CN1083526A

CN1083526A - The one-step affinity column purifying process of embolism-resisting enzyme

Info

Publication number: CN1083526A
Application number: CN93111074A
Authority: CN
Inventors: 单风平
Original assignee: Individual
Current assignee: Individual
Priority date: 1993-04-26
Filing date: 1993-04-26
Publication date: 1994-03-09

Abstract

The present invention relates to the production technique of a kind of medicine, i.e. the processing method of one-step affinity column purification Pheretima asiatica with embolism-resisting enzyme.This technological process is: fresh Pheretima washing → ultrasonic homogenate → high speed centrifugation → supernatant liquor thermally denature → mistake affinity post → dialysis → ultrafiltration is held back → degerming packing → survey activity, and it is lower to obtain molecular weight, relatively purer embolism-resisting enzyme product.

Description

The one-step affinity column purifying process of embolism-resisting enzyme

The present invention relates to the production technique of a kind of medicine, i.e. the processing method of one-step affinity column purification Pheretima asiatica with embolism-resisting enzyme.

Earthworm is used for the treatment of various diseases such as rheumatism etc. as medicinal in the history in existing more than 4,000 year of China, but all is to adopt decoction method, uses the composition of its non-protein part.Domestic in recent years existing report (1991 the 7th the 3rd phases of volume of journal of biological chemistry) about from earthworm, extracting the activated protein embolism-resisting enzyme, but for this embolism-resisting enzyme purifying is not then openly reported, the external cerebral thrombosis of treatment is at present mainly used the urokinase of genetically engineered manufactured and TPA etc., these medicines are very effective, but cost an arm and a leg and the time have serious toxic side effects to take place.Domestic still do not have this respect medicine at present and produce in batches to use.And earthworm embolism-resisting enzyme purification difficult, domestic do not have corresponding way again, and mixture difficulty again is used for clinical.

The object of the present invention is to provide one step of a kind of employing affinity post method with earthworm embolism-resisting enzyme purifying, promptly technology such as hold back through homogenate → centrifugal → cross affinity post → dialysis → ultrafiltration, it is lower to obtain molecular weight, relative purer embolism-resisting enzyme product.

The objective of the invention is to realize by following measure:

This technological process is: fresh Pheretima washing → ultrasonic homogenate → high speed centrifugation → supernatant liquor thermally denature → mistake affinity post → dialysis → ultrafiltration is held back → degerming packing → survey activity; Its detailed process is that a few hours are soaked with physiological saline in the washing back, the back is centrifugal with 15000 rev/mins, get supernatant liquor and handle through 60 ℃ of thermally denatures one hour, again through 16000 rev/mins centrifugal one hour, get supernatant liquor and cross Sigma affinity post, Tris-HCl washing with PH8.0, carry out wash-out with NaOH then, collect elution peak, the PH7.0 phosphate buffered saline buffer is dialysed with monitor monitors, use molecular weight 30000 membrane retentions then, filter with sterilization filter again.

Advantage of the present invention and positively effect are:

1. can simplify production stage with this processing method, particularly can obtain purer activated protein embolism-resisting enzyme product, and without any side effects.

2. with the embolism-resisting enzyme of this explained hereafter, can significantly reduce body inner fibrin content and can not cause the body internal hemorrhage, continue development and can make it become a kind of antithrombotic product that wide DEVELOPMENT PROSPECT is arranged.

Description of drawings is as follows:

Accompanying drawing of the present invention is preparation technology's flow diagram.

Below in conjunction with the accompanying drawing specific embodiment that develops simultaneously out, the present invention will be further described in detail:

The earthworm washing is in order to remove all earth, the intravital toxin of earthworm all to be told only in its technology.Ultrasonic homogenate is that earthworm polypide cell is all smashed, and all enzymes are all discharged.High speed centrifugation is advisable with 15000 rev/mins to 18000 rev/mins, and the equal sedimentation of the bigger part of all fragments and molecular weight is gone down.The supernatant liquor thermally denature is that some temperature-sensitive protein depositions are gone down.Crossing the affinity post is the agar affinity post that U.S. Sigma company produces, and is the key of embolism-resisting enzyme purifying.Cross that dialysis is in order to remove some trypsinase or other small molecular weight protein enzymes behind the post.It is in order to remove some high molecular weight protein enzymes that last ultrafiltration is held back.It is to determine unit of enzyme activity that activity is surveyed in the degerming packing, is medicinal definite index.

For example:

Get fresh Pheretima 20 grams, soaked three hours with triple physiological saline, do ultrasonic homogenate then and handle, get supernatant liquor after centrifugal one hour through 60 ℃ of thermally denatures one hour through 16000 rev/mins again, centrifugal one hour through 17000 rev/mins again, get supernatant liquor again and cross the affinity post, with the Tris-HCl washing of PH8.0, use the NaOH wash-out more then, behind monitor monitors collection elution peak, to the dialysis of PH7.0 phosphate buffered saline buffer, use the membrane retention of molecular weight 30000 then, draw refining embolism-resisting enzyme 450mg.

Claims

1, one-step affinity column purifying process of embolism-resisting enzyme is characterized in that this technological process is: fresh Pheretima washing → ultrasonic homogenate → high speed centrifugation → supernatant liquor thermally denature → mistake affinity post → dialysis → ultrafiltration is held back → degerming packing → survey activity; Its detailed process is, a few hours are soaked with physiological saline in the washing back, and the back is centrifugal with 15000 rev/mins, get supernatant liquor and handle in one hour through 60 ℃ of thermally denatures, centrifugal one hour through 16000 rev/mins again, get supernatant liquor and cross Sigma affinity post,, carry out wash-out with NaOH then with the Tris-HCl washing of PH8.0, collect elution peak with monitor monitors, the PH7.0 phosphate buffered saline buffer is dialysed, use molecular weight 30000 membrane retentions then, filter with sterilization filter again.