CN103214566A

CN103214566A - G factor separating and purifying method

Info

Publication number: CN103214566A
Application number: CN2013101076643A
Authority: CN
Inventors: 丁友玲
Original assignee: FUZHOU XINBEI BIOCHEMICAL INDUSTRY Co Ltd
Current assignee: FUZHOU XINBEI BIOCHEMICAL INDUSTRY Co Ltd
Priority date: 2013-03-30
Filing date: 2013-03-30
Publication date: 2013-07-24

Abstract

The invention discloses a G factor separating and purifying method. The method disclosed by the invention comprises a step of: separating and extracting hemocyte from limulus whole blood, a step of disintegrating the hemocyte and primarily separating and purifying various hemocyte components containing G factors in the limulus hemocyte through a affinity chromatography method, and a step of further separating and purifying combined components mainly containing the G factors acquired after the primarily separating and purifying step so as to prepare a high-purity G factor component without a coagulation source. Through the technical scheme adopted by the invention, the limulus resource can be fully used, and the problem of removing the coagulation source from the G factor component is overcome.

Description

A kind of separation purification method of G-factor

Technical field

The present invention relates to the separation purification method of the technical field of tachypleus amebocyte lysate, particularly a kind of G-factor.

Background technology

Nineteen sixty-eight Levin J and Bang F B have created since the tachypleus amebocyte lysate, make endotoxic detection become sensitivity, quick, easy method, by universally acknowledged, and by beautiful, day, in, European Pharmacopoeia records with " bacterial endotoxin test ".But Japanese scholar Kakinuma A in 1981 finds that non-pyrogen material (1-3)-callose also can make the tachypleus amebocyte lysate cohesion, this has greatly influenced common tachypleus amebocyte lysate and has detected endotoxic specificity, thereby detect endotoxic tachypleus amebocyte lysate at present and be mainly special tachypleus amebocyte lysate, the characteristics of its production technique are to utilize the affinity chromatography method can separate removal, " preparation technology of anti-interference tachypleus amebocyte lysate " who discloses among the Chinese patent CN1621841A as inventor's application with the G-factor of (1-3)-callose generation aggregation with what exist in the common tachypleus amebocyte lysate.

Because dextran is the main component of fungal cell wall, and patient's fungi infestation detection medically is being a difficult problem always, so the principle of dextran and G-factor generation aggregation is used in the detection of fungi infestation very soon, " a kind of direct fungus detection tachypleus amebocyte lysate box and the method " that the Chinese patent CN101880706A that applies for as the inventor discloses.This detection method is higher to the purity requirement of G-factor, especially solidifying of containing in the limulus blood cell former (coagulogen) can not mix wherein, and the isolating G-factor of conventional affinity chromatography and solidify former in same interval wash-out, therefore contain usually in the merging component of G-factor and solidify stock blend, the reagent that can not directly detect as fungi uses.So, with obtain high purity G-factor component be target developing a kind of from the king crab whole blood method of specific isolation purifying G-factor, or solidifying former G-factor with containing of producing in the special tachypleus amebocyte lysate production process merges the method that component is the further separation and purification of basis invention.Obviously, a kind of method in back more can make full use of valuable king crab resource, but from the G-factor component, remove character close to solidify former be a technical barrier.

Summary of the invention

The object of the present invention is to provide a kind of separation purification method of G-factor, it comprises from the king crab whole blood separation and Extraction hemocyte step and will utilize affinity chromatography to carry out the step of primary separation purifying to comprising the various hemocyte compositions of G-factor in the limulus blood cell after the hemocyte disintegration, it also comprises the step to the further separation and purification of merging component that mainly contains G-factor of primary separation purifying gained, and nationality is removed with preparation and solidified former high purity G-factor component.

The step of this further separation and purification is:

The first step: preparation ConA-Sepharose sepharose post, this ConA-Sepharose sepharose post specification is that post is Ф 2 * 16cm, carry out pre-equilibration with the 20 mM Tris-HCl solution that contain pH=8.0,0.25 mol/L NaCl, follow with the above-mentioned merging component of G-factor that mainly contains as sample on the sample;

Second step: fully wash and 0.5 mol/L methyl glucoside methyl-α-D-glucoside eluant solution with 0.5 mol/L NaCl solution, this 0.5 mol/L methyl glucoside methyl-α-D-glucoside solution is prepared with pH=8.0,20 mM Tris-HCl solution;

The 3rd step: collect the elutriant that second step produced, concentrate with Amicon PM-10 ultra-filtration membrane;

The 4th step: collect the concentrated solution that the 3rd step produced, further purify with Sephacryl S-200HR post, this Sephacryl S-200HR post is Ф 2.7 * 98cm, with pH8.0,50mM Na ₃PO ₄The damping fluid pre-equilibration;

The 5th step: collect the high purity G-factor merging component that the 4th step produced, use the freeze drier cryoconcentration.

Technical scheme of the present invention realized the making full use of of king crab resource, and overcome simultaneously to solidify the former difficult problem of removing of being difficult to from the G-factor component.

Description of drawings

Fig. 1 is a ConA-Sepharose sepharose post elution curve of the present invention.

Fig. 2 is a Sephacryl S-200HR post elution curve of the present invention.

Embodiment

The present invention discloses a kind of G-factor separation purification method, it comprises from the king crab whole blood separation and Extraction hemocyte step and will utilize affinity chromatography to carry out the step of primary separation purifying to comprising the various hemocyte compositions of G-factor in the limulus blood cell after the hemocyte disintegration, also comprise the step to the further separation and purification of merging component that mainly contains G-factor of primary separation purifying gained, nationality is removed with preparation and is solidified former high purity G-factor component.

Partly disclose as the embodiment among the Chinese patent application CN1632580A, typical from the king crab whole blood separation and Extraction hemocyte step and will utilize affinity chromatography to be after the hemocyte disintegration to the step that the various hemocyte compositions that comprise G-factor in the limulus blood cell carry out the primary separation purifying:

After the king crab that lives adds the antithrombotics blood sampling, centrifugal under 1000rpm, abandon supernatant liquor, collecting cell adds the washings washing that contains NaCl 3 mol/L and Tris-HCl 0.05 mol/L pH 8.0, abandoning supernatant again; The cell of collecting adds the disintegration liquid that contains NaCl 0.1 mol/L and Tris-HCl 0.02 mol/L pH 8.0 and carries out cell disruption, carries out centrifugally afterwards, collects extracting solution; Extracting solution carries out affinity chromatography, washes post with the NaCl and the Tris-HCl elutriant of different salt concn, collects elutriant; After elutriant differentiated, the elutriant of identical component is merged.

For another example, the step of primary separation purifying can also partly disclose as the embodiment among the Chinese patent application CN1621841A: adopt affinity chromatography, utilize the aseptic pyrogen-free affinity column of gel Dextran sulfate Sepharose CL-6B preparation and collect the equipment that separates composition, compositions such as each factor in the limulus blood cell such as the C factor, the B factor, G-factor, anti-endotoxin factor, proclotting enzyme are separated respectively.

After utilizing technique scheme or similar techniques scheme well known to those skilled in the art to obtain mainly to contain the merging component of G-factor, technical scheme of the present invention is carried out further purification procedures, and the concrete steps of taking in the present embodiment are:

The first step: preparation ConA-Sepharose sepharose post, this ConA-Sepharose sepharose post specification is that post is Ф 2 * 16cm, carry out pre-equilibration with the pH=8.0 that contains 0.25 mol/L NaCl, 20 mM Tris-HCl solution, follow with the above-mentioned merging component of G-factor that mainly contains as sample on the sample;

Second step: fully wash and 0.5 mol/L methyl glucoside methyl-α-D-glucoside eluant solution with 0.5 mol/L NaCl solution, this 0.5 mol/L methyl glucoside methyl-α-D-glucoside solution is prepared with pH=8.0,20 mM Tris-HCl solution.Consult ConA-Sepharose sepharose post elution curve shown in Figure 1 as can be known, G-factor with solidify former elution peak overlapping (shown in the elution peak that Fig. 1 hollow core circle is linked to be), the α-MG among Fig. 1 is writing a Chinese character in simplified form of methyl glucoside methyl-α-D-glucoside;

The 3rd step: collect the elutriant that second step produced, concentrate with Amicon PM-10 ultra-filtration membrane." elutriant that second step produced " refers to the elutriant of 0.5 mol/L methyl glucoside methyl-α-D-glucoside eluant solution among Fig. 1, do not comprise that 0.5 mol/L NaCl solution fully washes the solution of generation;

The 4th step: collect the concentrated solution that the 3rd step produced, further purify with Sephacryl S-200HR post, this Sephacryl S-200HR post is Ф 2.7 * 98cm, with pH 8.0,50mM Na ₃PO ₄The damping fluid pre-equilibration.Consult Sephacryl S-200HR post elution curve shown in Figure 2, in visible second step that is the elution peak eclipsed G-factor shown in Fig. 1 with solidify former two components and obviously separate (elution peak that Fig. 2 hollow core circle is linked to be is represented the G-factor component);

In the above-mentioned steps, ConA-Sepharose sepharose column packing is bought from U.S. Bio-Rad company, Amicon PM-10 ultra-filtration membrane is bought from U.S. Millipore company, Sephacryl S-200HR column packing is bought from U.S. GE company, and other reagent and consumptive material are those skilled in the art and know.

Above-mentioned only is specific embodiments of the invention, but design concept of the present invention is not limited thereto, and allly utilizes this design that the present invention is carried out the change of unsubstantiality, all should belong to the behavior of invading protection domain of the present invention.

Claims

1. the separation purification method of a G-factor, it comprises from the king crab whole blood separation and Extraction hemocyte step and will utilize affinity chromatography to carry out the step of primary separation purifying to comprising the various hemocyte compositions of G-factor in the limulus blood cell after the hemocyte disintegration, it is characterized in that: also comprise the step to the further separation and purification of merging component that mainly contains G-factor of primary separation purifying gained, nationality is removed with preparation and is solidified former high purity G-factor component;

The step of this further separation and purification is:

Second step: fully wash and 0.5 mol/L methyl glucoside methyl-α-D-glucoside eluant solution with 0.5 mol/L NaCl solution, this 0.5 mol/L methyl glucoside methyl-α-D-glucoside solution is prepared with pH=8.0,20 mM Tris-HCl () solution;

The 4th step: collect the concentrated solution that the 3rd step produced, further purify with Sephacryl S-200HR post, this Sephacryl S-200HR post is Ф 2.7 * 98cm, with pH 8.0,50mM Na ₃PO ₄The damping fluid pre-equilibration;