CN101089017A

CN101089017A - Process of separating and purifying melittin

Info

Publication number: CN101089017A
Application number: CN 200610046955
Authority: CN
Inventors: 张文礼; 孙井元
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-06-16
Filing date: 2006-06-16
Publication date: 2007-12-19

Abstract

The process of separating and purifying melittin includes the following steps: dip rinsing melittin with water, filtering, precipitating the filtrate in alcohol, extracting the precipitate with ammonium hydroxide and n-butanol, concentrating the extracted liquid and precipitating with acetone, dissolving the precipitate in buffering liquid A of urea acetate, stepped eluting in ionic exchange gel column, collecting the chromatographic component with highest hemolytic activity and absorption peak V, concentrating, desalting the concentrated solution in glucose gel G-10 column, precipitating in acetone, re-dissolving, desalting and precipitating, dissolving the precipitate in acetate buffering liquid B, eluting in glucose gel G-25 column with buffering liquid B, collecting component with absorption peak II, concentrating, desalting and freeze drying to obtain electrophoresis level melittin. The process has short production period and high yield and low cost.

Description

The separating and purifying method of mellitin

Technical field

The present invention relates to the separation-extraction technology of mellitin, particularly use the method for thick bee venom separation and Extraction mellitin.

Background technology

In numerous compositions of bee venom, the highest with the content of mellitin (melittin), biological activity is the strongest, has the most wide clinical prospect.Though the report of its separating and purifying method is arranged both at home and abroad, complex process, the condition harshness is difficult to mass production; And the production cycle is longer, generally needs 3 days at least; Product loss is serious, and mellitin yield only about 30% extracts purifying cost height.The disclosed patent No.: 92114271.4 " methods of sharp separation honeybee peptide from bee venom ", be after being dissolved in thick bee venom in the water, use ultra-filtration membrane and dialysis tubing that the honeybee peptide is separated, the molecular weight that dams of ultra-filtration membrane is more than 5000, the dialysis tubing molecular weight that dams is 1000, this method purpose is to isolate the non-proteic substance that macromolecule can cause allergic reaction, as adopt this method, the mellitin tetramer (molecular weight is 11360) was just dammed to fall at the separation initial stage, and the mellitin tetramer accounts for 10～20% in bee venom, so that the extraction yield of mellitin than theoretical extraction yield low 20～30%; This method is a separation method, and mellitin purity does not reach electrophoresis level purity, so Application Areas is restricted greatly.

Summary of the invention

The objective of the invention is to overcome the defective that prior art exists, existing chromatography partition method is improved, provide a kind of processing step few relatively, the mellitin loss is few in extracting purge process, it is the yield height, with short production cycle, the mellitin purity after the purification reaches the mellitin separating and purifying method of electrophoresis level.

The inventive method is that thick bee venom water is embathed, and the method for solvent extraction, gel filtration chromatography is extracted mellitin from bee venom, it is characterized in that:

1) thick bee venom is added water logging and wash, remove insolubles impurity, filtrate is used ethanol sedimentation, removes ethanol water liquid, and throw out 1 adds ammonium hydroxide and butanol solution extraction, the reject precipitation, and propyl carbinol is reclaimed in distillation, and enriched material adds acetone precipitation and gets throw out 2;

2) throw out 2 is dissolved among the acetate buffer A of urea, on ion-exchange gel CM-Sephrose.FF post, uses the buffer A gradient elution chromatography;

Chromatography fraction when 3) collecting the strongest absorption peak V of hemolytic activity under the ion-exchange gel post concentrates, and concentrated solution is by the desalination of sephadex G-10 post, and the desalination and concentration thing obtains throw out 3 with acetone precipitation;

4) throw out 3 is dissolved in Guanidinium hydrochloride, S-WAT and the parachloromercuribenzoate mixing solutions,, by the sephadex column desalination, gets throw out 4 again with acetone precipitation again with solution concentration;

5) throw out 4 is dissolved among the acetate buffer B, on sephadex G-25 post, use the buffer B gradient elution chromatography, the chromatography fraction when collecting the strongest absorption peak II of hemolytic activity under post concentrates, in sephadex column G-10 go up desalination, lyophilize promptly obtains mellitin.

The buffer A of using when carrying out chromatography on ion-exchange gel post and sephadex column and the PH of buffer B are 4.2～5.0, use gradient mix device to carry out gradient elution.Guanidinium hydrochloride, S-WAT and parachloromercuribenzoate mixing solutions are adjusted pH value with the ammonium hydroxide of 0.15M and are reached 7.0, and the concentration of Guanidinium hydrochloride, S-WAT and parachloromercuribenzoate is respectively 3M, 0.055M and 0.0023M.Filtrate when embathing thick bee venom is precipitated with 20 times dehydrated alcohol; Desalination in the pilot process, precipitation adopt the acetone of 3 times of amounts to precipitate.

The mellitin purity that adopts present method to produce can reach electrophoresis level purity, has satisfied the particularly technical requirements of clinical application of more areas; The extraction yield of mellitin all exceeds 7～8% than existing method, and processing step simplifies, and the production cycle of purifying reduces 1.5～2 days than original, and cost for purification reduces about 30% than original.

In conjunction with the embodiments the present invention is described in detail according to the technology diagram below.

Accompanying drawing is a process flow diagram of the present invention.

Get thick bee venom 20g adding 100ml distillation moisture and embathe for three times, use neutral filter paper filtering, insolubles impurity rejects such as honeybee sting.Filtrate is precipitated with 20 times of dehydrated alcohols, removes ethanol water liquid, and throw out 1 is dissolved in the ammonium hydroxide of 1200ml 0.15M, adds 800ml propyl carbinol jolting 2hr again.Separate with separating funnel, the throw out reject, propyl carbinol in the extraction liquid, enriched material triple acetone precipitation are reclaimed in distillation; Throw out 2 is dissolved in 1000mL and contains in the acetate buffer of 4mol/L urea, and PH is 4.75, and this is a buffer A, and last ion-exchange gel CM-Sephrose.FF post carries out gradient elution with buffer A+0.5mol/L sodium-chlor.Elutriant is followed the tracks of with the biological activity that hemolytic test carries out mellitin, and the chromatography fraction when collecting the strongest absorption peak V of hemolytic activity effect under the ionic gel post is 1000ml altogether, and evaporation concentration is to 100ml.Concentrated solution is by the desalination of sephadex G-10 post, desalination and concentration thing triple acetone precipitation.Throw out 3 is dissolved in the solution that contains 3mol/L Guanidinium hydrochloride, 55mmol/L S-WAT and 2.3mmol/L parachloromercuribenzoate of 2000mL, whole solution NH ₄OH is transferred to pH7.0, is incubated 2hr down at 37 ± 2 ℃, and evaporation concentration solution is to 200ml, again by sephadex column G-10 desalination, with 3 times acetone precipitation.Throw out 4 is dissolved in 500mL ammonium acetate buffer (0.05mol/L, pH4.75, this is a buffer B) in, last sephadex G-25 post carries out gradient elution (using gradient mixer) with buffer B, elutriant is followed the tracks of with the activity that hemolytic test carries out mellitin, chromatography fraction when post is collected down the strongest absorption peak II of hemolytic activity effect is 800ml altogether, is concentrated into 100ml, with the desalination of sephadex G-10 post, lyophilize obtains lyophilized products mellitin 9.4g at last.The yield of product mellitin is 4 7%.

Claims

1, a kind of separating and purifying method of mellitin is that thick bee venom water is embathed, and the method for solvent extraction, gel filtration chromatography is extracted mellitin from bee venom, it is characterized in that:

4) throw out 3 is dissolved in Guanidinium hydrochloride, S-WAT and the parachloromercuribenzoate mixing solutions,,, gets throw out 4 with acetone precipitation by the sephadex column desalination again with solution concentration;

2, in accordance with the method for claim 1, it is characterized in that PH=4.2～5.0 of buffer A and buffer B, Guanidinium hydrochloride, S-WAT and parachloromercuribenzoate mixing solutions are adjusted PH with 0.15M ammonium hydroxide and are reached 7.0.

3, in accordance with the method for claim 1, it is characterized in that embathing thick bee venom filtrate and precipitate with 20 times of dehydrated alcohols, desalination in the pilot process, precipitation adopt the acetone of 3 times of amounts to precipitate.