CN101585874B - Method for separating and purifying sea-mussel mucin by using salting out and dialyzing - Google Patents

Abstract

The invention relates to a method for separating and purifying the sea-mussel mucin by using salting out and dialyzing, the salting out and the dialyzing utilizes the proteinaceous physicochemical character to purify by a non- chromatograph method, and solves the problem of high apparatus and material cost in the chromatograph method for purifying the sea-mussel mucin, a quick, low-cost and high-concentration method for separating and purifying the sea-mussel mucin is provided. The strong acid is used for extracting, the salting out and the dialyzing are used for purifying, the sea-mussel mucin is determined by adopting the acetic acid-carbamide- polyacrylamide gel electrophoresis via the blue tetrazolium specificity colour development, and the purity is determined by the inversed phase chromatography quantitatively.

Description

A kind of use saltout and dialyse method of separating and purifying sea-mussel mucin

Technical field

The present invention relates to a kind of separation and purification method of protein, relate to a kind of use method with the dialysis method separating and purifying sea-mussel mucin of saltouing specifically.

Background technology

(Mussel adhesive protein MAP), also is Mytilus edulis byssus albumen (Mytilus edulisfoot protein to sea-mussel mucin; Mefp) from the seashells Mytilus edulis, Mytilus edulis, it has the ability of the tolerance wave effect in the coastal waters; It generates and stores a kind of albumin glue in special body of gland; Be discharged into through byssus on the solid surface of one type on rock, form water resisting combination, thus oneself is fixing.Show that in research on glass albumin glue has formed spot, extend from spot and open that very strong tensile strength (10 is arranged⁶-10⁷Newtonmeter^-2), and material has comprised sea-mussel mucin MAP in the spot.

Sea-mussel mucin contains L-3,4-dopa (L-DOPA), and it is formed by the effect of tyrosine oxidase to tyrosine residues.The L-DOPA residue is owing to oxidizing reaction is cross-linked with each other, and crosslinking reaction has caused strong and persistent splicing in mussel and the special surface fiber, and the mechanism of its bioadhesion is very interesting.Sea-mussel mucin adheres to does not have toxicity and immunogenicity to the people, and binding ability and life-span do not receive the influence of water, arouse attention as medical operation especially ophthalmologic operation glue.

Every year is carried out 1,100 ten thousand routine cataract operations in the whole world, and the cataractous sickness rate of China is very high, more than 50 years old among the crowd cataract incidence more than 29.64%, 70 years old crowd's sickness rate up to 60%.Along with the aggravation of social senilization, this numeral also can rise.At present the doctor can close with mode with two kinds and finish ophthalmologic operation: let wound heal voluntarily or use the nylon line suture otch, every kind of method of closing all has shortcoming: self-healing has to be infected and the danger of intraocular liquid leakage; Stitching has the danger of infection inflammation and angiodysplasia.And the sea-mussel mucin sea-mussel mucin can be crosslinked under lower concentration, and form the low viscosity liquid may be injected into irregularly shaped position, this solution solidifiable and be filled into designated space, but in several minutes closure of incisions, be less than the stitching required time.Sea-mussel mucin has also formed not destructible strip of paper used for sealing, under greater than the pressure of 12 times of normal people's intraocular pressures, intraocular liquid is revealed.Sea-mussel mucin has and the proximate specific refractory power of cornea, can disturbance not arrive retina.

In addition, the biological degradation character of sea-mussel mucin makes it very friendly to environment, also can be used for other field; Like the general bonding and sealing agent of biochemical bonding reagent, medical tissue, medical callus matrix, the bonding and sealing agent of dentist; Medical equipment top coat reagent, underwater operation equipment coating etc.

Saltout and dialysis is to utilize proteinic physicochemical property, carry out purifying with non-stratographic method, the target product concentration height that equipment cost is low, the experiment material cost is low, consuming time less, obtain need not concentrate.

Summary of the invention

The objective of the invention is to overcome chromatographic process purifying sea-mussel mucin equipment and the high problem of material cost, adopt the method saltout and dialyse, provide a kind of fast, the sea-mussel mucin separation purification method of low-cost, high density.

The objective of the invention is to realize through following technical scheme.

The method of saltouing and dialysing to the separating and purifying sea-mussel mucin of core of using provided by the invention comprises the steps:

1) with 0.5-2.5% perchloric acid or/and 1-10% acetate serves as to extract solvent, 100g mussel byssus adds 300-1000ml and extracts solvent, 10-25 ℃ of lixiviate 10-20min uses whisking appliance that refrigerated mussel byssus is smashed at a high speed and it evenly is suspended in extract in the solvent;

2) remove residue with the centrifugal 30-50min of 10000-16000r/min or with 45 μ m membrane filtrations, keep supernatant;

3) with the sodium-chlor of the 6-12M of the 1-6 times of volume 1-6h that under 15-25 ℃, saltouts;

4) 4 ℃, centrifugal 3-10min under the 10000-15000rpm, collecting precipitation is with the acetate dissolving of 1-3%;

5) 4 ℃, the wherein insoluble composition of the centrifugal 3-10min removal of 10000-15000rpm dissolves with the acetate of 1-3% once more;

6) with 4 ℃ of dialysis of acetate (dialysis tubing molecular weight cut-off 12000) 3-10h of 0.5-3%, 4 ℃ of preservations;

7) identify sea-mussel mucin from the angle of molecular weight: adopt 12-20% concentration sodium laurylsulfonate (SDS)-polyacrylamide gel electrophoresis (PAGE); Cma staining; 10-25 ℃ of dyeing 2-4h with HMW standard protein mark, confirms that the target protein molecular weight is 130kD;

8) blue (NBT) specific stain of tetrazole is identified sea-mussel mucin: adopt 12-20% strength acetic acid-urea (Acetic acid-Urea) polyacrylamide gel electrophoresis (Native PAGE); Staining fluid adopts tetrazole indigo plant-Padil solution (pH10); 10-25 ℃ of dyeing 1-3h, sea-mussel mucin will be developed the color by specificity;

9) analyze sea-mussel mucin purity with inverse bonded phase silica gel chromatographic column of C8 or Source polystyrene columns: with the acetonitrile-water is the moving phase isocratic elution, and 280nm detects.

The provided by the invention use to saltout and to dialyse is the method for the separating and purifying sea-mussel mucin of core, provide a kind of fast, the sea-mussel mucin separation purification method of low-cost, high density.

Embodiment

Embodiment 1, with perchloric acid extraction, saltout and separate sea-mussel mucin with dialysing

1) serve as to extract solvent with 0.5% perchloric acid, 100g mussel byssus adds 300ml and extracts solvent, and 25 ℃ of lixiviate 10min use whisking appliance that refrigerated mussel byssus is smashed at a high speed it evenly is suspended in the extraction solvent;

2) remove residue with the centrifugal 30min of 10000r/min, keep supernatant;

3) with the sodium-chlor of the 6-12M of the 2 times of volumes 1-6h that under 15-25 ℃, saltouts;

4) 4 ℃, centrifugal 3min under the 10000rpm, collecting precipitation, acetate dissolving with 2%;

5) 4 ℃, the wherein insoluble composition of the centrifugal 3min removal of 10000rpm dissolves with 2% acetate once more;

6) with 4 ℃ of dialysis of acetate (dialysis tubing molecular weight cut-off 12000) 5h of 2%, 4 ℃ of preservations;

7) identify sea-mussel mucin from the angle of molecular weight: adopt 15% concentration sodium laurylsulfonate (SDS)-polyacrylamide gel electrophoresis (PAGE); Cma staining; 25 ℃ of dyeing 2h with HMW standard protein mark, confirm that the target protein molecular weight is 130kD;

8) blue (NBT) specific stain of tetrazole is identified sea-mussel mucin: adopt 15% strength acetic acid-urea (Aceticacid-Urea) polyacrylamide gel electrophoresis (Native PAGE); Staining fluid adopts tetrazole indigo plant-Padil solution (pH10); 25 ℃ of dyeing 2h, sea-mussel mucin will be developed the color by specificity;

9) analyze sea-mussel mucin purity with the inverse bonded phase silica gel chromatographic column of C8: C8 chromatographic column 4.6 * 250mm; 5 μ m; is the moving phase isocratic elution with acetonitrile-water (10-90), and 280nm detects.

Embodiment 2, with acetic acid extraction, saltout and separate sea-mussel mucin with dialysing

1) serve as to extract solvent with 10% acetate, 100g mussel byssus adds 1000ml and extracts solvent, 25 ℃ of lixiviates, and 20min uses whisking appliance that refrigerated mussel byssus is smashed at a high speed it evenly is suspended in the extraction solvent;

2) remove residue with 45 μ m membrane filtrations, keep supernatant;

3) with the sodium-chlor of the 10M of the 3 times of volumes 6h that under 25 ℃, saltouts;

4) 4 ℃, centrifugal 3min under the 15000rpm, collecting precipitation, acetate dissolving with 3%;

5) 4 ℃, the wherein insoluble composition of the centrifugal 3min removal of 15000rpm dissolves with 3% acetate once more;

6) with 4 ℃ of dialysis of acetate (dialysis tubing molecular weight cut-off 12000) 10h of 3%, 4 ℃ of preservations;

7) identify sea-mussel mucin from the angle of molecular weight: adopt 12% concentration sodium laurylsulfonate (SDS)-polyacrylamide gel electrophoresis (PAGE); Cma staining; 25 ℃ of dyeing 2h with HMW standard protein mark, confirm that the target protein molecular weight is 130kD;

8) blue (NBT) specific stain of tetrazole is identified sea-mussel mucin: adopt 12% strength acetic acid-urea (Aceticacid-Urea) polyacrylamide gel electrophoresis (Native PAGE); Staining fluid adopts tetrazole indigo plant-Padil solution (pH10); 25 ℃ of dyeing 2h, sea-mussel mucin will be developed the color by specificity;

9) the Source polystyrene columns is analyzed sea-mussel mucin purity: with acetonitrile-water (15-85) is the moving phase isocratic elution, and 280nm detects.

Claims (1)

1. the use method of separating and purifying sea-mussel mucin of saltouing and dialyse comprises following step:

1) with 0.5-2.5% perchloric acid or/and 1-10% acetate serves as to extract solvent, 100g mussel byssus adds 300-1000ml and extracts solvent, 10-25 ℃ of lixiviate 10-20min uses whisking appliance that refrigerated mussel byssus is smashed at a high speed and it evenly is suspended in extract in the solvent;

2) remove residue with the centrifugal 30-50min of 10000-16000r/min or with 45 μ m membrane filtrations, keep supernatant;

3) with the sodium-chlor of the 6-12M of the 1-6 times of volume 1-6h that under 15-25 ℃, saltouts;

4) 4 ℃, centrifugal 3-10min under the 10000-15000rpm, collecting precipitation is with the acetate dissolving of 1-3%;

5) 4 ℃, the wherein insoluble composition of the centrifugal 3-10min removal of 10000-15000rpm dissolves with the acetate of 1-3% once more;

6) with 4 ℃ of dialysis of the acetate of 0.5-3% 3-10h,, wherein 12000,4 ℃ of preservations of dialysis tubing molecular weight cut-off;

7) identify sea-mussel mucin from the angle of molecular weight: adopt 12-20% concentration sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Cma staining; 10-25 ℃ of dyeing 2-4h with HMW standard protein mark, confirms that the target protein molecular weight is 130kD;

8) the blue specific stain of tetrazole is identified sea-mussel mucin: adopt 12-20% strength acetic acid-urea-polyacrylamide gel electrophoresis, staining fluid adopts tetrazole indigo plant-Padil solution of pH10,10-25 ℃ of dyeing 1-3h, and sea-mussel mucin will be developed the color by specificity;

9) analyze sea-mussel mucin purity with inverse bonded phase silica gel chromatographic column of C8 or Source polystyrene columns: with the acetonitrile-water is the moving phase isocratic elution, and 280nm detects.