medication group at random.Every group 10.Adopt intracerebroventricular injection to give the various dose medicine, the blank group gives equal-volume physiological saline.After 30 minutes, to 0.6% acetum 0.4ml of the new preparation of each group mouse peritoneal injection.After 5 minutes, that writes down mouse in 15 minutes subsequently turns round the body number of times.Turn round the body discrimination standard: the mouse web portion indent, trunk and back leg are upheld, hips up.
Behind the acetum of normal mouse abdominal injection 0.6%,, mainly show as symptom such as turn round body, draw the abdomen because of acetic acid produces stomachache to the stimulation of peritonaeum.Positive control drug and plan ω-MVIIA peptide A, B, C, D, E, F (icv) all can make the mouse writhing incidence reduce to handle, and good dosage correlation is arranged.At 0.1,0.2,0.4,0.8,1.6 μ gkg
-1In the dosage range, the body incidence of turning round of ω-MVIIA reduces 8.5～80.7%, and A, B, C, D, E, F group mouse writhing incidence reduce 10.8～90.9%, 16.4～89.9%, 30.6～95.9%, 18.1～86.0%, 15.3～87.9%, 13～93.7%.
Prompting is intended ω-MVIIA peptide and have very strong analgesic activity on mouse acetic acid twisting model, and analgesic activities is higher than ω-MVIIA.
Table 2. is intended ω-MVIIA peptide Dichlorodiphenyl Acetate and is turned round the analgesic activity of phantom type mouse
Compare with the physiological saline group,
*P＜0.05
*P＜0.01
Organize relatively with ω-MVIIA,
ΔP＜0.05
The Δ ΔP＜0.01
(2): 55 ℃ of hot plate methods of mouse are surveyed the pain test
Kunming mouse, female.At first measure the basis pain of every mouse and explain, promptly touch the time (s) that metapedes appears licking in mouse for the first time from mouse four limbs and 55 ℃ of hot plate table tops.Reject pain Fujian, basis value less than 5 seconds or greater than 30 seconds mouse.The administration group that qualified mouse is divided at random blank group and various dose.Every group 10.The administration group is the medicine of intracerebroventricular injection various dose respectively, the blank group gave equal-volume physiological saline after 0.5,1,1.5,3,6 hour, survey pain once more, relatively administration front and back mouse is to the variation of hot plate tolerance time, calculate after the administration may maximum analgesia percentage (PMAP), estimate the analgesic effect of medicine.
PMAP (%)=(threshold of pain after the administration-basic threshold of pain)/(60s-basis pain)
The mouse threshold of pain does not have significant difference after giving salt solution in advance.
Positive control drug and analogue are so that (0.1,0.2,0.4,0.8,1.6 μ gkg-1 icv) handle, and the PMAP of mouse all is the raising of dose-dependently.Compare with the salt solution group, have significant difference (table 3).
Prompting is intended ω-MVIIA peptide and have very strong analgesic activity on 55 ℃ of hot plates surveys of mouse pain model, and analgesic activities is higher than ω-MVIIA.
Table 3. is intended the analgesic activity of ω-MVIIA peptide to 55 ℃ of hot plate model mices
Compare with the physiological saline group,
*P＜0.05
*P＜0.01
(3): to the influence of the calcium channel of HEK293 cell transient expression
The patch-clamp whole cell recording mode is adopted in this experiment, by the HEK293 cell of the various high-voltage activated of transient expression calcium channel is studied, and the clear and definite ionic channel target and the selectivity of class omega-conotoxin peptides effect.The liposome transfection method is adopted in this research again, (α 1C (L), α 1B-1N, α 1A-2 (P/Q) or α 1E-3 (R)) mixes transfection HEK293 cell simultaneously with α 2b δ, β 1b subunit respectively with calcium channel α 1 subunit, successfully transient expression L, N, four kinds of calcium channel hypotypes of P/Q, R.This part Study adopts 5mMBa2+ to carry out calcium channel barium electric current (IBa) record as supported ionic.
I) plasmid transfection
The HEK293 cell places 37 ℃ of incubators that contain 5%CO2 to cultivate with the DMEM substratum that contains 10% foetal calf serum and 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, adopts the tryptic digestion method to carry out passage.Mix plasmid and comprise a kind of calcium channel α 1 subunit (α 1C (L type), α 1B-1 (N type), α 1A-2 (P/Q type) or α 1E-3 (R type)), calcium way α 2b δ, β 1b subunit and pEGFP-Cl plasmid, four 1: 1: 1 in molar ratio: 0.3 mixes.Cell transfecting adopts liposome Lipofectamin 2000, and (Invitrogen, Inc.) transfection reagent are operated in strict accordance with the transfection reagent specification sheets.24-72 hour cell is used for patch clamp experiments after the transfection.
The experiment of ii) full cell patch pincers
This part Study adopts 5mM Ba2+ to carry out calcium channel barium electric current (IBa) record as supported ionic.
All experiments are all carried out under room temperature (20 ℃), will cultivate or extracellular fluid rinsing 2 times of acute dispersive cell elder generation, and add the 1.5ml extracellular fluid.Under inverted phase contrast microscope, the recording electrode that will charge liquid in the electrode by little manipulation instrument is pressed to cell surface gently.When the sealing-in impedance increases, electrode is descended slightly, and apply controlled underbalance by recording electrode inside rapidly, make electrode and cell surface form tight sealing-in.Continue to apply negative pressure on this basis or, promptly form full cell record mode with the rupture of membranes of electricimpulse in short-term of Zap.All data adopts the Clmaptfit program of pCLAMP 8.2 softwares to carry out analyzing and processing.
Experimental result
I) intend ω-MVIIA peptide to the calcium channel of HEK293 cell transient expression influence
The result organizes relatively with ω-MVIIA as shown in Figure 2,
*P＜0.05
*P＜0.01.The result shows that on the HEK293 cell of transient expression N type voltage-dependent ca channel, 0.01,0.1,1 μ M intends ω-MVIIA peptide A, B, C are all inhibited to the N type calcium channel high-voltage activated barium electric current (HVA IBa) of expressing; Along with concentration increases, plan ω-MVIIA peptide A, B, C strengthen the restraining effect of N type HVA IBa; When 1 μ M, N type HVA is almost all suppressed by analogue.0.1 μ M, 0.01 μ M and the time, intend ω-MVIIA peptide A, B, C to the inhibiting rate of N type HVA IBa all significantly greater than ziconotide.
Results suggest, plan ω-MVIIA peptide A, B, C can suppress N type Calcium Current, and to the inhibition of N type Calcium Current, the effect of intending ω-MVIIA peptide is more obvious.
Ii) intend of the influence of ω-MVIIA peptide to the non-N type calcium channel of expression
The result as shown in Figure 3.The result shows, when 1 μ M, N type electric current is almost completely suppressed by analogue, but the HEK293 cell of transient expression L, P/Q, R type calcium channel gives after the 1 μ M analogue, the current density of above-mentioned calcium channel HVA IBa does not have considerable change (P＞0.05), explanation is when 1 μ M, and plan ω-MVIIA peptide does not have obvious influence to L, P/Q, the R type calcium channel of transient expression.
Conclusion
By above-mentioned research, as seen intend ω-MVIIA peptide and optionally suppress N type Calcium Current, can be used as a kind of potential N type voltage-dependent ca channel blocker performance analgesic activity.
(4) transformation period experiment in the cerebral tissue
Adopt chloramine-t method with
125I is mark ω-MVIIA, plan ω-MVIIA peptide A, B, C respectively.Use the C18 column separating purification
125The I marker is collected all cpds of single iodine labeling, measures emission ratio activity, radiochemicsl purity respectively, adds 5% trehalose freeze-drying in the marker, in-20 ℃ of preservations.Mix by a certain percentage during experiment
125The unlabelled medicine of the medicine of I mark and physiological saline solution is mixed with certain density solution injection, and radioactive concentration is 50 μ Ci/mL.
60 of Wistar rats, male and female half and half, be divided into 10 groups at random, adopt spinal cord sheath inner sleeve administration 2 μ g/kg, after the administration in 0.1,0.25,0.5,1,2,4,6,8,12 and 24h put to death a treated animal respectively, get cerebral tissue and make homogenate in 1: 5 ratio, get 400 μ L and measure gross activity, radiocounting is converted into the content of intending ω-MVIIA peptide A, B, C respectively with ultrapure water.Each time point is got 6 animals, male and female half and half.
Above concentration-time experimental data is carried out data processing with the 3P97 medicine for computation program, calculate the elimination transformation period in the cerebral tissue.
The result shows, intend ω-MVIIA peptide A, B, the administration of C single dose spinal cord sheath inner sleeve after, the elimination transformation period t in cerebral tissue
1/2Average out to 7.06h, 6.2h, 8.5h are all than the elimination transformation period t of ω-MVIIA
1/24.6h tangible prolongation is arranged.
08P103358.ST25.txt
SEQUENCE?LISTING
＜110〉Shenzhen Haiwang Pharmaceutical Co., Ltd
＜120〉a kind of selenium-containing active polypeptide and preparation method thereof and application
<130>SZ85-08P103358
<160>8
<170>PatentIn?version?3.1
<210>1
<211>25
<212>PRT
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(25)
<223>X＝Selenocysteine(U)or?X＝Cysteine(C)
<400>1
Xaa?Lys?Gly?Lys?Gly?Ala?Lys?Xaa?Ser?Arg?Leu?Met?Tyr?Asp?Xaa?Xaa
1???????????????5???????????????????10??????????????????15
Thr?Gly?Ser?Xaa?Arg?Ser?Gly?Lys?Xaa
20??????????????????25
<210>2
<211>25
<212>PRT
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(25)
<223>X＝Selenocysteine(U)
<400>2
Xaa?Lys?Gly?Lys?Gly?Ala?Lys?Cys?Ser?Arg?Leu?Met?Tyr?Asp?Cys?Xaa
1???????????????5???????????????????10??????????????????15
Thr?Gly?Ser?Cys?Arg?Ser?Gly?Lys?Cys
20??????????????????25
<210>3
<211>25
<212>PRT
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(25)
<223>X＝Selenocysteine(U)
<400>3
Cys?Lys?Gly?Lys?Gly?Ala?Lys?Xaa?Ser?Arg?Leu?Met?Tyr?Asp?Cys?Cys
1???????????????5???????????????????10??????????????????15
Thr?Gly?Ser?Xaa?Arg?Ser?Gly?Lys?Cys
20??????????????????25
<210>4
<211>25
<212>PRT
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(25)
<223>X＝Selenocysteine(U)
<400>4
Cys?Lys?Gly?Lys?Gly?Ala?Lys?Cys?Ser?Arg?Leu?Met?Tyr?Asp?Xaa?Cys
1???????????????5???????????????????10??????????????????15
Thr?Gly?Ser?Cys?Arg?Ser?Gly?Lys?Xaa
20??????????????????25
<210>5
<211>25
<212>PRT
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(25)
<223>X＝Selenocysteine(U)
<400>5
Xaa?Lys?G1y?Lys?Gly?Ala?Lys?Xaa?Ser?Arg?Leu?Met?Tyr?Asp?Cys?Xaa
1???????????????5???????????????????10??????????????????15
Thr?Gly?Ser?Xaa?Arg?Ser?Gly?Lys?Cys
20??????????????????25
<210>6
<211>25
<212>PRT
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(25)
<223>X＝Selenocysteine(U)
<400>6
Cys?Lys?G1y?Lys?Gly?A1a?Lys?Xaa?Ser?Arg?Leu?Met?Tyr?Asp?Xaa?Cys
1???????????????5???????????????????10??????????????????15
Thr?Gly?Ser?Xaa?Arg?Ser?Gly?Lys?Xaa
20??????????????????25
<210>7
<211>25
<212>PRT
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(25)
<223>X＝Selenocysteine(U)
<400>7
Xaa?Lys?Gly?Lys?Gly?Ala?Lys?Cys?Ser?Arg?Leu?Met?Tyr?Asp?Xaa?Xaa
1???????????????5???????????????????10??????????????????15
Thr?Gly?Ser?Cys?Arg?Ser?Gly?Lys?Xaa
20??????????????????25
<210>8
<211>25
<212>PRT
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(25)
<223>X＝Selenocysteine(U)
<400>8
Xaa?Lys?Gly?Lys?Gly?Ala?Lys?Xaa?Ser?Arg?Leu?Met?Tyr?Asp?Xaa?Xaa
1???????????????5???????????????????10??????????????????15
Thr?Gly?Ser?Xaa?Arg?Ser?Gly?Lys?Xaa
20??????????????????25
Claims (8)
1, a kind of selenium-containing active polypeptide is characterized in that it has the sequence shown in SEQ ID NO.1, and in described sequence,
(1) the 1st, 16, the 8th, 20, the 15th, 25 X is halfcystine in couples or be seleno-cysteine in couples;
(2) the 1st, 16, the 8th, 20, among the 15th, the 25 amino acids X, has a pair of seleno-cysteine that is simultaneously at least; And
(3) when forming the tertiary structure of peptide, the 1st and 16, the 8 and 20, the 15 and 25 halfcystine and/or seleno-cysteine form two selenium key and/or disulfide linkage respectively in couples.
2, selenium-containing active polypeptide as claimed in claim 1 is characterized in that described peptide has to be selected from following aminoacid sequence: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.
3, the described selenium-containing active polypeptide of claim 1 is characterized in that the amidation of described peptide chain C-terminal.
4, the pharmaceutical composition that contains the described selenium-containing active polypeptide of claim 1.
5, a kind of method for preparing the described selenium-containing active polypeptide of claim 1 may further comprise the steps:
1) according to the aminoacid sequence of selenium-containing active polypeptide, the synthesizing linear peptide;
2) linear peptides with the step 1) gained carries out the folding of peptide chain in the 0.1M of pH4.0～8.5 ammonium formate solution, forms two selenium keys;
3) separate and purification step 2) peptide that obtains, obtain the described polypeptide of claim 1.
6, preparation method as claimed in claim 5 wherein adopts solid-phase synthesis synthesis step 1) in linear peptides.
7, the application of the described selenium-containing active polypeptide of claim 1 in the preparation medicine relevant with N type calcium channel blocker.
8, the described application of claim 7, wherein said medicine are analgesic.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810067718 CN101602796B (en) | 2008-06-13 | 2008-06-13 | Selenium-containing active polypeptide, preparing method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810067718 CN101602796B (en) | 2008-06-13 | 2008-06-13 | Selenium-containing active polypeptide, preparing method and application thereof |
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| CN101602796A true CN101602796A (en) | 2009-12-16 |
| CN101602796B CN101602796B (en) | 2013-10-16 |
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| Application Number | Title | Priority Date | Filing Date |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160221941A1 (en) * | 2015-02-02 | 2016-08-04 | University Of Vermont And State Agricultural College | Selenocystine derivatives, alpha-methylselenocysteine, alpha-methylselenocysteine derivatives, and methods of making and using same |
| CN106167510A (en) * | 2016-03-11 | 2016-11-30 | 苏秀兰 | A kind of double selenium polypeptide and preparation method thereof |
| CN107043411A (en) * | 2017-02-23 | 2017-08-15 | 中国海洋大学 | Polypeptide and its synthetic method with potential analgesia property |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100531795C (en) * | 2006-07-11 | 2009-08-26 | 浙江大学 | Omega-conotoxin M VII A mutant and its preparation and uses |
-
2008
- 2008-06-13 CN CN 200810067718 patent/CN101602796B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160221941A1 (en) * | 2015-02-02 | 2016-08-04 | University Of Vermont And State Agricultural College | Selenocystine derivatives, alpha-methylselenocysteine, alpha-methylselenocysteine derivatives, and methods of making and using same |
| CN106167510A (en) * | 2016-03-11 | 2016-11-30 | 苏秀兰 | A kind of double selenium polypeptide and preparation method thereof |
| CN107043411A (en) * | 2017-02-23 | 2017-08-15 | 中国海洋大学 | Polypeptide and its synthetic method with potential analgesia property |
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| Publication number | Publication date |
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| CN101602796B (en) | 2013-10-16 |
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