CN101759779A - Active spider polypeptide, preparation method and application thereof - Google Patents
Active spider polypeptide, preparation method and application thereof Download PDFInfo
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- CN101759779A CN101759779A CN200810154588A CN200810154588A CN101759779A CN 101759779 A CN101759779 A CN 101759779A CN 200810154588 A CN200810154588 A CN 200810154588A CN 200810154588 A CN200810154588 A CN 200810154588A CN 101759779 A CN101759779 A CN 101759779A
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- polypeptide
- arginine
- spider venom
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- phenylalanine
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Abstract
The purpose of the invention is to provides a novel active polypeptide separated from spider venom, a preparation method thereof and the application of the active polypeptide in tumor treatment.
Description
Technical field
The present invention relates to a kind of new active polypeptide that separation obtains from spider venom and its production and application.
Background technology
Traditional medicine has the methods of treatment of " combatting poison with poison ", and spider venom has lethal effect as a kind of natural toxin to tumour cell.Put down in writing medicinal spider " main all malignant boils are swollen, sores such as pyogenic infection of the bone erosion, place wart knurl " according to the supplement to the Herbal of the CHEN Zang-Qi Tang Dynasty.Most document has reported that all spider poison have the composition that suppresses calcium channel.When tumour is invaded when organizing, normal cell distortion on every side, and discharge and can promote or quicken the somatomedin of tumor growth, and Ca
2+Passage may be the signal that normal cell discharges somatomedin, and therefore blocking this passage can help tumor treatment.In in vitro study, some composition of Zhao Weile report lycosa singoriensis poison is to the effect of causing injury of the human lung adenocarcinoma cell of cultivation.Other scholar also reported successively spider venom in vivo with the external growth that obvious inhibition tumour cell is all arranged, inducing apoptosis of tumour cell and necrocytosis effect.Containing a large amount of active polypeptide in the spider venom is the important source that new drug is found.
The contriver searches for comparison with active spider polypeptide complete sequence structure of the present invention through albumen database, finds no any identical polypeptide.
Summary of the invention
The object of the present invention is to provide a kind of separation obtains from spider venom new active polypeptide and its preparation method, and the application of this active polypeptide in oncotherapy.
This active spider polypeptide is a kind of single chain polypeptide, molecular weight 2188.4, iso-electric point 10.49, the polypeptide total order is classified as: L-Ala-Isoleucine-glycine-Threonine-arginine-glutamine-halfcystine-Serine-Serine-aspartic acid-aspartic acid-L-Ala-leucine-leucine-proline(Pro)-phenylalanine-phenylalanine-Methionin-arginine-arginine (AIGTRQCSSDDALLPFFKRR).
This active spider polypeptide preparation method is:
A. the centrifugal 15-25min of spider venom 10000-12000r/min, the supernatant liquor lyophilize;
B. gel-filtration: above-mentioned lyophilized powder 100mg is dissolved in 5mL water, and the centrifugal 8-15min of 10000-12000r/min removes precipitation, and SephadexG-50 gel-filtration column (long 1000mm, diameter 26mm) is used 0.1mol/LNa
2HPO
4-NaH
2PO
4PH6.0 damping fluid balance, fully sample is gone up in the back, with same elutriant wash-out, collects active peak.
C. reverse phase HPLC: contain the abundant balance C18 reversed-phase column of ultrapure water (long 300mm, diameter 5mm) of 1 ‰ trifluoroacetic acids,, collect active peak with the above-mentioned elutriant of the fine buffer solution for gradient elution of second that contains 1 ‰ trifluoroacetic acids.
The fast atom bombardment mass spectroscopy(FABMS) method is adopted in the peptide molecule flow measurement: with glycerine: m-nitrobenzyl alcohol: methyl-sulphoxide (1: 1: 1, volume ratio) is a substrate, Cs
+As projectile, electric current 1 μ A, emission voltage 25Kv.
The polyacrylamide gel isoelectric focusing method is measured polypeptide iso-electric point (Wei and equality, Biochemistry Experiment and guidance, 250-253, Chinese Medicine science and technology press, 2003)
Determined amino acid sequence: sample is placed the hydrolysis pipe, add the hydrochloric acid soln of 6mol/L, vacuum seal.At 110 ℃ of following hydrolysis 24h, cooled and filtered and evaporate to dryness, the hydrochloric acid soln that adds 0.02mol/L is again placed 30min in air.Adopt 835-50 automatic analyzer for amino acids (Japanese Hitachi company) to measure aminoacid sequence.
This polypeptide is used for the treatment of the purposes of tumour by cultured tumor cells in vitro and animality transplantation tumor Growth Inhibition experimental example are illustrated.
Test example one: to the restraining effect of cultured tumor cells in vitro
1, experiment material
96 orifice plates (available from Costar company); The RMPI1640 substratum is available from Gibco company; New-born calf serum is available from Hangzhou folium ilicis chinensis company; Bromination tetrazole indigo plant (MTT) is available from AMRESCO company; DMSO is available from Amresco company.Cell strain: A549 human lung carcinoma cell, SMMC-7721 human liver cancer cell, SK-OV-3 Proliferation of Human Ovarian Cell.
2 experimental programs
Measure bromination tetrazole indigo plant (MTT) method that adopts routinely
(1) get and be in exponential phase of growth, one bottle in cell in good condition, tryptic digestion comes off attached cell, is made into cell suspension with the RMPI1640 nutrient solution that contains 10% calf serum, and counting is inoculated in 96 orifice plates cell density: 0.5-2 * 10
4Individual/mL;
(2) inoculation go into 96 orifice plates cell at 37 ℃, 5%CO
2Incubator was cultivated 24 hours;
(3) medicine is diluted to 10 with nutrient solution
-4Mol/L, 5 * 10
-5Mol/L, 10
-5Mol/L, 10
-6Mol/L, 10
-7Five different concns dosage of mol/L group, 100 μ L/ holes, and set up the solvent contrast, put 37 ℃, 5%CO
2Incubator was cultivated 48 hours;
(4) add MTT after hatching 48h, 20 μ L/ holes continued to hatch 4 hours at incubator;
(5) abandon liquid in the clear opening, every hole adds 200 μ L DMSO again, and shaking table 10 minutes is mixing gently;
(6) on microplate reader, detect, detect wavelength 570nm, with reference to wavelength 630nm.Detect the OD value in every hole, calculate inhibiting rate.
3 experimental results
Experimental studies results shows: the spider venom polypeptide has the obvious suppression effect to A549 human lung carcinoma cell, SMMC-7721 human liver cancer cell, SK-OV-3 Proliferation of Human Ovarian Cell, its IC
50Be respectively 7.18 * 10
-7Mol/L, 9.36 * 10
-7Mol/L, 8.94 * 10
-6Mol/L.
The spider venom polypeptide is to the restraining effect of the tumour cell of vitro culture
Test example two: to the restraining effect of animality transplantation tumor
1. to the restraining effect of mice-transplanted tumor S180
1.1 experimental technique
Get 50 of ICR mouse and inoculate back 24 hours and claim mouse heavy, and be divided into 5 groups at random by transplanted tumor organon inoculation S180 solid-type, 10 every group, male and female half and half.Inoculate administration after 24 hours, intravenous administration, every other day once, administration is 4 times altogether, and the 2nd day mouse weighed after drug withdrawal, puts to death tumor-bearing mice and separates the knurl piece, claims knurl heavy, and the gained data are carried out statistical procedures (t check).
1.2 experimental result
The result shows, compares with the blank group, and 10mg/kg, 5mg/kg, 1mg/kg group spider venom polypeptide and 10mg/kg paclitaxel injection, group all can suppress S180 tumor growth (P＜0.01) significantly and see Table 1.
Table 1 spider venom polypeptide is to the restraining effect of mice-transplanted tumor S180
* compare with the blank group P＜0.05 * * P＜0.01
2. to the restraining effect of mice-transplanted tumor EC
2.1 experimental technique
Get 50 of ICR mouse and inoculate back 24 hours and claim mouse heavy, and be divided into 5 groups at random by transplanted tumor organon inoculation EC solid-type, 10 every group, male and female half and half.Inoculate administration after 24 hours, intravenous administration, every other day once, administration is 4 times altogether, and the 2nd day mouse weighed after drug withdrawal, puts to death tumor-bearing mice and separates the knurl piece, claims knurl heavy, and the gained data are carried out statistical procedures (t check).
2.2 experimental result
The result shows, compares with the blank group, and 10mg/kg, 5mg/kg, 1mg/kg group spider venom polypeptide all can suppress the growth (P＜0.01) of mice transplanted tumor EC significantly.(seeing Table 2).
Table 2 spider venom polypeptide is to the restraining effect of mice-transplanted tumor EC
* compare with the blank group P＜0.05 * * P＜0.01
Embodiment
Embodiment:
The active spider polypeptide preparation method is:
A. the centrifugal 15-25min of spider venom 10000-12000r/min, the supernatant liquor lyophilize;
B. gel-filtration: above-mentioned lyophilized powder 100mg is dissolved in 5mL water, and the centrifugal 8-15min of 10000-12000r/min removes precipitation, and SephadexG-50 gel-filtration column (long 1000mm, diameter 26mm) is used 0.1mol/LNa
2HPO
4-NaH
2PO
4PH6.0 damping fluid balance, fully sample is gone up in the back, with same elutriant wash-out, collects active peak.
C. reverse phase HPLC: contain the abundant balance C18 reversed-phase column of ultrapure water (long 300mm, diameter 5mm) of 1 ‰ trifluoroacetic acids,, collect active peak with the above-mentioned elutriant of the fine buffer solution for gradient elution of second that contains 1 ‰ trifluoroacetic acids.
The fast atom bombardment mass spectroscopy(FABMS) method is adopted in the peptide molecule flow measurement: with glycerine: m-nitrobenzyl alcohol: methyl-sulphoxide (1: 1: 1, volume ratio) is a substrate, Cs
+As projectile, electric current 1 μ A, emission voltage 25Kv.
The polyacrylamide gel isoelectric focusing method is measured polypeptide iso-electric point (Wei and equality, Biochemistry Experiment and guidance, 250-253, Chinese Medicine science and technology press, 2003)
Determined amino acid sequence: sample is placed the hydrolysis pipe, add the hydrochloric acid soln of 6mol/L, vacuum seal.At 110 ℃ of following hydrolysis 24h, cooled and filtered and evaporate to dryness, the hydrochloric acid soln that adds 0.02mol/L is again placed 30min in air.Adopt 835-50 automatic analyzer for amino acids (Japanese Hitachi company) to measure aminoacid sequence.
The spider venom polypeptide that obtains by aforesaid method is a kind of single chain polypeptide, molecular weight 2188.4 iso-electric points 10.49, the polypeptide total order is classified as: L-Ala-Isoleucine-glycine-Threonine-arginine-glutamine-halfcystine-Serine-Serine-aspartic acid-aspartic acid-L-Ala-leucine-leucine-proline(Pro)-phenylalanine-phenylalanine-Methionin-arginine-arginine (AIGTRQCSSDDALLPFFKRR).
Claims (4)
1. one kind is separated the peptide species obtain from spider venom, it is characterized in that this polypeptide total order classifies as: L-Ala-Isoleucine-glycine-Threonine-arginine-glutamine-halfcystine-Serine-Serine-aspartic acid-aspartic acid-L-Ala-leucine-leucine-proline(Pro)-phenylalanine-phenylalanine-Methionin-arginine-arginine.
2. polypeptide according to claim 1 is characterized in that the molecular weight 2188.4 of this polypeptide, iso-electric point 10.49.
3. polypeptide according to claim 1 is characterized in that the extracting method of this polypeptide is,
A. the centrifugal 15min-25min of spider venom 10000-12000r/min, lyophilize;
B. gel-filtration: above-mentioned lyophilized powder 100mg is dissolved in 5mL water, and the centrifugal 8-15min of 10000-12000r/min removes precipitation, gel-filtration column 0.1mol/LNa
2HPO4-NaH
2PO
4PH6.0 damping fluid balance, fully sample is gone up in the back, with same elutriant wash-out, collects active peak.
C. reverse phase HPLC: reverse phase HPLC: contain the abundant balance C18 reversed-phase column of ultrapure water of 1 ‰ trifluoroacetic acids,, collect active peak with the above-mentioned elutriant of the fine buffer solution for gradient elution of second that contains 1 ‰ trifluoroacetic acids.
4. polypeptide according to claim 1 is characterized in that this polypeptide is used for oncotherapy.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810154588A CN101759779A (en) | 2008-12-26 | 2008-12-26 | Active spider polypeptide, preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810154588A CN101759779A (en) | 2008-12-26 | 2008-12-26 | Active spider polypeptide, preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103638057A (en) * | 2013-12-01 | 2014-03-19 | 大理学院 | Preparation method and medical application of nephila spider extract anticancer part |
| CN103655631A (en) * | 2013-12-01 | 2014-03-26 | 大理学院 | Preparation method for effective part of nephila spider for suppressing fungi growth and application of nephila spider |
| CN105777885A (en) * | 2016-04-11 | 2016-07-20 | 中南大学 | Antibacterial peptide of Xinjiang Lycosa singoriensis and application |
-
2008
- 2008-12-26 CN CN200810154588A patent/CN101759779A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103638057A (en) * | 2013-12-01 | 2014-03-19 | 大理学院 | Preparation method and medical application of nephila spider extract anticancer part |
| CN103655631A (en) * | 2013-12-01 | 2014-03-26 | 大理学院 | Preparation method for effective part of nephila spider for suppressing fungi growth and application of nephila spider |
| CN105777885A (en) * | 2016-04-11 | 2016-07-20 | 中南大学 | Antibacterial peptide of Xinjiang Lycosa singoriensis and application |
| CN105777885B (en) * | 2016-04-11 | 2019-07-09 | 中南大学 | A kind of Xinjiang lycosa singoriensis Lycosa singoriensis antibacterial peptide and application |
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Open date: 20100630 |