CN105949300A - Method for extracting and separating and purifying protein - Google Patents

Abstract

The invention discloses a method for extracting and separating and purifying protein. The method includes an ovomucin separating and purifying method, an ovotransferrin separating and purifying method and an egg immunoglobulin separating and purifying method. The method is characterized in that the method performs dilution before homogenization, egg white is diluted by normal saline and then homogenized, the viscosity of the egg white is lowered, the complex structure of the egg white is destroyed, and accordingly the egg white can be filtered fast, easy membrane cleaning is achieved, membrane pollution degree is lowered, and the membrane service life is prolonged; when operation pressure is 0.15MPa, a relatively-stable circulation state is achieved, and filtrate concentration polarization is inhibited to a certain degree; the method is based on a dilution method and uses polyethylene glycol and DEAE-Toyopearl 650M one-step elution ion-exchange column chromatography to perform purification, and high-yield and high-purity chicken egg yolk IgY.

Description

A kind of protein extraction and isolation and purification method

Technical field

The invention belongs to glycoprotein technical field, particularly relate to a kind of protein extraction and isolation and purification method.

Background technology

Avian ovomucoid is a kind of glycoprotein prepared in clear by ovum gallinaceum, has the tryptic work of strong suppression With, it is usually used in tryptic zymology Quality Research and is prepared by tryptic affinitive layer purification.Molecular weight: 28000Da, Molecule is formed glycoprotein by the subunit that four molecular weight are close and forms, i.e. D-MANNOSE, D-galactose, glucamine, sialic acid； Chemical stability, heat-resisting: under the conditions of 80 DEG C, physicochemical property does not changes, organic solvent-resistant: at the acetone soln of 50% In, good dissolubility can be kept, resistance to precipitant: do not precipitate in the solution of the TCA of 10%.Isoelectric point, IP character, waits electricity Point: pI between 3.9-4.5, the state in solution: stable in the solution of pH3.0, easily decompose in the solution of pH8.0. Chicken yolk immunoglobulin, is called for short IgY, also known as chicken yolk antibody.There is immunogenicity, in egg yolk with α-, β-and γ-yolk Three kinds of forms of globulin exist.

The IgY isolation and purification method used in the world at present mainly includes water dilution method, the organic solvent extracting sedimentation method, surpasses The physical-chemical process such as filter, supercritical extraction and freeze thawing and saltouing and the method such as DEAE chromatograph combines.Be related to both at home and abroad from The existing more research of separation and Extraction active substance and report in Ovum Gallus domesticus album, and the industrialized production be correlated with creating well Economic benefit, such as lysozyme, immunoglobulin etc..And the research extracting ovotransferrin from Ovum Gallus domesticus album the most also rests on Laboratory scale, and unrealized industrialized production, progress is the slowest.

Summary of the invention

It is an object of the invention to provide a kind of protein extraction and isolation and purification method, it is intended to solve described in background technology Problem.

The present invention is achieved in that a kind of protein extraction and isolation and purification method, described protein extraction and isolated and purified It is pure that method includes that ovomucin isolation and purification method, egg ovotransferrin isolation and purification method and egg immunoglobulin extract Change method；

Described ovomucin isolation and purification method uses GaCL₂Or MgCl₂Prepare ovomucin；

Described egg ovotransferrin isolation and purification method includes: the clear stock solution of Fresh Egg, 0.9 normal saline dilution, 3000r/min is centrifuged 15min and removes ovomucin, then saltouts removing ovalbumin by isoelectric point, IP；By centrifugation and isoelectric precipitation Sample microfiltration remove 0.1-100 μ m impurity, and then use ultrafiltration, with polysulfone resin derivatives membrane remove 100kDa with Upper macro-molecular protein, then with the polysulfone resin derivatives membrane molecular cut off protein more than 50kDa, obtain concentrated solution, obtain Concentrated solution carry out dialysis treatment, and lyophilization；

Described egg immunoglobulin method for extraction and purification uses water dilution method to combine polyethylene glycol precipitation and DEAE- Toyopearl 650M one-step elution ion-exchange chromatography is purified, and obtains highly purified Ovum Gallus domesticus Flavus IgY of high-recovery.

Further, described ovomucin isolation and purification method comprises the following steps:

Shell egg adds the saline solution of 5 times of volumes, 0.05mol/LMgCl after stirring clearly₂Or 0.1mol/L NaCl, homogenizing Adjusting pH=6.0 with 0.1mol/L HCl afterwards, 4 DEG C stand overnight, and 15,000g are centrifuged 10min, 4 DEG C, precipitation 0.5mol/L NaCl is resuspended, places 4h for 4 DEG C, and 15,000g are centrifuged 10min, 4 DEG C, and to supernatant, the precipitation distilled water washing obtained is not contained egg White matter, sediment fraction is ovomucin crude extract；

Crude extract is dissolved in 10mmol/L borate acid buffer pH=9.6 and is made into 5mg/ml solution, and 4 DEG C stirred At night, carrying out gel chromatography after 0.45 μm filtering with microporous membrane, eluent is containing 0.2mol/LMgCl₂Tris-HCl buffer, 20mmol/L, pH=8.4, flow velocity 0.5ml/min；The lyophilizing sample collecting each eluting peak is identified through SDS-PAGE, target -20 DEG C of preservations after component lyophilization.

Further, described ovomucin is respectively 0.05mg/mL to the minimal inhibitory concentration of escherichia coli and Salmonella And 0.2mg/mL.

Further, described egg ovotransferrin isolation and purification method includes:

Connect ultrafiltration apparatus, continue 1h by 0.2MPa ultra-pure water pressure, after water flux density is stable, measure ultrafilter membrane Initial pure water flux, after ultrafiltration, takes out ultrafilter membrane and carries out distilled water flushing, and soak with 0.1M NaOH and 0.1M HCl 30min, finally rinses repeatedly with distilled water, again measures film stream of pure water flux；

The clear stock solution of Fresh Egg, 0.9 normal saline dilution, 3000r/min is centrifuged 15min and removes ovomucin, then uses Electricity puts removing ovalbumin of saltouing；

Sample microfiltration with isoelectric precipitation removes the impurity of 0.1-100 μ m by centrifugation, and then uses hyperfiltration technique, More than 100kDa macro-molecular protein is removed with polysulfone resin derivatives membrane, bigger with polysulfone resin derivatives membrane molecular cut off In the protein of 50kDa, obtaining concentrated solution, the concentrated solution obtained afterwards carries out dialysis treatment, and lyophilization.

Further, the ultrafiltration apparatus pressure of described protein extraction and isolation and purification method is 0.15MPa, 0.9 normal saline Extension rate is 10 times, and stir speed (S.S.) is 125r/min, and now membrane flux value is 39.80, and pH is 7.

Further, described egg immunoglobulin method for extraction and purification includes:

Choose Fresh Egg, use egg-white egg-yellow separator, isolated egg yolk, with distilled water, egg yolk is cleaned, be placed in Blot on filter paper, puncture membrane of yolk, collect egg yolk liquid, with volume percentage, with distilled water, egg yolk is carried out different multiples Dilute 8 times to stir, the different pH value 5.0～5.6 of regulation, 4 DEG C of standings different time (2h, 4h, 6h, 8h, 10h), 5000 × After frozen centrifugation 30min, obtain clarified supernatant water-soluble component WSF Han IgY；

Adding the PEG6000 of different quality mark in the WSF of gained, magnetic stirrer 30min, 22000 × g is cold Freeze centrifugal, collect precipitation；

Dialysis, collects precipitation and adds distilled water, stirring, load bag filter, and molecular cut off is in 8000-14000KD, and 4 DEG C dialysis remove part residual PEG6000, lyophilization i.e. obtains thick IgY component；

Ion exchange chromatography IgY, uses Na₂HPO₄-NaH₂PO₄Buffer balances, and arranging pH value is 6.0,7.0, Different buffer ionic strength (0.03mol/L, 0.05mol/L, 0.1mol/L) loading balances, isorheic elution under the conditions of 8.0 (0.075mol/L, 0.1mol/L), eluting effluent is collected automatically by often pipe 5ml/3min；Component collected by chromatography is loaded Bag filter molecular cut off is 8000-14000KD, is placed in distilled water, 4 DEG C of dialysis desalinations.

The protein extraction of present invention offer and isolation and purification method, use GaCL₂With MgCl₂In conjunction with method prepare ovum glue Albumen best results, the ovomucin purity 97% of preparation, the ovomucin purity using the method to prepare is more than 97%, eventually Yield 408.65 ± 4.32mg/100g Ovum Gallus domesticus album, the response rate is (85.3 ± 1.08) %.Separate through gel filtration, be successfully separated Being 302.1mg to pure ovomucin, total yield is 57.03%.

The present invention uses the method for homogenizing after first diluting, and egg white solution is homogenizing after normal saline dilution, not only reduces egg The viscosity of clear liquid, and destroy the structure of its complexity, so that the egg white solution rate of filtration is very fast, Membrane cleaning is relatively easy to, fall The low pollution level of film, extends the service life of film when operating pressure at 0.15MPa, is in the shape that circulates the most smoothly State, and filtrate concentration polarization phenomenon the most always.

The present invention is based on water dilution method, and combines polyethylene glycol precipitation and DEAE-Toyopearl650M mono-step Elution ionic displacement chromatography is purified, and obtains highly purified Ovum Gallus domesticus Flavus IgY of high-recovery.

Accompanying drawing explanation

Fig. 1 is protein extraction and the isolation and purification method flow chart of embodiment of the present invention offer.

Fig. 2 is that the ovomucin that the embodiment of the present invention provides affects schematic diagram to Escherichia coli Growth curve.

Fig. 3 is that the ovomucin that the embodiment of the present invention provides affects schematic diagram to Salmonella growth curve.

Fig. 4 is that the ovomucin that the embodiment of the present invention provides affects schematic diagram to staphylococcus aureus growth curve.

Fig. 5 is the process chart of the ovotransferrin ultra-filtration and separation that the embodiment of the present invention provides.

Fig. 6 is that the extension rate that the embodiment of the present invention provides affects schematic diagram to Ovum Gallus domesticus album liquid relative viscosity.

Fig. 7 is that the shear rate that the embodiment of the present invention provides affects schematic diagram to Ovum Gallus domesticus album liquid relative viscosity.

Fig. 8 is that the temperature that the embodiment of the present invention provides affects schematic diagram to Ovum Gallus domesticus album liquid relative viscosity.

Detailed description of the invention

In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, to the present invention It is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to Limit the present invention.

Below in conjunction with the accompanying drawings the application principle of the present invention is explained in detail.

One, ovomucin

1.2 ovomucins are isolated and purified

1.2.1 the technological process that ovomucin is isolated and purified, as shown in Figure 1.

S101: shell egg adds the saline solution (0.05mol/LMgCl of 5 times of volumes after stirring clearly₂Or 0.1mol/L NaCl), adjusting pH=6.0 with 0.1mol/L HCl after homogenizing, 4 DEG C stand overnight, and 15,000g are centrifuged 10min (4 DEG C), and precipitation is used 0.5mol/L NaCl is resuspended, places 4h for 4 DEG C, and 15,000g are centrifuged 10min (4 DEG C), by supreme for the precipitation distilled water washing obtained Without protein in Qing, sediment fraction is ovomucin crude extract；

S102: crude extract is dissolved in 10mmol/L borate acid buffer (pH=9.6) and is made into 5mg/ml solution, 4 DEG C Being stirred overnight, carry out gel chromatography (Sephacry IS-300HR, 2.0*7cm) after 0.45 μm filtering with microporous membrane, eluent is Containing 0.2mol/LMgCl₂Tris-HCl buffer (20mmol/L, pH=8.4), flow velocity 0.5ml/min.Collect each eluting peak Lyophilizing sample identify through SDS-PAGE ,-20 DEG C of preservations after target components lyophilization.

1.2.2 substep is saltoutd and is prepared ovomucin crude product

Shell egg adds the saline solution (0.05mol/LMgCl of 5 times of volumes after stirring clearly₂Or 0.1mol/L NaCl), homogenizing Adjusting pH=6.0 with 0.1mol/L HCl afterwards, 4 DEG C stand overnight, and 15,000g are centrifuged 10min (4 DEG C), precipitation 0.5mol/L NaCl is resuspended, places 4h for 4 DEG C, and 15,000g are centrifuged 10min (4 DEG C), and to supernatant, the precipitation distilled water washing obtained is not contained egg White matter, sediment fraction is ovomucin crude extract.

1.2.3 gel permeation chromatography

Crude extract is dissolved in 10mmol/L borate acid buffer (pH=9.6) and is made into 5mg/ml solution, and 4 DEG C stirred At night, carrying out gel chromatography (Sephacry IS-300HR, 2.0*7cm) after 0.45 μm filtering with microporous membrane, eluent is for containing 0.2mol/LMgCl₂Tris-HCl buffer (20mmol/L, pH=8.4), flow velocity 0.5ml/min.Collect each eluting peak Lyophilizing sample is identified through SDS-PAGE ,-20 DEG C of preservations after target components lyophilization.

1.2.4 polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE uses Tris-HCl buffer system to carry out vertical electrophoresis, and resolving gel concentration is 10%, concentrates gum concentration Being 5%, coomassie brilliant blue R_250 (2%) and Schifft reagent dye respectively.Protein lysates be containing SDS (1%, And 0.01mol/L Tris-HCl (pH=7.0) buffer of 2 mercapto ethanol (10 μ l/mL) w/v).Running voltage 8V/cm.

Target stripe is carried out after gel imaging system imaging by PAGE glue through Gelpro.32 protein image analysis software Analyze.

1.2.5 purity testing

Using HPLC method to measure the purity of ovomucin, target components is configured to the solution sample introduction of 1mg/mL, and RP-HPLC adopts Use C₄Post, flowing is volume fraction 0.05% trifluoroacetic acid aqueous solution mutually, column temperature 30 DEG C, sample size 20 μ l, 15-95% acetonitrile ladder Degree eluting, detects wavelength 280nm, and flow velocity is 1mL/min.

1.2.6 yield and the mensuration of yield

In the content 100g albumen protein of ovomucin, the milligram number of ovomucin represents.Computing formula is:

Wherein, the powder quality (g) after W=100g Ovum Gallus domesticus album is lyophilization prepared by raw material；

The yield of ovomucin is prepares content and the ratio of content in Ovum Gallus domesticus album raw material in sample, and computing formula is:

Wherein, W₁For the quality of dried sample, W₂For Ovum Gallus domesticus album quality

1.2.7 ovomucin Antibacterial Property

In-vitro antibacterial performance with escherichia coli, Salmonella, staphylococcus aureus as experimental subject, to ovomucin It is evaluated.

1) actication of culture and inoculation

Being inoculated in nutrient broth on the strain rearmounted aseptic operating platform of defrosting, 37 DEG C of isothermal vibrations cultivate 12h, make clump count Reach 10¹⁰And make bacteria suspension.

2) mensuration (MIC) of minimal inhibitory concentration

MIC uses doubling dilution to measure.Experimentation reference literature (left beautiful and Ma Meihu 2010).

3) mensuration of growth curve

It is 10 that the nutrient broth of the strain sterilizing of activation is diluted to cell concentration⁸CFU/m1, and then take 100 μ L and be added to 96 In the ELISA Plate of hole, add the 100 μ L ovomucin solution (0.05mg/ through the biofilter filtration of 0.45 μm at sample well ML) mixing, measures OD in 37 DEG C of constant incubators in microplate reader after cultivating 24h₆₀₀nm.With OD₆₀₀Incubation time mapping is painted Thalli growth curve processed, sets blank, positive control and negative control simultaneously, and 5, every hole is parallel, and experiment is repeated 3 times.

4) mensuration of bacteriostasis rate

(0.45 μm is thin to be sequentially added into activation bacteria suspension 100 μ L, the 0.05mg/mL ovomucin solution of dilution in 96 orifice plates Bacterium filter filters) 100 μ L, cultivate 24h in 37 DEG C of constant incubators, microplate reader measure OD after mixing₆₀₀Nm is blank right Adding 100 μ L normal saline according to for 100 μ L culture medium, matched group is the ovomucin solution that normal saline replaces sample sets, antibacterial Rate calculates (Kodama, Kimura, 2001) according to below equation.

1.2.8 ovomucin Anti-viral activity in vitro is evaluated

1.2.8.1 viral activation and the preparation of viral suspension

Taking out the test Strain of cryopreservation, 37 DEG C of tepidariums are melted, and add 1-2mL cell maintenance medium, then inoculate In the Tissue Culture Flask having covered with cell monolayer, put and 37 DEG C of incubators make and cell absorption, growth.The most under the microscope Observe pathological changes, when pathological changes occurs in 3/4 cell, collect culture fluid, and multigelation host cell, releasing virus.6000rpm from Heart 15min, removes precipitation (predominantly cell debris), and supernatant is required viral suspension.Subpackage is stored in-70 DEG C of refrigerators In, titration titer is standby.

1.2.8.2 the preparation of chicken red blood cell

Sterilizing syringe draws normal health Sanguis Gallus domesticus after washing with the sodium citrate of 3.8% in advance, is put in and is previously added 3 times of bodies In the sterile centrifugation tube of long-pending A Shi liquid, it is centrifuged 10min through 1800r/min after mixing gently, abandons supernatant.Add sterilizing again PBS suspension blood cell, 1800r/min is centrifuged 10min, abandons supernatant.Repeat this step 3 time, fully draw leukocyte and platelet After Deng, the erythrocyte of precipitation is made into 1% concentration according to institute's expense normal saline.

1.2.8.3 the mensuration of virus virulence

1) Hemagglutination titer of influenza virus

Taking plastics 96 orifice plate, 30 μ L are in the second hole in song addition from left to right, and mixing, with this doubling dilution to 11 holes, is drawn 30 μ L inhale and abandon to disinfectant solution.The dilution factor in each hole 1:2,1:4,1:8 respectively after dilution ... last hole is comparison.Each hole adds Enter chicken RBC30 μ L, the shake 1min on satellite blender of 1%, make blood cell be sufficiently mixed with virus.37 DEG C of incubators act on 15- 30min, treats the erythroprecipitin observable result of control wells.Titer judge time control tube should not coagulation, making in developmental tube The viral highest dilution that " ++ " coagulation occurs is agglutination titer, and this pipe dilution factor is 1 HAU.

2) mensuration of the TCID50 of influenza virus

Taking out one piece of Tissue Culture Plate, each hole about passes 8000-10000 cell, the cell in each hole about 70% Abundance gets final product virus inoculation.With 10 times of doubling dilution virus stock solution useds of serum-free Incubating Solution in EP pipe.Suck with multiple tracks sample injector Culture fluid in 97 orifice plates, absorption Incubating Solution is added in every Kong Zhongzai and blows and beats gently once, then sucking-off Incubating Solution.By diluted Virus liquid is added on 96 orifice plates, every hole 100 μ L, arranges normal cell controls.37℃CO₂Incubator is hatched 1h, takes out training Support plate, basis of microscopic observation cytopathy.Observe CPE, find out the viral dilution multiple that half cell bottle or pipe can be caused to infect, The TCID50 of this virus liquid is calculated by Reed-Muench method.

Difference between lgTCID50=distance proportion × dilution logarithm+higher than the dilution logarithm of 50% pathological changes rate

TCID50=Antilog is higher than the logarithm+distance proportion of the viral highest dilution of 50% pathological changes

1.2.8.4 the toxicity to chicken red blood cell

With PBS, ovomucin is made into the stock solution of 10mg/mL, with PBS by ovomucin in 96 hole V-type Microhemagglutination plates Stock solution doubling dilution successively, every hole 30 μ L.Then every hole adds 1% red cell suspension 30 μ L, and is allowed to fully shake up, quiet in greenhouse Put 30-60min, observe erythrocytic cohesion situation.

2 results and analysis

2.1 ovomucins rough standby

Use GaCL₂With MgCl₂In conjunction with method prepare ovomucin best results.Ovomucin prepared by the method is pure Degree 97%, uses ovomucin purity prepared by the method more than 97%, and end, yield 408.65 ± 4.32mg/100g Ovum Gallus domesticus album, returned Yield is (85.3 ± 1.08) %.

The chromatography purification of 2.2 ovomucins

Ovomucin crude extract eluting on Sephacry S-300 after saltouing, eluent is containing 0.05mol/ LMgCl₂Tris-HCl (20mmol/L, pH8.4), crude extract has 4 albumen eluting peaks behind gel level west.By difference eluting The albumen at peak carries out SDA-PAGE electrophoretic analysis, there are about 385.87 ± 5.32mg during 100g shell egg is clear, and yield is about 73.83%, Separating through gel filtration, be successfully separated that to obtain pure ovomucin be 302.1mg, total yield is 57.03%.

2.3 ovomucins inhibition to different bacterium

Use constant broth dilution method have studied Salmonella that this laboratory preserves by the ovomucin of variable concentrations, big Enterobacteria and the fungistatic effect of staphylococcus aureus.Result is as shown in table 1.

Table 1 ovomucin minimal inhibitory concentration (MIC) to Salmonella, escherichia coli and staphylococcus aureus

Note: "-" represents glucose-non-variable color of phenol red broth bouillon；"+", represents that glucose-phenol red broth bouillon becomes Yellowly, has colony growth

Note: "-" represent that the color of Broth medium does not change, no bacterial grow；"+" represent that the color of Broth medium changed to yellow, bacterial grow.

From table 1 it follows that ovomucin is to the antibiotic property of three kinds of antibacterials, there were significant differences, wherein to escherichia coli and Salmonella all has inhibitory action in various degree, and when ovomucin concentration reaches more than 0.2mg/mL, Salmonella is cultivated Base invariant color, shows that Salmonella growth is substantially suppressed.And when ovomucin concentration reaches more than 0.050mg/mL, greatly Enterobacteria culture medium invariant color, shows that now Escherichia coli Growth is substantially suppressed.Therefore, ovomucin to escherichia coli and The minimal inhibitory concentration of Salmonella is respectively 0.05mg/mL and 0.2mg/mL.And in the range of experimental concentration degree, ovomucin Impact on staphylococcus aureus is then contrary, and the experimental group culture medium adding ovomucin all becomes yellow, the most each experimental group All there is colony growth, and culture medium color be relatively deeper than blank group color, shows that now thalli growth is the most not suppressed, Promoted on the contrary, but the difference on effect of each concentration group is not notable (P ＞ 0.05).Therefore ovomucin is to staphylococcus aureus Without obvious inhibiting effect.

The impact on growth curve of bacteria of 2.4 ovomucins

Using the turbidimetry for Determination variable concentrations ovomucin impact on three kinds of growths curve of bacteria, result is respectively such as figure Shown in 2, Fig. 3, Fig. 4:

By above-mentioned each figure it can be seen that ovomucin is more significant with the impact ratio of three kinds of growths curve of bacteria on experiment.And In fig. 2 it can be seen that colibacillary growth curve is respectively positioned at whole growth cycle after adding 0.05mg/mL ovomucin Under matched group.Show that escherichia coli are the most substantially suppressed during whole growth cycle.In figure 3, Salmonella It is suppressed in laundering period and logarithmic (log) phase.Therefore, ovomucin is delay thalline right to the Salmonella mode of playing a role The number phase, without obvious bactericidal activity.Ovomucin to staphylococcus aureus (Fig. 4) then without obvious inhibiting effect.

2.5 ovomucin Anti-viral activity in vitro are evaluated

The virulence of the most tested Strain

By H₁N₁The metamorphosis of observation of cell under every day inverted microscope after tested virus sensitive cells, with 7d The cytopathic sample well of interior appearance is positive hole, the TCID of experiment with computing Strain₅₀, and measure the hemagglutinative titer of strain.Knot Fruit finds H₁N₁The hemagglutinative titer of tested Strain is 1:320, TCID₅₀It is 10^-3.4。

2.5.2 the different sample toxicity to chicken red blood cell

Being diluted ovomucin respectively, research variable concentrations diluent is to H₁N_1-The toxicity of mdck cell, result is such as Table 2.

The table 2 egg ovomucin cytotoxicity to chicken red blood cell

Note: "-" represents generation blood clotting, does not deposits；"+" slight blood clotting；There is not blood clotting in " ++ ", cell deposits completely

It is shown in table, when ovomucin concentration is 5mg/mL or is less than 5mg/mL, free of toxic effects to chicken red blood cells, But after ovomucin concentration reaches 10mg/mL, cell can not occur normal deposition, and is laid in Microhemagglutination plate V shaped hole in, it is taken as that 10mg/mL ovum colloblast has cytotoxic effect to erythrocyte.

The hemagglutinative titer of Avian pneumo-encephalitis virus is raised with concentration and reduces, to Avian pneumo-encephalitis virus by variable concentrations ovomucin Hemagglutinative titer suppression ratio increases with concentration and rises.Illustrate that ovomucoid has the strongest inhibitory action to Avian pneumo-encephalitis virus.

Two, egg ovotransferrin (ovoconalbumin) is isolated and purified and in-vitro antibacterial is studied

It is related to from Ovum Gallus domesticus album the existing more research of separation and Extraction active substance and report both at home and abroad, and be correlated with Industrialized production also creates good economic benefit, such as lysozyme, immunoglobulin etc..And from Ovum Gallus domesticus album, extract ovum turn ferrum The research of albumen the most also rests on laboratory scale, and unrealized industrialized production, and progress is the slowest.This experiment is adopted With two step ultrafiltration separation and Extraction ovotransferrin, freeze-dried after obtain product, provide reference for commercial production.

1 materials and methods

1.1 material

1.1.1 raw material

Fresh hen egg Jin Yi egg product company limited provides

Ovotransferrin standard substance sigma Chinese companies

For examination strain:

Staphylococcus aureus (Staphylovovvus aure, ATCC 29213)

Escherichia coli (Escherichia coli, ATCC 25922)

Salmonella (Salmonella, CICC 21382) is preserved by this laboratory.

1.1.2 main agents

1.2 experimental technique

1.2.1 the technological process of ovotransferrin ultra-filtration and separation, as shown in Figure 5.

1.2.2 ultrafiltration apparatus and key points for operation

Connect ultrafiltration apparatus, continue 1h beforehand through 0.2MPa ultra-pure water pressure, after water flux density is stable, measure super Filter membrane initial pure water flux, after ultrafiltration, takes out ultrafilter membrane and carries out distilled water flushing, and with 0.1M NaOH and 0.1M HCl Soak 30min, finally repeatedly rinse with distilled water, again measure film stream of pure water flux, it is ensured that its initial flux reach 98% with On.

The clear stock solution of Fresh Egg, 0.9 normal saline dilution, 3000r/min is centrifuged 15min and removes ovomucin, then uses Electricity puts removing ovalbumin of saltouing.Sample microfiltration with isoelectric precipitation removes the impurity of 0.1-100 μ m by centrifugation, and then Use hyperfiltration technique, remove more than 100kDa macro-molecular protein with polysulfone resin derivatives membrane (molecular cut off 100kDa), It is more than the protein of 50kDa again with polysulfone resin derivatives membrane (molecular cut off 50kDa) molecular cut off, obtains concentrated solution, it After the concentrated solution that obtains carry out dialysis treatment, and lyophilization

1.2.3 the pre-treatment impact on egg white solution viscosity

1.2.3.1 the extension rate impact on egg white solution viscosity

Taking appropriate egg white solution, the NaCl solution with 0.9% dilutes, and extension rate is respectively 5 times, 10 times, 15 times, 20 times, 25 times, 3000r/min frozen centrifugation 10min, take supernatant and measure Ovum Gallus domesticus album viscosity.

1.2.3.2 the shear rate impact on egg white solution viscosity

Ovum Gallus domesticus album after dilution is respectively at 4000r/min, and 5000r/min, 6000r/min, 7000r/min, 8000r/min are high Carry out homogenizing on speed homogeneous dispersion device, measure egg white solution viscosity respectively, investigate the impact of all confrontation egg white solution viscositys.

1.2.3.3 the temperature impact on egg white solution viscosity

30 DEG C will be heated separately to through the egg white solution of dilution, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, measure different temperatures and lay eggs The viscosity of clear liquid, studies the temperature impact on egg white solution viscosity.

1.2.4 the research of ultrafiltration technology condition

1.2.4.1 the impact that ovotransferrin is separated by material extension rate

Egg liquid stir speed (S.S.) is fixed as 125r/min, and operation pressure is 0.10MPa, and pH value is 6 with volume ratio 5 times, 10 times, 15 times, 20 times are diluted, and with unit interval membrane flux as evaluation index, investigate egg white solution extension rate and ovum is turned ferrum The impact of albuminous coat separating effect.

1.2.4.2 the impact that ovotransferrin is separated by stir speed (S.S.)

Fixing egg clear liquid extension rate is 10 times (v/v), and ultrafiltration apparatus operation pressure is 0.10MPa, and solution ph is 6.Stir speed (S.S.) is respectively 50r/min, 125r/min, 200r/min, with unit interval membrane flux as evaluation index, and research stirring The speed impact on Ovum Gallus domesticus album ovotransferrin membrane separating effect.

1.2.4.3 the impact that ovotransferrin is separated by operation pressure

Fixing egg clear liquid extension rate is 10 times (v/v), and stir speed (S.S.) is 125r/min, and pH value is 6, and operation pressure divides Not Wei 0.05MPa, 0.10MPa, 0.15MPa, within the unit interval, membrane flux is as evaluation index, research operation pressure to Ovum Gallus domesticus album ovum The impact of transferrins membrane separating effect.

1.2.4.4pH the impact that ovotransferrin is separated it is worth

Fixing egg clear liquid extension rate is 10 times (v/v), and stir speed (S.S.) is 125r/min, and ultrafiltration apparatus operation pressure is 0.10MPa.Respectively the pH value of egg white solution is adjusted to 6,7,8 with 0.1mol/L NaOH and 0.1mol/L HCl, with in the unit interval Membrane flux is evaluation index, studies the pH value impact on ovotransferrin separating effect.

1.2.5 the mensuration of relative viscosity

Use Ubbelohde viscometer measures.List of references Yin Ye is flat, Wang Hui constitution 2007.

1.2.6 the mensuration of membrane flux

It is used for characterizing the speed of ultrafiltration membrance filter feed liquid, is the important indicator weighing film properties.

Membrane flux rate computing formula:

$J=\frac{Q}{At}$

J in formula: membrane flux, L (m^-2H^-1) Q: throughput, L；T: the operating time, h；A: membrane area, m²

1.3SDS-PAGE electrophoresis

Prepared by gel: configuration resolving gel concentration is 10%, concentration gum concentration is 5%.

Sample treatment: sample buffer is made into 1mg/mL concentration, boiling water heating 3～5min before electrophoresis.

Deposition condition: concentrate constant voltage 80V in glue, treats that sample enters separation gel and adjusts voltage to 120V, constant voltage 1h.

1.4 reverse high performance liquid chromatography (RT-HPLC) detection ovotransferrin

1mg/mL ovotransferrin does standard specimen, and chromatographic column selects Vydac214TP54C₄Post.

The qualification of 1.5 ovotransferrin and purity analysis

Ovotransferrin is carried out by the SDS-PAGE electrophoresis that the collection liquid after the egg white solution of dialysis, chromatography first passes through 10% Identify, then measure purity by HPLC.

The pretreatment of protein example: take protein sample liquid and mix with sample treatment liquid equal-volume, locate in 100 DEG C of water-baths Reason 2min, standby after cooling room temperature.

1.6 data analysis

Use Excel to set up data base, draw with Origin7.5 version software, process soft by DPS200, SAS8.1 data Part carries out data analysis.

2 experimental results and analysis

2.1 experiment of single factor reducing Ovum Gallus domesticus album viscosity

2.1.1 extension rate affects result to egg white solution viscosity

Using the method for homogenizing after first diluting, egg white solution is homogenizing after normal saline dilution, not only reduces egg white solution Viscosity, and destroy the structure of its complexity, so that the egg white solution rate of filtration is very fast, Membrane cleaning is relatively easy to, and reduces film Pollution level, extend the service life of film

As seen from Figure 6, the relative viscosity of egg white solution declines with the increase of extension rate, and extension rate is big from 5 times 15 times, viscosity degradation is obvious.And then slowly, level off to time shaft, determine optimal multiple 15 times.

2.1.2 shear rate affects result to egg white solution viscosity

Ovum Gallus domesticus album is after dilution, and the stickiness of egg liquid, it is also possible to block pollution ultrafilter membrane of living, therefore uses high speed dispersion equal Matter machine reduces egg white solution viscosity.Albumen after high speed dispersion homogenizing, relative viscosity declines quickly, as shown in Figure 7:

2.1.3 temperature affects result to egg white solution viscosity

Temperature raises and can reduce egg white solution viscosity, but the too high protein denaturation that can cause again of temperature, therefore investigate temperature pair The impact of egg white solution viscosity.As shown in Figure 8:

As shown in Figure 8, along with the rising of temperature, egg white solution viscosity degradation is clearly.But when temperature is too high, more than 45 DEG C After, the viscosity of ovotransferrin increases rapidly, and it is relevant that this changes structure with albumen in egg white solution because of high temperature.Therefore experimental selection 45 DEG C is adequate operation temperature.

2.1.4 the single factor experiment of membrane flux

2.1.4.1 material extension rate affects result to separate ovotransferrin membrane flux

Membrane flux is gradually lowered along with the prolongation of ultrafiltration time, and in front 10min, flux depression is all very fast, after 10min, Flux depression is the mildest.When extension rate is defined as 15 times, the concentration polarization phenomenon of filtrate is shallower and equipment runs More stable, therefore extension rate controls within 15 times (v/v).

2.1.4.2 stir speed (S.S.) affects result to separate ovotransferrin membrane flux

Membrane flux, along with the carrying out of egg white solution ultrafiltration, declines the mildest.Ultrafiltration system fortune when stir speed (S.S.) is 50r/min Row is steady, but membrane flux is low.When stir speed (S.S.) is 125r/min, circulation declines relatively slow, and phase convective flux is bigger.Stir speed (S.S.) Very fast for flux depression during 200r/min, and the decline of ultrafiltration later stage circulation is very fast.Consider, stir speed (S.S.) is controlled About 125r/min.

2.1.4.3 operation pressure affects result to separate ovotransferrin membrane flux

Along with the prolongation of filtrate ultrafiltration time, egg white solution concentration polarization phenomenon increases the weight of, and membrane flux diminishes.When operation pressure exists During 0.15MPa, it is in circulation status relatively smoothly, and filtrate concentration polarization phenomenon the most always.

2.1.4.4pH it is worth and affects result to separate ovotransferrin membrane flux

Along with the prolongation of ultrafiltration time, the membrane flux of filtrate declines rapidly, and when pH is 7, overall ultrafiltration system is in phase To steady statue.

2.1.5 the optimum results of ultrafiltration optimum condition

Experiment selection operation pressure, extension rate, 3 factors of stir speed (S.S.) carry out orthogonal experiment, with unit interval inner membrance Flux is inspection target, is optimized ultrafiltration technology condition.Combined experiment finds, affects the primary and secondary order of ultrafilter membrane flux For operation pressure dilution multiple stir speed (S.S.), optimum combination be ultrafiltration apparatus pressure be 0.15MPa, solution extension rate 10 Times, stir speed (S.S.) is 125r/min.Now membrane flux value is 39.80.

2.1.6SDS-PAGE electrophoresis result

The ovotransferrin sample obtained after lyophilizing is mainly the ovotransferrin of about 78KDa and the ovum of about 45KDa Albumin, a little foreign protein band, reason is probably part ovotransferrin and exists with combined state form with other albumen, no The most separately.

2.1.7RT-HPLC analysis result

2.1.8 in-vitro antibacterial research

The bacteriostatic activity of table 3 ovotransferrin and preservative compares

Note: Odontothrips loti is also known as pipe butterfly method, and antibacterial circle diameter 20mm is extremely sensitive " +++ ", 15mm antibacterial circle diameter 20mm is high sensitive " ++ ", 10mm antibacterial circle diameter 15mm be middle sensitivity "+", antibacterial circle diameter 10mm be hypoallergenic or Invalid "-"

As seen from table, ovotransferrin is the strongest to escherichia coli fungistatic effect, takes second place Salmonella, to golden yellow Fructus Vitis viniferae Coccus fungistatic effect is relatively weak.

Three, egg immunoglobulin extraction purification and In Vitro Bacteriostasis effect

Chicken yolk immunoglobulin, is called for short IgY, also known as chicken yolk antibody.There is immunogenicity, in egg yolk with α-, β- Exist with three kinds of forms of γ-livitin.The IgY isolation and purification method used in the world at present mainly include water dilution method, The physical-chemical process such as the organic solvent extracting sedimentation method, ultrafiltration, supercritical extraction and freeze thawing and saltouing combines with DEAE chromatograph Etc. method.This experiment operates in first laboratory, and therefore based on early-stage Study water dilution method, and it is heavy to combine Polyethylene Glycol Form sediment and DEAE-Toyopearl 650M one-step elution ion-exchange chromatography is purified, obtain the highly purified chicken of high-recovery Egg yolk IgY.

1 method

Prepared by 1.1 thick IgY

1.1.1 water dilution method obtains water-soluble component (WSF)

Choose Fresh Egg, use egg-white egg-yellow separator, isolated egg yolk, with distilled water, egg yolk is cleaned, be placed in Blot on filter paper, puncture membrane of yolk, collect egg yolk liquid, with volume percentage, with distilled water, egg yolk is carried out different multiples Dilution (2 times, 4 times, 6 times, 8 times, 10 times) stir, the different pH value (4.6,4.8,5.0,5.2,5.4) of regulation, 4 DEG C of standings Different time (2h, 4h, 6h, 8h, 10h), after 5000 × frozen centrifugation 30min, obtains clarified supernatant water-soluble component Han IgY (WSF)。

1.1.2PEG protein precipitation

Adding the PEG6000 of different quality mark in the WSF of gained, magnetic stirrer 30min, 22000 × g is cold Freeze centrifugal, collect precipitation.

1.1.3 dialysis

Collect precipitation and add a certain amount of distilled water, stirring, load bag filter (molecular cut off is 8000-14000KD) In, 4 DEG C of dialysis remove part residual PEG6000, and lyophilization i.e. obtains thick IgY component.

1.1.4 ion exchange chromatography IgY

Use Na₂HPO₄-NaH₂PO₄Buffer balances, and arranging pH value is 6.0, different buffer ions under the conditions of 7.0,8.0 Intensity (0.03mol/L, 0.05mol/L, 0.1mol/L) loading balance, isorheic elution (0.075mol/L, 0.1mol/L), pass through Comparative study determines optimized buffer liquid pH and eluent ionic strength.Eluting effluent is collected automatically by often pipe 5ml/3min.Will Component collected by chromatography loads bag filter (molecular cut off is 8000-14000KD), is placed in distilled water, and 4 DEG C of dialysis remove Salt.

1.1.5 protein content determination

1.1.5.1 crude protein content measures with bovine serum albumin as standard protein, measures egg with Coomassie Brilliant Blue White matter content.

1.1.5.2 thick IgY assay

Measure light absorption value under sample 280nm.

1.1.6IgY purity testing

PAGE gel electrophoretic analysis, the concentration glue of 5%, the separation gel of 10%, concentrate gel electrophoresis voltage 80V, separate Glue voltage 120V, fades according still further to standardization program after electrophoresis Sample G250 coomassie brilliant blue staining.Utilize Lab-Image software Calculate IgY purity.

1.1.7IgY determination of recovery rates

IgY response rate formula calculates:

2 experimental results

The 2.1 different extension rate impacts on IgY separating effect

Taking 15mL egg yolk liquid respectively, dilute with different amounts of sterilized water, stir, extension rate is 2,4,6,8,10 Times, Coomassie Brilliant Blue measures protein content, determines that optimum diluting multiple is 8 times, and protein content is 375.27mg/mL.

The 2.2 different pH impacts on IgY separating effect

In supernatant, protein content response rate when pH5.0-5.6 is higher, all at more than 300mg/mL, and wherein pH5.2 Time reach peak 321.57mg/mL.

2.3SDS-PAGE gel electrophoresis

2.4IgY antibacterial experiment effect

IgY to escherichia coli, Salmonella, staphylococcus aureus growth curve antibacterial effect:

After adding IgY and your albumen excellent in antibacterial liquid, IgY and excellent that albumen are to Bacillus subtilis and escherichia coli The OD value having obvious inhibitory action, test group and matched group at early growth period has obvious difference.And after 24 hours Inhibitory action declines, thus it is speculated that be owing to temperature causes the inactivation of IgY to its impact, so that the nitrogen source that IgY becomes bacterial strain has It is beneficial to bacterial strain must grow.IgY is inconspicuous for staphylococcus aureus inhibitory action, the value of the OD value relatively experimental group of matched group Little.

The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention Any amendment, equivalent and the improvement etc. made within god and principle, should be included within the scope of the present invention.

Claims (6)

1. a protein extraction and isolation and purification method, it is characterised in that described protein extraction and isolation and purification method include ovum Mucin isolation and purification method, egg ovotransferrin isolation and purification method and egg immunoglobulin method for extraction and purification；

Described ovomucin isolation and purification method uses GaCL₂Or MgCl₂Prepare ovomucin；

Described egg ovotransferrin isolation and purification method includes: the clear stock solution of Fresh Egg, 0.9 normal saline dilution, 3000r/ Min is centrifuged 15min and removes ovomucin, then saltouts removing ovalbumin by isoelectric point, IP；By centrifugation with the sample of isoelectric precipitation Microfiltration removes the impurity of 0.1-100 m scope, and then uses ultrafiltration, removes more than 100kDa with polysulfone resin derivatives membrane and divides greatly Sub-protein, then with the polysulfone resin derivatives membrane molecular cut off protein more than 50kDa, obtain concentrated solution, the concentration obtained Liquid carries out dialysis treatment, and lyophilization；

Described egg immunoglobulin method for extraction and purification uses water dilution method to combine polyethylene glycol precipitation and DEAE- Toyopearl 650M one-step elution ion-exchange chromatography is purified, and obtains highly purified Ovum Gallus domesticus Flavus IgY of high-recovery.

2. protein extraction as claimed in claim 1 and isolation and purification method, it is characterised in that described ovomucin is isolated and purified Method comprises the following steps:

Shell egg adds the saline solution of 5 times of volumes, 0.05mol/LMgCl after stirring clearly₂Or 0.1mol/L NaCl, use after homogenizing 0.1mol/L HCl adjusts pH=6.0, and 4 DEG C stand overnight, and 15,000g are centrifuged 10min, 4 DEG C, precipitation 0.5mol/L NaCl weight Outstanding, place 4h, 15,000 g for 4 DEG C and be centrifuged 10min, 4 DEG C, the precipitation distilled water washing obtained is not contained protein to supernatant, Sediment fraction is ovomucin crude extract；

Crude extract is dissolved in 10mmol/L borate acid buffer pH=9.6 and is made into 5mg/ml solution, and 4 DEG C are stirred overnight, and 0.45 Carrying out gel chromatography after m filtering with microporous membrane, eluent is containing 0.2 mol/LMgCl₂Tris-HCl buffer, 20mmol/ L, pH=8.4, flow velocity 0.5ml/min；The lyophilizing sample collecting each eluting peak is identified through SDS-PAGE, and target components is freezing -20 DEG C of preservations after drying.

3. protein extraction as claimed in claim 2 and isolation and purification method, it is characterised in that described ovomucin is to large intestine bar The minimal inhibitory concentration of bacterium and Salmonella is respectively 0.05mg/mL and 0.2mg/mL.

4. protein extraction as claimed in claim 1 and isolation and purification method, it is characterised in that described egg ovotransferrin divides Include from purification process:

Connect ultrafiltration apparatus, continue 1h by 0.2MPa ultra-pure water pressure, after water flux density is stable, measure ultrafilter membrane initial Pure water flux, after ultrafiltration, takes out ultrafilter membrane and carries out distilled water flushing, and soak with 0.1M NaOH and 0.1M HCl 30min, finally rinses repeatedly with distilled water, again measures film stream of pure water flux；

The clear stock solution of Fresh Egg, 0.9 normal saline dilution, 3000r/min is centrifuged 15min and removes ovomucin, then uses isoelectric point, IP Saltout removing ovalbumin；

Sample microfiltration with isoelectric precipitation removes the impurity of 0.1-100 m scope by centrifugation, and then uses hyperfiltration technique, with poly- Sulphone resin derivatives membrane removes more than 100kDa macro-molecular protein, then is more than with polysulfone resin derivatives membrane molecular cut off The protein of 50kDa, obtains concentrated solution, and the concentrated solution obtained afterwards carries out dialysis treatment, and lyophilization.

5. protein extraction as claimed in claim 4 and isolation and purification method, it is characterised in that described protein extraction and separation are pure The ultrafiltration apparatus pressure of change method is 0.15MPa, and 0.9 normal saline dilution multiple is 10 times, and stir speed (S.S.) is 125r/min, Now membrane flux value is 39.80, and pH is 7.

6. protein extraction as claimed in claim 1 and isolation and purification method, it is characterised in that described egg immunoglobulin carries Take purification process to include:

Choose Fresh Egg, use egg-white egg-yellow separator, isolated egg yolk, with distilled water, egg yolk is cleaned, be placed in filter paper On blot, puncture membrane of yolk, collect egg yolk liquid, with volume percentage, with distilled water, egg yolk is carried out the dilution 8 of different multiples Stir again, different pH value 5.0 ~ 5.6, the 4 DEG C standing of regulation, after 5000 × frozen centrifugation 30min, obtain clarified supernatant and contain IgY water-soluble component WSF；

In the WSF of gained, add the PEG6000 of different quality mark, magnetic stirrer 30min, 22000 × g freezing from The heart, collects precipitation；

Dialysis, collects precipitation and adds distilled water, stirring, load bag filter, and molecular cut off is in 8000-14000KD, and 4 DEG C thoroughly Analysis removes part residual PEG6000, and lyophilization i.e. obtains thick IgY component；

Ion exchange chromatography IgY, uses Na₂HPO₄-NaH₂PO₄Buffer balances, buffer ionic strength loading balance, perseverance Stream eluting, eluting effluent is collected automatically by often pipe 5ml/3min；Component collected by chromatography is loaded bag filter and retains molecule Amount is 8000-14000KD, is placed in distilled water, 4 DEG C of dialysis desalinations.