CN1546171A - Huwen tarantula protease inhibitor - Google Patents
Huwen tarantula protease inhibitor Download PDFInfo
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- CN1546171A CN1546171A CNA2003101106081A CN200310110608A CN1546171A CN 1546171 A CN1546171 A CN 1546171A CN A2003101106081 A CNA2003101106081 A CN A2003101106081A CN 200310110608 A CN200310110608 A CN 200310110608A CN 1546171 A CN1546171 A CN 1546171A
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- 229940124158 Protease/peptidase inhibitor Drugs 0.000 title claims abstract description 8
- 239000000137 peptide hydrolase inhibitor Substances 0.000 title claims abstract description 8
- 241000239292 Theraphosidae Species 0.000 title claims 4
- 239000002299 complementary DNA Substances 0.000 claims abstract description 12
- 239000003053 toxin Substances 0.000 claims abstract description 5
- 231100000765 toxin Toxicity 0.000 claims abstract description 5
- 239000002435 venom Substances 0.000 claims description 8
- 231100000611 venom Toxicity 0.000 claims description 8
- 210000001048 venom Anatomy 0.000 claims description 8
- 241000282326 Felis catus Species 0.000 claims description 3
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 claims 2
- SKHCUBQVZJHOFM-NAKRPEOUSA-N Ala-Arg-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SKHCUBQVZJHOFM-NAKRPEOUSA-N 0.000 claims 1
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- DQNLFLGFZAUIOW-FXQIFTODSA-N Arg-Cys-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O DQNLFLGFZAUIOW-FXQIFTODSA-N 0.000 claims 1
- NMRHDSAOIURTNT-RWMBFGLXSA-N Arg-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N NMRHDSAOIURTNT-RWMBFGLXSA-N 0.000 claims 1
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- MCAVASRGVBVPMX-FXQIFTODSA-N Gln-Glu-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MCAVASRGVBVPMX-FXQIFTODSA-N 0.000 claims 1
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- SLQDSYZHHOKQSR-QXEWZRGKSA-N Met-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCSC SLQDSYZHHOKQSR-QXEWZRGKSA-N 0.000 claims 1
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- XOHJOMKCRLHGCY-UNQGMJICSA-N Phe-Pro-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O XOHJOMKCRLHGCY-UNQGMJICSA-N 0.000 claims 1
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Abstract
本发明涉及一种从蜘蛛毒液中获得的蛋白酶抑制剂，它的cDNA全长592bp，一级化学结构由55个氨基酸残基组成，该毒素的理化性状为纯化冻干粉为白色或类白色疏松体，无气味，极易溶解于水，水溶液近于无色透明，该蛋白质的最大紫外吸收波长为280nm，等电点为9.45；它对胰蛋白酶有很强的抑制作用，抑制常数为5.54×10－13M，因此是一种很强的胰蛋白酶抑制剂。The invention relates to a protease inhibitor obtained from spider venom. Its cDNA has a full length of 592bp and its primary chemical structure consists of 55 amino acid residues. The physicochemical properties of the toxin are that the purified freeze-dried powder is white or off-white loose solid, odorless, easily soluble in water, the aqueous solution is nearly colorless and transparent, the maximum ultraviolet absorption wavelength of the protein is 280nm, and the isoelectric point is 9.45; it has a strong inhibitory effect on trypsin, and the inhibition constant is 5.54× 10 -13 M, so it is a strong inhibitor of trypsin.
Description
Technical field:
The present invention relates to a kind of enzyme inhibitor, be specifically related to a kind of protease inhibitor that from spider venom, obtains.
Background technology:
Often contain abundant bioactive molecule in the biological venom, comprise protease inhibitor.They can optionally act on some protease, as trypsin, chymase, plasmin, plasma kallikrein etc.Have and to be developed to the new drug application prospect that helps human health.As, Kunitz type serpin BPTI (bovine pancreatic trypsin inhibitor), it is one of the strongest active protease inhibitor in the world of up to the present finding, it is by combining and the activity of Profilin enzyme with the avtive spot of enzyme, thereby in shock, postpartum hemorrhage, acute circulatory failure, hemorrhagic cerebral edema, the operation and the hemorrhage fields such as control of hyperfibrinolysis that occur after the wound all show better curative effect behind treatment pancreatitis, burn, and certain clinical practice is also being arranged aspect the treatment of AIDS.Found multiple Kunitz type serpin at present in animals and plants, it combines with trypsin at 1: 1, has only an action site.Contain in the spider venom to have in a large number and cause death or benumb active neurotoxin composition, when predation, play an important role; And containing a large amount of digestive enzyme in the Aranea saliva, hydrolysis neurotoxin composition reduces its effect in the venom thereby penetrate into sometimes.Therefore how obtaining a kind of enzyme inhibitor from spider venom is good problem to study.
Summary of the invention:
The present invention is intended to obtain the stronger protease inhibitor of a kind of inhibitory action from spider venom.
The foregoing invention purpose realizes by following scheme:
The cDNA sequence of HWTX-XI is:
ccttgtggag gaagttttct tcagaccccg gtattgtctc gccaggaatc ttggcgcttg 60
tctgtcctga gataccagcg gcggcccgac ttacgcatcc ttccccagga aacctttgaa 120
ggactgaagg cggtgaaaca gcaagct?atg?gga?ata?gca?aga?att?ctc?agt?gca?gtg?ttg 180
Met?Gly?Ile?Ala?Arg?Ile?Leu?Ser?Ala?Val?Leu
-33 -30
tcc?ctc?agc?gtt?ctt?ttc?gtg?gtg?aca?ttt?cct?gcc?ctt?ctc?tca?gcg?gat?cac?cat?gac?240
Phe?Leu?Ser?Val?Leu?Phe?Val?Val?Thr?Phe?Pro?Ala?Leu?Leu?Ser?Ala?Asp?His?His?Asp
-20 -10
gga?aga
ata?gat?aca?tgc?cgt?
ttg?ccc?tct?gac?cgt?ggg?aga?tgc?
aag?gca?tcc?ttt?gaa300
Gly?Arg?Ile?Asp?Thr?Cys?Arg?Leu?Pro?Ser?Asp?Arg?Gly?Arg?Cys?Lys?Ala?Ser?Phe?Glu
-1 1 10
cgt?tgg?tac?ttc?aat?ggc?aga?aca?tgc?
gct?aag?ttt?att?tat?gga?ggc?tgc?ggt?ggc?aac360
Arg?Trp?Tyr?Phe?Asn?Gly?Arg?Thr?Cys?Ala?Lys?Phe?Ile?Tyr?Gly?Gly?Cys?Gly?Gly?Asn
20 30
ggt?aat?aag?ttc?cca?act?
caa?gag?gcc?tgc?atg?aaa?aga?tgt?gca?aaa?gca?taa?414
Gly?Asn?Lys?Phe?Pro?Thr?Gln?Glu?Ala?Cys?Met?Lys?Arg?Cys?Ala?Lys?Ala?End
40 50 55
cggaacacag?acagagcatt?atcgtcaaca?cctacttgag?cgcagccctg?ttcaccaggg?474
gtttcgatga?agtttcgtca?agacctcaac?atggatgttt?taaagtagtg?ctctcaatgt?534
aatgttaaat?acatgtctca?gcaataaaga?tttatacaac?gaaaaaaaaa?aaaaaaaa 592
Below in conjunction with accompanying drawing this research is further described.
Description of drawings:
Fig. 1 is the separation and purification collection of illustrative plates of HWTX-XI (HWTX-XI)
Wherein 1-I is the ion exchange collection of illustrative plates, E represents that purpose peak, 1-II are reversed-phase HPLC collection of illustrative plates for the first time, and it is that the reversed-phase HPLC collection of illustrative plates second time, the 1-IV of purpose toxin is that the mass spectrum of HWTX-XI (HWTX-XI) is identified that 12.5min expresses peak time, 1-III
Fig. 2 is the one-level chemical constitution of HWTX-XI (HWTX-XI)
Fig. 3 is the Tricine SDS-PAGE collection of illustrative plates of the HWTX-XI (HWTX-XI) of expression
Wherein 1 is that low-molecular-weight standard, 2 is the HWTX-XI (HWTX-XI) of natural HWTX-XI (HWTX-XI), 3 for expressing
Fig. 4 is that HWTX-XI (HWTX-XI) suppresses tryptic concentration dependent collection of illustrative plates
Fig. 5 is HWTX-XI (HWTX-XI) and tryptic interaction of biomacromolecules collection of illustrative plates
Fig. 6 is the interaction of biomacromolecules collection of illustrative plates of HWTX-XI (HWTX-XI) and chymase
HWTX-XI (HWTX-XI) can obtain by two kinds of approach: extract from the thick poison of Selenocosmiahuwena and the approach acquisition by gene expression, concrete operations are as follows:
1. the isolation and purification method of the collection of venom and HWTX-XI
(1) acquisition method of venom
With the 3-4 root long for 4cm, internal diameter be that the transparent plastic hose of 1.5mm is tied into a branch of as luring thing, clamp the chest of Selenocosmiahuwena from the side with big tweezers, Aranea is just opened the chela melon, at this moment hose bundle is lured thing to send under the chela melon, it promptly embraces hose bundle with pedipalps, effectively thrusts the chela melon in the flexible pipe and penetrates venom.Penetrate the poison back and under the chela melon, slowly extract hose bundle out, venom is wherein extracted out, behind lyophilization, can obtain dry powder, be thick poison with light golden rod yellow with microsyringe.
(2) isolation and purification method of HWTX-XI
Carry out separating for twice according to proteinic electrically charged character and proteinic hydrophobicity, in conjunction with cation exchange and reversed-phase HPLC chromatography.5 milligrams of above-mentioned rugosity are dissolved in 6 milliliters of distilled waters, last sample is to using in advance in the good Water Protein PakCM 8HR cation exchange column (5 * 50 millimeters) of 0.1M phosphate buffer balance, with 0-81% sodium chloride linear gradient elution, the time is 60 minutes, and flow velocity is 3mL/min.Collect the further reversed-phase HPLC purification in last peak.With the anti-phase analytical column of C18 acetonitrile (containing 0.1% trifluoroacetic acid) linear gradient elution with 20-40% in 40min, collecting retention time is the further reversed-phase HPLC in peak of 12.5min, obtain single goal peak (Fig. 1), mass spectrograph detects to one-component and to record accurate molecular weight be 6166.23 (Fig. 1).
HWTX-XI obtains its one-level protein sequence (Fig. 2) through the 491-A sequenator, is made up of 55 amino acid residues, contains 6 cysteine.According to the difference of theoretical molecular and mass spectroscopy molecular weight, can infer that 6 cysteine have formed three pairs of disulfide bond.The physicochemical properties of this toxin are: purified lyophiled powder is the loose body of white or off-white color, and odorlessness very easily is dissolved in water, and aqueous solution is bordering on water white transparency.This proteinic uv-absorption maximum wavelength is 280nm, and isoelectric point, IP is 9.45, is basic protein.
Separate the Kunitz type serpin that obtains from the thick poison of Selenocosmiahuwena, it has very strong inhibitory action to trypsin, and suppressing constant is 5.54 * 10
-13M also has certain inhibitory action to other some serine proteases simultaneously.
2. genetic engineering clonal expression HWTX-XI (HWTX-XI)
(1) acquisition of the full length cDNA sequence of HWTX-XI
①3′RACE：
Quick extracted total RNA from Selenocosmiahuwena poison gland at first, the total RNA of reverse transcription obtains total cDNA.Aminoacid sequence according to known HWTX-XI designs gene specific degenerate primer p1-a:5 '-TT (T/C) GA (A/G) (A/C) G (A/T/C/G) TGG TA (T/C) TT (T/C) AA (C/T)-3 ' and p1-b:5 '-TG (T/C) GC (A/T/C/G) AA (G/A) TT (T/C) AT (A/C/T) TA (T/C) GG-3 '.Carry out pcr amplification with gene-specific primer and AUAP and a small amount of cDNA chain and obtain the one section cDNA chain of size about 300bp, the purification rear clone is gone into pGEM Teasy carrier (available from Promega company), checks order.
②5′RACE
Design and synthesize antisense primer p2-a:5 '-AATGCTCTGACTGTGTTCCG-3 ' and the p2-b:5 '-TCTTTTCATGCAGGCCTCTTG-3 ' of 5 ' RACE according to the 3 ' RACE sequencing result that has obtained.Do pcr amplification with gene-specific primer and Chao Shi primer, the product purification rear clone is gone into pGEM Teasy carrier (available from Promega company) and is checked order.
3. full-length cDNA
3 ' RACE and 5 ' RACE sequencing result are spliced, obtain the full length cDNA sequence of HWTX-XI.It comprises the pro sequence of the open reading-frame of 3 ' non-translated sequence, 5 ' non-translated sequence, coding molecule precursor, a sophisticated toxin sequence and an insertion.
(2) expression of HWTX-XI
Maturation protein sequential design primer p3-a:5 '-CGTCTAGATAAGAGAATAGATACATGC-3 ' and p3-d:5 '-CCGAAGC TTTATGCTTTTGCACATC-3 ' according to HWTX-XI carry out PCR, obtain the cDNA of maturation protein, cloning and sequencing, the expression of preparing HWTX-XI.Be connected cDNA and transform DH5 α with pVT102U carrier (Chinese Academy of Sciences's Shanghai biochemistry is given with the positive military academician of relative of RESEARCH ON CELL-BIOLOGY institute), change over to again in the host cell S-78 yeast and carry out cell culture.Express 500mL with ammonium sulfate precipitation proPVT, get supernatant after centrifugal to leave standstill half an hour altogether with the DE52 cellulose of handling well, sucking filtration, filter liquor is the plain supernatant that discolors.Filter liquor is transferred pH to 4.2 with acetic acid, go up the CM-Sepharose32 post then, the classification eluting is got the further reversed-phase HPLC purification of target peak.With the anti-phase analytical column of C18 acetonitrile (containing 0.1% trifluoroacetic acid) linear gradient elution with 20-40% in 40min, collecting retention time is the further reversed-phase HPLC in peak of 13min.Identify with MALDI-TOF mass spectrum and Tricine SDS-PAGE then.(Fig. 3).
Can obtain expressing output in this way is 12.5mg/L.
In order to illustrate and disclose the effect of this lps molecule better, we utilize spectrophotometer and interaction of biomacromolecules instrument to measure natural HWTX-XI and express HWTX-XI to tryptic inhibition activity, and the active size with bovine pancreatic trypsin inhibitor BPTI compares simultaneously.
1. main material and instrument
HWTX-XI obtains by above-mentioned two kinds of methods, has identified that by HPLC and MALDI-TOF mass spectrograph its purity is more than 99% simultaneously; Trypsin TPCK handles) be the Sigma product, benzoyl-DL-spermine acyl paranitroanilinum (BAPNA), chymase are Shanghai Bai Ao biotech firm product, other chemical reagent all reach analytical pure.
Experimental apparatus: U-2000 type ultraviolet-visible spectrophotometer (Japanese HITACHI product), surface plasma resonance biological sensor (BIAcore X, Sweden Uppsala product).
2. experimental technique and result
2.1 with the spectrophotometric determination HWTX-XI to trypsin inhibition activity
With BAPNA (5 * 10
-4M) be substrate, add a certain amount of trypsin, get not commensurability HWTX-XI and react with it, entire reaction is carried out in pH8.0, concentration are the Tris-HCL buffer system of 0.05mol/L, and reaction temperature is 25 ℃.Measure absorbance value at 405nm.The result shows: HWTX-XI has very strong inhibitory action to trypsin, and presents concentration dependent relation, and when the ratio of HWTX-XI and tryptic amount of substance was 1: 1, trypsin was suppressed fully, sees Fig. 4.
2.2 surface plasma resonance is measured interaction of biomacromolecules
Coupled to the CM5 chip HWTX-XI, the trypsin of usefulness variable concentrations and chymase flow of solution are through chip surface.Found that HWTX-XI presents very significant concentration dependency relationships with combining of trypsin and chymase.Select for use the BIA data processing software to handle the gained data automatically, can get HWTX-XI and trypsin is that 25 ℃, buffer pH value are to combine very by force under 7.4 the condition at system temperature, and dissociation constant is 5.54 * 10
-13M.The dissociation constant of HWTX-XI and chymase is 1.07 * 10
-7M.(Fig. 5, Fig. 6).
2.3 the active size of the inhibition of HWTX-XI and bovine pancreatic trypsin inhibitor BPTI relatively
With BAPNA (5 * 10
-4M) be substrate, add a certain amount of trypsin, getting not commensurability natural HWTX-XI, expression HWTX-XI and bovine pancreatic trypsin inhibitor BPTI respectively reacts with it, entire reaction is carried out in the Tris-HCL of pH8.0,0.05mol/L buffer system, and reaction temperature is 25 ℃.Measure absorbance value at 405nm.The result shows: the bonded dissociation constant of natural HWTX-XI and trypsin is 3.07 * 10
-8M, expressing HWTX-XI and the bonded dissociation constant of trypsin is 2.91 * 10
-8M, the bonded dissociation constant of bovine pancreatic trypsin inhibitor BPTI and trypsin is 6.85 * 10
-8M.
Above-mentioned three experimental results show that HWTX-XI is a kind of very strong trypsin inhibitor, and its inhibition activity is higher than BPTI.Natural HWTX-XI and the active basically identical of expressing HWTX-XI show that this expression system can produce correct molecular folding.
Selenocosmiahuwena protein inhibitor of the present invention can separation and purification from crude venom of the spider, also can clone acquisition, and suppresses activity and be higher than BPIT, is a kind of very strong trypsin inhibitor.
The cDNA sequence of HWTX-XI is:
ccttgtggag gaagttttct tcagaccccg gtattgtctc gccaggaatc ttggcgcttg 60
tctgtcctga gataccagcg gcggcccgac ttacgcatcc ttccccagga aacctttgaa?120
ggactgaagg cggtgaaaca?gcaagct?atg?gga?ata?gca?aga?att?ctc?agt?gca?gtg?ttg?180
Met?Gly?Ile?Ala?Arg?Ile?Leu?Ser?Ala?Val?Leu
-33 -30
tcc?ctc?agc?gtt?ctt?ttc?gtg?gtg?aca?ttt?cct?gcc?ctt?ctc?tca?gcg?gat?cac?cat?gac?240
Phe?Leu?Ser?Val?Leu?Phe?Val?Val?Thr?Phe?Pro?Ala?Leu?Leu?Ser?Ala?Asp?His?His?Asp
-20 -10
gga?aga
ata?gat?aca?tgc?cgt?
ttg?ccc?tct?gac?cgt?ggg?aga?tgc?
aag?gca?tcc?ttt?gaa300
Gly?Arg?Ile?Asp?Thr?Cys?Arg?Leu?Pro?Ser?Asp?Arg?Gly?Arg?Cys?Lys?Ala?Ser?Phe?Glu
-1 1 10
cgt?tgg?tac?ttc?aat?ggc?aga?aca?tgc?
gct?aag?ttt?att?tat?gga?ggc?tgc?ggt?ggc?aac360
Arg?Trp?Tyr?Phe?Asn?Gly?Arg?Thr?Cys?Ala?Lys?Phe?Ile?Tyr?Gly?Gly?Cys?Gly?Gly?Asn
20 30
ggt?aat?aag?ttc?cca?act?
caa?gag?gcc?tgc?atg?aaa?aga?tgt?gca?aaa?gca?taa?414
Gly?Asn?Lys?Phe?Pro?Thr?Gln?Glu?Ala?Cys?Met?Lys?Arg?Cys?Ala?Lys?Ala?End
40 50 55
cggaacacag?acagagcatt?atcgtcaaca?cctacttgag?cgcagccctg?ttcaccaggg?474
gtttcgatga?agtttcgtca?agacctcaac?atggatgttt?taaagtagtg?ctctcaatgt?534
aatgttaaat?acatgtctca?gcaataaaga?tttatacaac?gaaaaaaaaa?aaaaaaaa?592
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979411A (en) * | 2010-10-21 | 2011-02-23 | 湖南师范大学 | Huwen tarantula protease inhibitor |
| CN102978228A (en) * | 2012-11-19 | 2013-03-20 | 厦门北大之路生物工程有限公司 | Recombination tiger gram pancreatic peptide supporter and preparation method and application thereof |
| CN102978211A (en) * | 2012-11-19 | 2013-03-20 | 厦门北大之路生物工程有限公司 | Interest protein preparation method and purpose thereof |
| CN103014048A (en) * | 2012-11-19 | 2013-04-03 | 厦门北大之路生物工程有限公司 | Preparation method and application of target protein |
-
2003
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979411A (en) * | 2010-10-21 | 2011-02-23 | 湖南师范大学 | Huwen tarantula protease inhibitor |
| CN102978228A (en) * | 2012-11-19 | 2013-03-20 | 厦门北大之路生物工程有限公司 | Recombination tiger gram pancreatic peptide supporter and preparation method and application thereof |
| CN102978211A (en) * | 2012-11-19 | 2013-03-20 | 厦门北大之路生物工程有限公司 | Interest protein preparation method and purpose thereof |
| CN103014048A (en) * | 2012-11-19 | 2013-04-03 | 厦门北大之路生物工程有限公司 | Preparation method and application of target protein |
| CN102978211B (en) * | 2012-11-19 | 2015-04-08 | 未名生物医药有限公司 | Interest protein preparation method and purpose thereof |
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