CN102174097B - Actinoporin and application thereof - Google Patents
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- CN102174097B CN102174097B CN 201110027250 CN201110027250A CN102174097B CN 102174097 B CN102174097 B CN 102174097B CN 201110027250 CN201110027250 CN 201110027250 CN 201110027250 A CN201110027250 A CN 201110027250A CN 102174097 B CN102174097 B CN 102174097B
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Abstract
The invention relates to the technical field of marine organisms, in particular to a coding gene, an amino acid sequence and application of actinoporin. The actinoporin is a protein family which has the molecular weight of 20KD or so and specificity preference on membrane sphingomyelin and has quite wide physiological activities comprising myocardium poison, cytolytic effect, pulmonary edema causing, antibiosis, tumor resistance, and the like. The invention aims to provide novel actinoporin, and a nucleotide sequence of the coding gene of the actinoporin is shown as SEQ ID NO:1. The actinoporin disclosed by the invention has strong hemolytic activity and the activity of sphingomyelinase.
Description
Technical field
The present invention relates to the marine biotechnology field, be specifically related to a kind of encoding gene, aminoacid sequence and application thereof of sea anemone cytolysin.
Background technology
In the marine organisms, much can produce toxin, such as sea snake, jellyfish, sea anemone, cone shell etc.These toxin are unique, have widely that neural system is active, the various biological such as cardiovascular system activity and cytoactive are active, hold out broad prospects in research and the application facet of biomedical, molecular biology and pharmacy.
The tentacle of sea anemone and health all contain a large amount of thread cells, the organoid that a special cryptomere is arranged in each thread cell, stinging capsule by name, the interior venom that storing, thread tube is tortuous in stinging capsule, the ecthoaeum tip is pricker, when being subject to machinery or chemical stimulation, can discharging ecthoaeum and thrust the prey health and by thread tube toxin is injected in the prey body.To the research of congestin from 20 century 70s, from about 40 kinds of sea anemones, be separated at present and surpassed 300 kinds of toxin, according to the difference of molecular weight and biological function, can be divided into sea anemone synocytotoxin and sea anemone polypeptide class neurotoxin two large classes.
Sea anemone cytolysin (actinoporin) be the molecule amount about 20KD, sphingophospholipid on the film is had the protein family of a class of specificity preference.At present, from more than 20 kinds of sea anemones isolation identification more than 30 kind of cytolysin.The Equinatoxin-2 that separates from sea anemone Actinia equina is the two kinds of the most deep actinoporin that study at present with the Sticholysins-2 that separates from sea anemone Stichodactylahelianthus.Their aminoacid sequence is measured, and has made mensuration (Lanio, the M.E. of gene clone, expression and functional study and crystalline structure, Morera, V., Alvarez, C., Tejuca, M., Gomez, T., Pazos, F., Besada, V., Martinez, D., Huerta, V., Padron, G., de los Angeles Chavez, M., 2001.Purificationand characterization of two hemolysins from Stichodactyla helianthus.Toxicon 39,187-194; Macek, P., Lebez, D., 1988.Isolation and characterization of three lethaland hemolytic toxins from the sea anemone Actinia equina L.Toxicon 26,441-451.).
The sea anemone cytolysin is the most frequently used model proteins of research eukaryote pore-forming toxoid and membrane interaction.At present research thinks that the machine-processed process that fenestra forms is as follows: the negative charge on the positive charge of toxin and phospholipid bilayer surface attracts each other and close, is reversing process; Then, the N of convertible conformation holds amphiphilic helical region to insert in the film, and certain density toxin protein polymerization formation three or the tetramer form cationic channel, and this process is non-reversible process.
The sea anemone cytolysin has very widely physiologically active, comprise myocardium toxicity, molten cytosis, the pulmonary edema that causes, antibiotic, antitumor etc., wherein most typical is to form perforation at cytolemma, makes cytoclasis, dissolving, at antibiotic, desinsection, anti-tumor aspect using value is arranged.Sticholysins-2 is 30ng/ml to erythrocytic half concentration of ordinary dissolution.To studies show that of mouse isolated heart, Equinatoxin-2 reduces the left ventricle blood pressure, cause the release of irregular pulse and serum lactic dehydrogenase (LDH), finally cause heart failure (Bunc, M., Drevensek, G., Budihna, M., Suput, D., 1999.Effects of equinatoxin II from Actiniaequina (L.) on isolated rat heart:the role of direct cardiotoxic effects in equinatoxin IIlethality.Toxicon 37,109-123.).
Along with the growing interest to oceanic resources, newfound sea anemone cytolysin can be on the increase, and research also will deepen continuously to the structure and function of sea anemone cytolysin.Extensive and deep research helps rationally and fully to utilize the sea anemone resource of China's abundant to Actinia neurotoxin, for the sting treatment measure of hindering of new drug development and sea anemone all significant.
Summary of the invention
The object of the invention provides a kind of new sea anemone cytolysin encoding gene, aminoacid sequence, carrier, and the application in pharmacy.
Used sea anemone is Stichodactyla mertensii (Heteractis magnifica) among the present invention, picks up from South China Sea.Its morphological specificity is as follows: Stichodactyla mertensii, claim again huge different spoke sea anemone, and cylinder is red, tentacle is brown.Tentacle is suitable to terminal diameter from base portion, is straight-tube shape.The diameter of whole strain sea anemone is about 1m.
Carry out protein separation through the tentacle to this Stichodactyla mertensii, obtained the synocytotoxin Cytolysin-4 of the about 19KD of molecular weight, Cytolysin-4 is carried out the order-checking of N end, obtain 20 aminoacid sequences of N end.On this basis, the design primer, the cDNA sequence of amplification Cytolysin-4 from the total RNA of sea anemone tentacle, obtained the cDNA full length sequence of Cytolysin-4, its cDNA total length is 629bp, sequence is submitted Genbank carry out the homologous sequence comparison, the result shows with the sea anemone cytolysin nucleotide sequence of having reported incomplete same, is a new toxin protein.CLUSTLW aminoacid sequence comparison result shows, Cytolysin-4 and the sea anemone cytolysin Cytolysin-3, RTX-A, Sticholysin-1 and the Sticholysin-2 that have reported, higher homology is arranged, and similarity is respectively 90%, 88%, 83%, 87%.
The invention provides the encoding gene of a kind of sea anemone cytolysin Cytolysin-4, its nucleotide sequence is as follows:
CTCATCGTCTTATTTCTGATCGTTACCATGATATGTGCGACCATCGCTGTGCC
ATCTGGAAAGGAATTCCAAGGCAGAAAAGAGGACAAGAAGAGTGCAGCA
CTCGCTGGCACAATTATCGAAGGGGCAAGTCTAGGTTTCCAAATCCTTGAC
AAAGTACTCACAGAACTTGGTAAAGTGTCTCGGAAGATTGCTATCGGTATC
GACAACGAGTCAGGAGGGTCATGGACAGCAATGAATGCATATTTCCGTTCT
GGTACTACAGACGTCATTCTACCAGAGTTTGTCCCAAACCAAAAAGCGCTA
CTCTACAGCGGTCGGAAGGACACAGGCCCTGTTGCAACGGGCGCTGTGGG
TGCCCTTGCCTATTACATGAGCGATGGAAACACTCTTGCCGTTATGTTCAGC
GTTCCCTTTGACTACAACCTGTACAGCAACTGGTGGGATGTCAGAGTCTAT
AGTGGGAAGAGGAGAGCCGACCAAAAGATGTACGAAGACCTCTATAATGG
CTCTCCATTTCGAGGGGACAATGGATGGCACCAGAAGAATCTTGGATATGG
ACTGAGGATGAAGGGAATCATGACAAGTGCTGGCGAAGCAAAACTGCAA
ATTAGGATTTCACGC
TAATA(SEQ ID NO:1, the line part is not translated)
The present invention also provides the aminoacid sequence of a kind of sea anemone cytolysin Cytolysin-4 as follows:
LIVLFLIVTMICATIAVPSGKEFQGRKEDKKSA
ALAGTIIEGASLGFQILDKVLT
ELGKVSRKIAIGIDNESGGSWTAMNAYFRSGTTDVILPEFVPNQKALLYSGRK
DTGPVATGAVGALAYYMSDGNTLAVMFSVPFDYNLYSNWWDVRVYSGKRR
ADQKMYEDLYNGSPFRGDNGWHQKNLGYGLRMKGIMTSAGEAKLQIRISR
(SEQ ID NO:2, the line part is maturation protein)
The present invention also provides the application of sea anemone cytolysin Cytolysin-4 in preparation treatment sea anemone stings vulnerary thing, haemolysis medicine, antitumor drug.
The present invention finds that it has very strong hemolytic activity, and has the activity of sphingomyelinase by the property research to sea anemone cytolysin Cytolysin-4, can be hydrolyzed specifically the sphingophospholipid on various cytolemma and the liposome.
The invention provides sequence information and the mechanism of action of a kind of new synocytotoxin PROTEIN C ytolysin-4, can be the treatment sea anemone stings to hinder new thinking is provided, simultaneously, thereby the sphingophospholipid that Cytolysin-4 can be hydrolyzed on the cytolemma specifically destroys the cell membrane integrity, but the film of wearing of ancillary drug absorbs, and might bring into play certain effect in oncotherapy.
Description of drawings
Fig. 1 is the sphingomyelinase activity experiment result of sea anemone cytolysin Cytolysin-4 of the present invention.
Embodiment
Describe the present invention below in conjunction with embodiment.But the following example should not regarded limitation of the scope of the invention as.
Embodiment 1: the separation and purification of sea anemone cytolysin Cytolysin-4
Get Stichodactyla mertensii (picking up from South China Sea, 17.3 ° of N of north latitude, 113 ° of E of east longitude) tentacle and organize 5g to shred, add the cold deionized water homogenate of 20ml, 15000rpm removed residue in centrifugal 20 minutes.0.45 the filter of μ m filters the thick poison that namely obtains congestin, is stored in-75 ℃.Get HitrapCapto S cationic exchange coloum on the thick malicious sample of 1ml, this post is used ammonium acetate buffer (pH 5.5) balance of 0.05mol/L in advance.The complete rear usefulness 0%, 11%, 21%, 30% of loading and 100%1mol/L sodium-chlor damping fluid (ammonium acetate that contains 0.05mol/L, pH 5.5) gradient elution, each gradient elution time is 5min, flow velocity is 1ml/min.Collecting elution time is component and the lyophilize of 11-14min.The freeze-drying sample is redissolved with 0.15mol/L ammonium acetate buffer (pH 5.5), get 2ml and cross HiLoad 16/60Superdex 75prep grade gel chromatography column, with same damping fluid balance and wash-out, flow velocity is 1ml/min.Collecting elution volume is component and the lyophilize of 83-88ml, namely obtains the sterling of Cytolysin-4, Cytolysin-4 is kept at-75 ℃.Other takes a morsel and carries out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), shows that its molecular weight is about 19KD.
Embodiment 2: the total RNA of Stichodactyla mertensii tentacle extracts and cDNA synthesizes
Preparation is without the utensil of RNA enzyme, and the extraction of the total RNA of Stichodactyla mertensii tentacle is according to the specification sheets operation of the RNAiso Plus of TaKaRa company; The synthetic specification sheets operation according to Fermentas RevertAid First StrandcDNA Synthesis Kit of cDNA.The total RNA that extracts illustrates that through the visible 18S of electrophoresis detection and 28S two bands the integrity of total RNA is better.
Embodiment 3:Cytolysin-4 gene cloning, sequencing and analysis
According to two primers of CDS sequences Design of the higher sea anemone cytolysin Hmg III of Cytolysin-4 homology,
Upstream primer is: 5 ' CGTCTCATCGTCTTATTT 3 ' (SEQ ID NO:3);
Downstream primer is: 5 ' TGTCTTCTTATTAGCGTG 3 ' (SEQ ID NO:4).
The cDNA that obtains take embodiment 2 carries out pcr amplification as template, and agarose electrophoresis detects, and the specific amplification band is arranged near 630bp.Reclaiming this band delivers to Shanghai Ying Jun Bioisystech Co., Ltd (Invitrogen) and carries out determining nucleic acid sequence, the sequence that obtains is carried out the analysis of blast homology, the result shows with the sea anemone cytolysin nucleotide sequence of having reported incomplete same, is a kind of new toxin protein.CLUSTLW aminoacid sequence comparison result shows, Cytolysin-4 and the sea anemone cytolysin Cytolysin-3, RTX-A, Sticholysin-1 and the Sticholysin-2 that have reported, higher homology is arranged, and similarity is respectively 90%, 88%, 83%, 87%.
Embodiment 4: the hemolytic activity of sea anemone cytolysin Cytolysin-4 of the present invention
Ripe human blood is limpid to supernatant several times with the PBS rinsing, abandon supernatant.Red corpuscle and PBS be by dilution in 1: 50, reaction cumulative volume 1.5ml, red cell suspension 0.5ml wherein, the common 1ml of PBS and Cytolysin-4.Establish in addition negative control (not adding toxin), positive control (0.5%Triton X-100) for every group.37 ℃ of vibration 30min behind the application of sample, then the centrifugal 5min of 3000 * g gets supernatant and detects the 420nm absorbance, repeats 3 times, calculates the haemolysis mark.
Haemolysis mark=[(sample hose absorbance-negative control pipe absorbance)/positive control pipe absorbance] * 100%.
The result shows when the concentration of Cytolysin-4 is 40ng/ml, can cause 50% erythrocyte hemolysis, illustrates that this cytolysin has very strong hemolytic activity.Its hemolytic activity is with basically identical from the Sticholysins-1 that separates among the sea anemone Stichodactylahelianthus and Sticholysins-2, and they cause that the concentration of 50% erythrocyte hemolysis is 30-45ng/ml approximately.
Embodiment 5: the sphingomyelinase of sea anemone cytolysin Cytolysin-4 of the present invention is active
TNPAL-sphingomyelin (the amino lauroyl sphingophospholipid of trinitrobenzene is available from Sigma company) is the sphingophospholipid derivative with the colour developing group of synthetic, is the specific substrate of sphingomyelinase.After this substrate and the sphingomyelinase reaction, hydrolysis produces TNPAL colour developing group, and this group has absorption value at the 330nm place.Cytolysin-4 and the TNPAL-sphingomyelin of different concns (0.15 μ g, 0.3 μ g, 0.6 μ g, 1.2 μ g, 2.4 μ g) are reacted 2min at 37 ℃, and then termination reaction detects absorbance at 330nm.Positive control is commercial sphingomyelinase from streptococcus aureus.The result shows that the Cytolysin-4 of 0.3 μ g just can be hydrolyzed the substrate of 3nmol, and it is active to have shown more significant sphingomyelinase, as shown in Figure 1.
Claims (3)
1. sea anemone cytolysin, the nucleotide sequence of its encoding gene is shown in SEQ ID NO:1.
2. sea anemone cytolysin, its aminoacid sequence is shown in SEQ ID NO:2.
3. the application of sea anemone cytolysin according to claim 1 and 2 in preparation haemolysis medicine or antitumor drug.
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| CN103275911B (en) * | 2013-01-02 | 2015-11-25 | 温州医学院 | A kind of intestinal bacteria containing pET-28a (+)-protein400 recombinant plasmid and preparation method thereof |
| CN105294848B (en) * | 2014-06-23 | 2019-02-01 | 中国人民解放军第二军医大学 | A kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody and its preparation and application |
| CN105399835B (en) * | 2015-12-02 | 2019-07-12 | 华东理工大学 | Recombinant immunotoxin Gigantoxin-4-4D5 scFv and its preparation method and application |
| CN114163508A (en) * | 2020-09-11 | 2022-03-11 | 王月志 | Amino acid sequences capable of destroying cells and related nucleotide sequences and related uses |
| CN113980970B (en) * | 2021-09-28 | 2024-06-18 | 中国人民解放军海军特色医学中心 | Aptamer specifically binding with sea anemone cytolysin GT-4 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1594573A (en) * | 2004-07-09 | 2005-03-16 | 中山大学 | Sea anemone neurotoxin gene Sr1 and application thereof |
| CN101538330A (en) * | 2009-04-15 | 2009-09-23 | 广东药学院 | Modified product of reconstructed sea-anemone neurotoxin rhk2a, modifying method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1594573A (en) * | 2004-07-09 | 2005-03-16 | 中山大学 | Sea anemone neurotoxin gene Sr1 and application thereof |
| CN101538330A (en) * | 2009-04-15 | 2009-09-23 | 广东药学院 | Modified product of reconstructed sea-anemone neurotoxin rhk2a, modifying method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Maria Eliana Lanio et al.Purification and characterization of two hemolysins from Stichodactyla helianthus.《Toxicon》.2001,第39卷187-194. * |
| Peter Macek and Drago Lebez.Isolation and characterization of three lethal and hemolytic toxins from the sea anemone Actinia equina L..《Toxicon》.1988,第16卷(第5期),441-451. * |
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