CN102533694A - Method for extracting phospholipase A2 from trimeresurus albolabris venin - Google Patents
Method for extracting phospholipase A2 from trimeresurus albolabris venin Download PDFInfo
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- CN102533694A CN102533694A CN2012100611006A CN201210061100A CN102533694A CN 102533694 A CN102533694 A CN 102533694A CN 2012100611006 A CN2012100611006 A CN 2012100611006A CN 201210061100 A CN201210061100 A CN 201210061100A CN 102533694 A CN102533694 A CN 102533694A
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Abstract
The invention discloses a method for extracting phospholipase A2 from trimeresurus albolabris venin. The method comprises the following steps of: pretreating the trimeresurus albolabris venin; performing CM-Sephadex C-50 cation-exchange chromatography; performing Sephadex G-75 gel chromatography; and performing Q-Sepharose FF pre-packed column chromatography to obtain a pure product. The phospholipase A2 is separated and extracted from the trimeresurus albolabris venin; the preparation method is simple, convenient to implement and low in cost; and the obtained phospholipase A2 has high purity.
Description
Technical field
The invention belongs to the biological extraction field, specifically, relate to from a kind of Trimeresurus albolabris (Gray) snake venom and extract phospholipase A
2Method.
Background technology
Phospholipase A
2(phospholipase A
2, PL A
2) extensively be present in the animal venom and mammiferous pancreas liquid of snake venom, bee venom and scorpion venom, it is a kind of Ca
2+Dependent enzyme, the hydrolysis of second acyl key of catalyzing glycerol phosphatide that can be single-minded generates lysophospholipid and lipid acid.This enzyme is mainly similar by a class formation, the protein family of molecular weight about 14KDa formed, and generally contains 6 to 7 pairs of disulfide linkage.Phospholipase A
2Almost be present in the venom of all poisonous snakes; Except enzymic activity, also have multiple physiology, pharmacological action, after one's own heart dysentery; Cell toxicant; Muscle poison, neural poison and NGFF etc., and anticoagulation, haemolysis, hypotensive, platelet aggregation-against are arranged, induce the formation oedema and cause effect such as convulsions.In addition, phospholipase A
2Also having the function that bacteria growing inhibiting, cell death inducing and resistance HIV virus gets into host cell, is the important tool enzyme of the heterogeneous catalysis mechanism of structure of phospholipid and enzyme in research microbial film, the lipoprotein.From the 1950's first to the phospholipase A of cobra-venom
2Study beginning (Wittcoff et al, 1951), existing so far more than 100 kind of phospholipase A
2Come out by purifying, various experiments show the phospholipase A that different types of poisonous snake contains
2Be not quite similar, even the poisonous snake of same kind, also possible difference owing to living environment, and cause the phospholipase A in the venom
2Structure with active bigger difference arranged.Phospholipase A
2This distinctive chemical property and complex physiological functions are not only carried out biological study, constitutional features and toxicological study to it and are had the important in theory meaning, and at aspects such as treatment tumour, cardiovascular disordeies important application prospects are arranged also as medicine.
Trimeresurus albolabris (Gray) is mainly seen in ground such as Guangzhou, Sichuan, Guizhou, Guangxi, Fujian, Yunnan for poisonous snake is arranged.The Trimeresurus albolabris (Gray) snake venom belongs to the blood circulation poison, because its toxin expelling amount is less, can be at once not fatal after the people is bitten, if but untimelyly taking the treatment measure, also can be in peril of one's life.After being bitten by Trimeresurus albolabris (Gray), the wounded shows as wound more and has an intense pain, and it is more than to bleed, and suffers from around the limb red and swollenly, and the local area also blister, blood blister can occur, severe patient symptom such as also might occur spitting blood, have blood in stool.Research report to its venom mainly contains fiber eliminating enzyme, Thrombin-like enzyme, 5 '-phosphonuclease etc. in recent years, and the Shang Weiyou report separates the preparation phospholipase A from the Trimeresurus albolabris (Gray) snake venom
2Method and phospholipase A thereof
2The research of zymologic property.
Summary of the invention
For solving above technical problem, the object of the present invention is to provide in a kind of Trimeresurus albolabris (Gray) snake venom and extract phospholipase A
2Method.
The present invention seeks to realize like this: extract phospholipase A in a kind of Trimeresurus albolabris (Gray) snake venom
2Method, it is characterized in that: carry out as follows:
(1) Trimeresurus albolabris (Gray) snake venom pre-treatment: collect venom, the venom lyophilize is powdered subsequent use, get thick malicious lyophilized powder 0.3～1g, be dissolved in 5ml pH 5.2-5.8, in 0.025～0.05mol sodium-acetate buffer, frozen centrifugation, it is for use to get supernatant;
(2) CM-Sephadex C-50 cation-exchange chromatography: use pH 5.2-5.8,0.025～0.05mol/L sodium-acetate buffer balance CM-Sephadex C-50 positively charged ion chromatography post; With appearance on the supernatant in the step (1), with the above-mentioned buffer solution for gradient elution of concentration range, detect wavelength: 280nm at 0～0.5mol/L NaCl, flow velocity: 0.5～1ml/min collects the 3ml/ pipe, collects the strongest active peak I, lyophilize;
(3) Sephadex G-75 gel chromatography: with pH 7.5～8.0,0.01～0.03mol/L Tris-HCl buffer balance Sephadex G-75 post is gone up appearance with the freeze-drying sample of collecting in the step (2) with same buffer dissolving back; Detect wavelength: 280nm; Flow velocity 0.15ml/min collects the 2ml/ pipe, surveys each peak phospholipase activity; Collect the strongest active peak IV, lyophilize;
(4) Q-Sepharose FF prepacked column chromatography: with pH 7.5～7.8; 0.02 the Tris-HC1 damping fluid balance Q-Sepharose FF prepacked column of～0.05mol/L is gone up appearance with making the freeze-drying sample in the step (3) with above-mentioned damping fluid dissolving back, with the above-mentioned buffer solution for gradient elution of concentration range at 0～0.5mol/L NaCl; Flow velocity 1ml/min; The 1ml/ pipe is collected active peak II, and lyophilize promptly gets pure article.
Adopt in the above-mentioned steps (1) and sting ware extrusion process collection venom, wherein cool off the centrifugal 10～15min of centrifugal employing 3000～5000r/min.
All detect the OD280nm light absorption value in the chromatography process, adopt the egg plate method to measure phospholipase activity.Adopt SDS-polyacrylamide gel electrophoresis determining molecular weight size.
Adopt egg plate to measure phospholipase A
2Active: take by weighing agarose 0.15g, be added to 100ml and contain 1mol/L NaCl, in the solution of 0.005M/L CaCl2, high-temperature sterilization 15min gets new fresh hen egg, the aseptic egg yolk liquid of getting of ethanol.After having sterilized, wait be cooled to 50 ℃ after, add the abundant mixing of egg yolk liquid 4ml by every 100ml substratum after, bed board, treat that gelling is solid after, the punch tool punching, every hole adds the 10ul testing sample, places 20h for 50 ℃, observes flat board and transparent circle whether occurs, if phospholipase A is arranged
2Transparent circle will appear in activity; If no, then transparent circle can not occur.
Beneficial effect: the present invention's separation and Extraction from the Trimeresurus albolabris (Gray) snake venom has gone out a kind of phospholipase A
2, the preparation method is simple, be convenient to operation, with low cost, and the gained phospholipase A
2Purity is higher.
Figure of description
Fig. 1 is a CM-Sephadex C-50 cation-exchange chromatography collection of illustrative plates;
Fig. 2 is a Sephadex G-75 gel chromatography collection of illustrative plates;
Fig. 3 is a Q-Sepharose FF prepacked column chromatography collection of illustrative plates;
Fig. 4 prepares phospholipase A for the present invention
2The SDS-PAGE collection of illustrative plates.
Embodiment
Embodiment 1
(1) Trimeresurus albolabris (Gray) snake venom pre-treatment: adopt and sting ware extrusion process collection venom; The venom lyophilize is powdered subsequent use; Get thick malicious lyophilized powder 0.3g, be dissolved in 5ml pH 5.2, in the 0.025mol/L sodium-acetate buffer; 3000～5000r/min frozen centrifugation, 10～15min, it is for use to get supernatant.
(2) CM-Sephadex C-50 cation-exchange chromatography: with pH 5.2,0.025mol/L sodium-acetate buffer balance CM-Sephadex C-50 positively charged ion chromatography post; With appearance on the supernatant in the step (1), with the above-mentioned buffer solution for gradient elution of concentration range at 0～0.5mol/L NaCl.Detect wavelength: 280nm, flow velocity: 1ml/min collects 3ml/ pipe (referring to Fig. 1).Collect the strongest active peak I, lyophilize.
(3) Sephadex G-75 gel chromatography: with pH 7.5; 0.25mol/L Tris-HCl buffer balance Sephadex G-75 post; The freeze-drying sample of collecting in the step (2) is gone up appearance with same buffer dissolving back; Detect wavelength: 280nm, flow velocity 0.15ml/min collects 2ml/ pipe (referring to Fig. 2).Survey each peak phospholipase activity, collect the strongest active peak IV, lyophilize.
(4) Q-Sepharose FF prepacked column chromatography: with pH 7.5, the Tris-HC1 damping fluid balance Q-Sepharose FF prepacked column of 0.25mol/L.Go up appearance with making the freeze-drying sample in the step (3) with above-mentioned damping fluid dissolving back, with the above-mentioned buffer solution for gradient elution of concentration range at 0～0.5mol/L NaCl, flow velocity 1ml/min, 1ml/ manages (referring to Fig. 3).Collect active peak II, lyophilize promptly gets pure article.
(5) molecular-weight determination: use the SDS-polyacrylamide gel electrophoresis, resolving gel concentration is 12%.The result is as shown in Figure 4, and through above-mentioned purification step, sample is shown as a protein band, and molecular weight is 17200 dalton.
Embodiment 2
(1) Trimeresurus albolabris (Gray) snake venom pre-treatment: adopt and sting ware extrusion process collection venom, the venom lyophilize is powdered subsequent use, get thick malicious lyophilized powder 1g; Be dissolved in 5ml pH 5.8; 0.03mol/L in the sodium-acetate buffer, 5000r/min frozen centrifugation 15min, it is for use to get supernatant.
(2) CM-Sephadex C-50 cation-exchange chromatography: with pH 5.8,0.03mol/L sodium-acetate buffer balance CM-Sephadex C-50 positively charged ion chromatography post; With appearance on the supernatant in the step (1), with the above-mentioned buffer solution for gradient elution of concentration range at 0～0.5mol/L NaCl.Detect wavelength: 280nm, flow velocity: 0.5ml/min collects the 3ml/ pipe.Collect the strongest active peak I, lyophilize.
(3) Sephadex G-75 gel chromatography: with pH 7.8; 0.2mol/L Tris-HCl buffer balance Sephadex G-75 post is gone up appearance with the freeze-drying sample of collecting in the step (2) with same buffer dissolving back, detects wavelength: 280nm; Flow velocity 0.15ml/min collects the 2ml/ pipe.Survey each peak phospholipase activity, collect the strongest active peak IV, lyophilize.
(4) Q-Sepharose FF prepacked column chromatography: with pH 7.8, the Tris-HC1 damping fluid balance Q-Sepharose FF prepacked column of 0.4mol/L.Go up appearance with making the freeze-drying sample in the step (3) with above-mentioned damping fluid dissolving back, with the above-mentioned buffer solution for gradient elution of concentration range at 0～0.5mol/L NaCl, flow velocity 1ml/min, 1ml/ pipe.Collect active peak II, lyophilize promptly gets pure article.
Claims (2)
1. extract phospholipase A in a Trimeresurus albolabris (Gray) snake venom
2Method, it is characterized in that: carry out as follows:
(1) Trimeresurus albolabris (Gray) snake venom pre-treatment: collect venom, the venom lyophilize is powdered subsequent use, get thick malicious lyophilized powder 0.3～1g, be dissolved in 5ml pH 5.2-5.8, in 0.025～0.05mol sodium-acetate buffer, frozen centrifugation, it is for use to get supernatant;
(2) CM-Sephadex C-50 cation-exchange chromatography: use pH 5.2-5.8,0.025～0.05mol/L sodium-acetate buffer balance CM-Sephadex C-50 positively charged ion chromatography post; With appearance on the supernatant in the step (1), with the above-mentioned buffer solution for gradient elution of concentration range, detect wavelength: 280nm at 0～0.5mol/L NaCl, flow velocity: 0.5～1ml/min collects the 3ml/ pipe, collects the strongest active peak I, lyophilize;
(3) Sephadex G-75 gel chromatography: with pH 7.5～8.0,0.01～0.03mol/L Tris-HCl buffer balance Sephadex G-75 post is gone up appearance with the freeze-drying sample of collecting in the step (2) with same buffer dissolving back; Detect wavelength: 280nm; Flow velocity 0.15ml/min collects the 2ml/ pipe, surveys each peak phospholipase activity; Collect the strongest active peak IV, lyophilize;
(4) Q-Sepharose FF prepacked column chromatography: with pH 7.5～7.8; 0.02 the Tris-HC1 damping fluid balance Q-Sepharose FF prepacked column of～0.05mol/L is gone up appearance with making the freeze-drying sample in the step (3) with above-mentioned damping fluid dissolving back, with the above-mentioned buffer solution for gradient elution of concentration range at 0～0.5mol/L NaCl; Flow velocity 1ml/min; The 1ml/ pipe is collected active peak II, and lyophilize promptly gets pure article.
2. according to extracting phospholipase A in the said a kind of Trimeresurus albolabris (Gray) snake venom of claim 1
2Method, it is characterized in that: adopt in the said step (1) and sting the ware extrusion process and collect venom, wherein cool off the centrifugal 10～15min of centrifugal employing 3000～5000r/min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109715176A (en) * | 2016-06-24 | 2019-05-03 | 金容隨 | The method of the purification bee venom of removal allergic component and the purification bee venom of the removal allergic component prepared by the method are prepared using yolk |
| CN109929020A (en) * | 2017-12-15 | 2019-06-25 | 浙江京新药业股份有限公司 | A kind of purification process of cobra venom and products thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1846713A (en) * | 2005-04-13 | 2006-10-18 | 上海医药工业研究院 | Snake venom extract for preventing and treating pyemia |
| CN101265469A (en) * | 2008-04-07 | 2008-09-17 | 重庆师范大学 | Method for preparing trimeresurus albolabris snake venom 5'-nucleotidase |
-
2012
- 2012-03-09 CN CN2012100611006A patent/CN102533694A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1846713A (en) * | 2005-04-13 | 2006-10-18 | 上海医药工业研究院 | Snake venom extract for preventing and treating pyemia |
| CN101265469A (en) * | 2008-04-07 | 2008-09-17 | 重庆师范大学 | Method for preparing trimeresurus albolabris snake venom 5'-nucleotidase |
Non-Patent Citations (2)
| Title |
|---|
| 李顺意等: "竹叶青蛇毒五种磷脂酶A2的分离纯化和氨基酸序列分析", 《湖北大学学报（自然科学版）》 * |
| 邓疆渝邓: "竹叶青属毒蛇的毒素研究进展", 《生物学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109715176A (en) * | 2016-06-24 | 2019-05-03 | 金容隨 | The method of the purification bee venom of removal allergic component and the purification bee venom of the removal allergic component prepared by the method are prepared using yolk |
| CN109929020A (en) * | 2017-12-15 | 2019-06-25 | 浙江京新药业股份有限公司 | A kind of purification process of cobra venom and products thereof |
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Application publication date: 20120704 |