CN1291616A - Process for separating and purifying high-purity high-activity phycocyanin - Google Patents

Abstract

A process for separating and purifying high-purity high-activity phycocyanin includes such steps as preparing mashed ambly spirulina, extracting, salting out, dialysis, concentrating, sephadex G200, DEAE, Sephadex G25, SephadexG200 and freeze drying. Its advantages include simple process, easy operation and low cost, Obtained phycocyanin can be used as fluorescent label substance for immunochemical analysis.

Description

The separation purification method of high-purity high-activity phycocyanin

The present invention relates to a kind of separation method of Phycocyanins, C-, especially a kind of from spirulina plalensis (Spiruina Platensis) method of separating and purifying high-purity high reactivity Phycocyanins, C-.

Chinese patent application 94109295.X discloses a kind of " Obtusatus arthrospira phycocyanin and application thereof ", its technical process is dry powder → extraction → DEAE52 → cellulose → saltout → dialyse → SephadexG75 → lyophilize, acquisition is as the Phycocyanins, C-of protective foods, without any purity and active requirement and index.

Purpose of the present invention just provide a kind of from spirulina plalensis separation and purification obtain the method for high-purity high-activity phycocyanin.

The present invention can be realized by following mode: fresh spirulina plalensis algae mud, add 0.001～0.003M, and the phosphoric acid buffer of PH=7 (PBS) after the freeze thawing 1～2 time, places ice bath ultrasonication cell; At 4～10 ℃, with 9000～11000 revolutions per seconds centrifugal 50～70 minutes, abandon precipitation; Get supernatant, add 30～60% saturation ratio ammonium sulfate at 4～10 ℃, the lucifuge standing over night, at 4～10 ℃, with 13000～18000 revolutions per seconds centrifugal 20～40 minutes, get precipitation; With 0.01～0.03M, the phosphoric acid buffer dissolution precipitation of PH=7 is at 4～10 ℃, lucifuge leaves standstill, with 0.01～0.03M, and the dialysis of PH=7 phosphoric acid buffer, molecular retention amount 12000Da, after concentrating fast, last SephadexG200 chromatography column is with 0.01～0.03M, PH=7 phosphoric acid buffer wash-out, collect the peak value component, last DEAE-SephadexA25 post is with 0.005～0.007M, the phosphoric acid buffer of PH=7 and 0.2～0.4M, the sodium-chlor wash-out of PH=7 is collected the peak value component, last SephadexG200 post, with 0.01～0.03M, the phosphoric acid buffer wash-out of PH=7, whole column chromatography carry out under the lucifuge condition at 8～10 ℃; Get the Phycocyanins, C-sample detection A620nm/A280nm value of purifying, ultraviolet-visible spectrogram, fluorescence spectrum figure and PAGE electrophoresis (manifest single blue electrophoresis band, do not have other foreign protein); Phycocyanins, C-is frost drying immediately, and gained dry powder keeps in Dark Place at 4～10 ℃.

The present invention has simplified the separation and purification program, easy handling, the purity of the Phycocyanins, C-that obtains and active high, and cost is lower, can be used as a kind of fluorescent marker and is used for immunochemical analyses, can accomplish double-colored or tricolor marker, has potential applicability in clinical practice, as the early diagnosis of cancer cells.

Below in conjunction with embodiment, the invention will be further described.

Embodiment one

Spirulina plalensis algae mud 100 grams of gathering fresh issue change matter or damage at natural temperature when avoiding spirulina to gather, and add 0.001M, and the phosphoric acid buffer of PH=7 freezes (20 ℃) and melts (18 ℃, lucifuge) 2 times, places ice bath ultrasonication cell; At 5 ℃, with 10000 revolutions per seconds centrifugal 60 minutes, abandon precipitation; Get supernatant, add 60% saturation ratio ammonium sulfate at 4 ℃, the lucifuge standing over night, at 4 ℃, with 15000 revolutions per seconds centrifugal 30 minutes, get precipitation; Use 0.02M, the phosphoric acid buffer dissolution precipitation of PH=7, at 8 ℃, lucifuge leaves standstill, with 0.01M, the dialysis of PH=7 phosphoric acid buffer, molecular retention amount 12000Da; After concentrating fast, last SephadexG200 chromatography column is used 0.01M, PH=7 phosphoric acid buffer wash-out is collected the peak value component, last DEAE-SephadexA25 post, use 0.005M, the phosphoric acid buffer of PH=7 and 0.2M, the sodium-chlor wash-out of PH=7, collect peak value component S ephadexG200, use 0.01M, the phosphoric acid buffer wash-out of PH=7, whole column chromatography carries out under 8 ℃, lucifuge condition, the gained Phycocyanins, C-is frost drying immediately, and gained dry powder keeps in Dark Place at 4 ℃.

The Phycocyanins, C-product detects key instrument to be had: UV, visible light scanning spectrometer, UV, visible light spectrophotometer, fluorescent scanning spectrograph, electrophoresis apparatus etc., and the significant parameter of Phycocyanins, C-and proterties:

(1) purified product is pure sky blue, has red fluorescence;

(2) purity ratio (A620nm/A280nm) 〉=4;

(3) uv-vis spectra presents main peak at the 620nm place;

(4) polyacrylamide gel electrophoresis (PAGE) manifests single blue electrophoresis band;

(5) the fluorescence excitation peak lays respectively at 590nm and 635nm, and fluorescence emission peak is positioned at 650nm, all manifests the main peak collection of illustrative plates.

The high-purity high-activity phycocyanin of the present invention's preparation can single-mindedly combine with DNA, in the flow cytometer detection system, can demonstrate the heteroploid nuclear gene group that overlength such as human body cervical cancer cell are expanded clear and delicately, thereby carry out the diagnosis of cancer cells quickly and accurately, have potential applicability in clinical practice.

Claims (4)

1, a kind of separation purification method of high-purity high-activity phycocyanin is characterized in that:

(1) fresh spirulina plalensis algae mud adds 0.001～0.005M, and the phosphoric acid buffer of PH=7 after the freeze thawing 1～2 time, places ice bath ultrasonication cell;

(2) at 4～10 ℃, with 9000～11000 revolutions per seconds centrifugal 50～70 minutes, abandon precipitation;

(3) get supernatant, add 30～60% saturation ratio ammonium sulfate at 4～10 ℃, the lucifuge standing over night, at 4～10 ℃, with 13000～18000 revolutions per seconds centrifugal 20～40 minutes, get precipitation;

(4) with 0.01～0.03M, the phosphoric acid buffer dissolution precipitation of PH=7, at 4～10 ℃, lucifuge leaves standstill, with 0.01～0.03M, the dialysis of PH=7 phosphoric acid buffer, molecular retention amount 12000Da;

(5) after concentrating fast, last SephadexG200 chromatography column is with 0.01～0.03M, PH=7 phosphoric acid buffer wash-out is collected the peak value component, last DEAE-SephadexA25 post, with 0.005～0.007M, the phosphoric acid buffer of PH=7 and 0.2～0.4M, the sodium-chlor wash-out of PH=7, collect the peak value component, last ScphadcxG200 post, with 0.01～0.03M, the phosphoric acid buffer wash-out of PH=7, whole column chromatography carries out under the lucifuge condition at 8～10 ℃;

(6) Phycocyanins, C-frost drying immediately, gained dry powder keeps in Dark Place at 4～10 ℃.

2,, it is characterized in that Phycocyanins, C-purity and activity index A620nm/A280nm 〉=4 according to the separation purification method of the described Phycocyanins, C-of claim 1;

3,, it is characterized in that Phycocyanins, C-fluorescence excitation peak lays respectively at 590nm and 635nm according to the separation purification method of the described Phycocyanins, C-of claim 1;

4,, it is characterized in that the Phycocyanins, C-fluorescence emission peak is positioned at 650nm according to the separation purification method of the described Phycocyanins, C-of claim 1.