CN101822692B

CN101822692B - Method for removing hemolytic toxicity from physaliatoxin

Info

Publication number: CN101822692B
Application number: CN2010101497795A
Authority: CN
Inventors: 肖良; 王倩倩; 张黎明; 刘四化
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2010-04-16
Filing date: 2010-04-16
Publication date: 2012-02-01
Anticipated expiration: 2030-04-16
Also published as: CN101822692A

Abstract

The invention relates to the technical field of medicines, in particular to a method for removing hemolytic toxicity from physaliatoxin. The method includes the steps that: (1) jellyfish tentacles are autolyzed in self-prepared seawater with the temperature of 4 DEG C for four days, a magnetic stirrer carries out stirring, tentacle-autolyzed liquid is collected, and is centrifugated under 10000g for 15 minutes, and supernate is crude jellyfish toxin extract; (2) the pH of the crude jellyfish toxin extract is regulated to 10.5 by 0.1mol/L of NaOH, the crude jellyfish toxin extract is kept still for 4 hours, and is centrifugated under 10000g for 15 minutes to remove precipitate, the supernate is dialyzed by a dialysis bag, the permeable molecular weight of which is 10kDa, 0.02mol/L of acetic acid buffer solution with the pH of 6.0 is first used for 24-hour dialysis, buffer solution is then replaced by 0.02mol/L of Tris-acetic acid buffer solution with the pH of 9.5 to continue dialysis for 24 hours, centrifugation is carried out under 10000g for 15 minutes to remove precipitate, and supernate is physaliatoxin soilution without hemolytic toxicity. The invention provides a simple and effective method for removing hemolytic toxicity from physaliatoxin.

Description

A kind of medusocongestin goes the method for hemolytic toxicity

Technical field

The present invention relates to medical technical field, is the method that a kind of medusocongestin removes hemolytic toxicity.

Background technology

Medusocongestin is that a class formation is novel, the extremely strong peptide toxoid of toxicity, has multiple biological activitys such as cardiovascular, haemolysis, nerve, breathing.Have characteristics such as responsive to temperature, instability, easy adhesion, easy inactivation owing to medusocongestin, the purification of its toxic component is slow with the evaluation work progress, lags behind other common biotoxins generally.

Medusocongestin Cardiovascular Toxicity and hemolytic toxicity are stung in the poisoning Jellyfish and are played a significant role, and also are two types of maximum toxic components of research report in the medusocongestin.Wherein Cardiovascular Toxicity component abundance is lower; But the cardiovascular function that causes damage is the lethal main cause of medusocongestin (Xiao L; Liu GS, Wang QQ, et al.; The lethality of tentacle-only extract from jellyfish Cyanea capillata is primarily attributed to cardiotoxicity in anaesthetized SD rats [J] .Toxicon.2010,55 (4): 838-845).Hemolytic toxicity component abundance is higher; Haemolysis can cause blood potassium to raise, and possibly play synergism (Xiao L, Zhang J to the damage cardiovascular function; Wang QQ; Et al., In vitro and in vivo haemolytic studies of tentacle-only extract from jellyfish Cyanea capillata [J] .Toxicol In Vitro.2010, doi:10.1016/j.tiv.2010.02.009).Therefore, further investigate medusocongestin Cardiovascular Toxicity mechanism, must remove abundant hemolytic toxicity component in the thick poison of Jellyfish, be beneficial to the proteic separation and purification of Cardiovascular Toxicity.

Research shows, Cardiovascular Toxicity ratio of component hemolytic toxicity component more alkaline-resisting (Nie Fei, Xiao Liang, Zhang Jing, etc. send out the comparison and the analysis of Influential Factors [J] thereof of shape rosy clouds medusocongestin separated product hemolytic activity. the journal .2008 of The 2nd Army Medical College, 29 (1): 83-86; Xiao L; He Q; Guo Y; Et al., Cyanea capillata tentacle-only extract as a potential alternative of nematocyst venom:Its cardiovascular toxicity and tolerance to isolation and purification procedures [J] .Toxicon.2009,53 (1): 146-152).But do not see the relevant report of removing high abundance hemolytic toxicity component in the thick poison of Jellyfish with alkali condition so far.

Summary of the invention

The present invention provides a kind of medusocongestin to go the method for hemolytic toxicity, and step is following:

1. prepare the thick malicious extracting solution of Jellyfish

1) sampling: the Jellyfish that collects is cut tentacle rapidly, immediately that tentacle dry ice is freezing, transport that to be placed on-70 ℃ of ultra cold storage freezers frozen subsequent use back;

2) preparation Jellyfish thick malicious extracting solution: with frozen tentacle with the autogamy sea water in 4 ℃ of self-dissolvings 4 days, magnetic stirrer, the collection tentacle is from solution body, the centrifugal 15min of 10000g, supernatant are the thick malicious extracting solution of Jellyfish;

2. the medusocongestin solution of hemolytic toxicity is removed in preparation

1) destroy hemolytic toxicity albumen with alkali condition: the thick malicious extracting solution of Jellyfish of above-mentioned preparation is transferred pH to 10.5 with 0.1mol/L NaOH, leave standstill 4h, the centrifugal 15min of 10000g goes deposition, gets supernatant;

2) purification: select suitable bag filter and dialysis buffer liquid for use, adopt the dialysis process can be, and keep the Cardiovascular Toxicity component of medusocongestin with ruined hemolytic toxicity albumen or other foreign proteins and Impurity removal.

It is the bag filter of 10kDa that the present invention selects for use through molecular weight; Above-mentioned supernatant is first with 0.02mol/L, pH6.0 acetate buffer solution dialysis 24h; Be that 0.02mol/L, pH 9.5Tris-acetate buffer solution continue dialysis 24h with buffer exchange again; The centrifugal 15min of 10000g abandons deposition, and supernatant is the medusocongestin solution of hemolytic toxicity.

The present invention goes protein concentration, hemolytic toxicity and the Cardiovascular Toxicity of hemolytic toxicity front and back to carry out the contrast experiment medusocongestin; The result shows that the protein concentration of the prepared medusocongestin that removes hemolytic toxicity is significantly less than slightly poison of Jellyfish, and hemolytic toxicity is lost basically; And Cardiovascular Toxicity is only slightly malicious a little less than Jellyfish; Explain that the inventive method can effectively remove the hemolytic toxicity of medusocongestin, and can preserve its Cardiovascular Toxicity, help furtheing investigate medusocongestin Cardiovascular Toxicity mechanism.

The inventive method is simple, for further investigation medusocongestin Cardiovascular Toxicity mechanism provides a kind of simple and effective method of removing hemolytic toxicity.

Description of drawings

Fig. 1. thick poison of Jellyfish and the hemolytic activity comparison diagram that removes the hemolytic toxicity medusocongestin.

Fig. 2. thick poison of Jellyfish and the cardiac vascular activity comparison diagram that removes the hemolytic toxicity medusocongestin.

The specific embodiment

Combine accompanying drawing and embodiment that the present invention is described in detail at present.

The hemolytic toxicity medusocongestin is removed in embodiment 1. preparations

1. prepare the thick malicious extracting solution of Jellyfish

Send out shape rosy clouds Jellyfish and pick up from Sanmen Wan marine site, Zhejiang Province, get tentacle 10g, add autogamy sea water (NaCl 28g, MgCl ₂6H ₂O 5g, KCl 0.8g, CaCl ₂1.033g, add water to 1000ml) and 10ml, self-dissolving 4 days, magnetic stirring apparatus stirs 2 times every day, each 10min.100 order cell screen filtrations 3 times, centrifugal 3 times of filtrating 10000g, each 15min, collecting supernatant is the thick malicious extracting solution of Jellyfish (cTOE, crude tentacle-only extract).The medusocongestin of hemolytic toxicity is removed in preparation

Adopt 0.1mol/L NaOH to transfer pH to 10.5 the thick malicious extracting solution of above-mentioned Jellyfish, leave standstill 4h, the centrifugal 15min of 10000g goes deposition, gets supernatant; Supernatant uses through molecular weight and dialyses as the bag filter of 10kDa; Earlier with 0.02mol/L, pH 6.0 acetate buffer solutions dialysis 24h; Be that 0.02mol/L, pH 9.5Tris-acetate buffer solution continue dialysis 24h with buffer exchange again, the centrifugal 15min of 10000g abandons deposition; Supernatant is the medusocongestin solution (tTOE, treated tentacle-only extract) of hemolytic toxicity.

All operations all carries out under frozen water or 4 ℃ of conditions.Adopt 0.01mol/L, the pH7.4PBS 8h that dialyses before using.

Medusocongestin goes hemolytic toxicity front and back protein concentration, hemolytic toxicity and Cardiovascular Toxicity contrast experiment

1) protein concentration relatively

Determination of protein concentration adopts the Bradford method to carry out, and recording the cTOE protein concentration is 1.5mg/ml, and the tTOE protein concentration is 0.5mg/ml, is 1/3 of cTOE protein concentration.Explanation can reduce the foreign protein concentration among the cTOE greatly through this method.

2) hemolytic toxicity relatively

1 of male SD rat (the The 2nd Army Medical College animal center provides, down together), body weight 250g with 25% urethane intraperitoneal injection of anesthesia, is 1.2g/kg by the body weight anaesthesia dosage.Femoral arteriography is got rat artery blood, the anticoagulant of equal-volume 50u heparin-saline, and PBS rinsing several, the centrifugal 5min of 3000g abandons supernatant after each rinsing, till supernatant is limpid.Erythrocyte and PBS 1: 50 by volume (2%) are diluted to red cell suspension.Reaction cumulative volume 1.5ml, red cell suspension 0.5ml wherein, experimental group cTOE and tTOE respectively establish 5 dose groups, add 0.2,0.4,0.6,0.8 respectively, 1.0ml, and reuse PBS adds to and reacts cumulative volume 1.5ml; Negative control group does not add toxin (being 0ml), and positive controls adds 0.4ml 0.2mg/ml Saponin, adds to reaction cumulative volume 1.5ml with PBS yet.37 ℃ of water-bath 30min, the centrifugal 5min of 3000g detects A respectively with spectrophotometer ₅₄₅, calculate the haemolysis mark, haemolysis mark=[(sample cell A ₅₄₅)-(negative control A ₅₄₅)]/[(positive control A ₅₄₅)-(negative control A ₅₄₅)] * 100%, the result sees Fig. 1.Visible by Fig. 1, the cTOE hemolytic toxicity is very strong, is dose dependent, and the hemolytic toxicity of tTOE is almost completely lost, and explains in the process for preparing tTOE and has successfully removed the hemolytic toxicity among the cTOE.

3) Cardiovascular Toxicity relatively

12 of male SD rats, body weight 200～250 grams are divided into two groups of cTOE and tTOE at random, and 6 every group, 25% urethane intraperitoneal injection of anesthesia is by body weight anaesthesia dosage 1.2g/kg.The left side femoral arteriography connects pressure receptor, the variation of monitoring of MPA bio signal analytical system and record SD rat femoral blood pressure.The right side external jugular vein is put pipe, and by the body weight administration, dosage is respectively cTOE 10mg/kg, and tTOE 3.3mg/kg (being equivalent to cTOE 10mg/kg) observes the SD rat blood pressure and changes, and the result sees Fig. 2.From the A of Fig. 2, B, C, can find out; Though the tTOE after handling through the inventive method compare with cTOE Cardiovascular Toxicity slightly a little less than; But still has very strong Cardiovascular Toxicity; Explain that this method when removing non-cardiovascular toxic component such as hemolytic toxicity, can preserve the Cardiovascular Toxicity composition preferably.

Claims

1. send out the method that shape rosy clouds medusocongestin removes hemolytic toxicity for one kind, step is following:

1) the thick malicious extracting solution of shape rosy clouds Jellyfish is sent out in preparation

The shape rosy clouds Jellyfish that sends out that collects is cut tentacle rapidly, immediately that tentacle dry ice is freezing, place-70 ℃ of ultra cold storage freezers frozen subsequent use again; With frozen tentacle with the autogamy sea water in 4 ℃ of self-dissolvings 4 days, magnetic stirrer is collected tentacle from solution body, the centrifugal 15min of 10000g, supernatant are and send out the thick malicious extracting solution of shape rosy clouds Jellyfish; Said autogamy sea water is: NaCl 28g, MgCl ₂6H ₂O 5g, KCl 0.8g, CaCl ₂1.033g, add water to 1000ml;

2) a shape rosy clouds medusocongestin solution of hemolytic toxicity is removed in preparation

The thick malicious extracting solution of shape rosy clouds Jellyfish of sending out of above-mentioned preparation is transferred pH to 10.5 with 0.1mol/L NaOH, leave standstill 4h, the centrifugal 15min of 10000g goes deposition, gets supernatant; Supernatant uses through molecular weight and dialyses as the bag filter of 10kDa; Earlier with 0.02mol/L, pH 6.0 acetate buffer solutions dialysis 24h; Be that 0.02mol/L, pH 9.5Tris-acetate buffer solution continue dialysis 24h with buffer exchange again; The centrifugal 15min of 10000g abandons deposition, and supernatant is a shape rosy clouds medusocongestin solution of hemolytic toxicity.