CN103525785A - Method for separating and purifying neuraminidase (avian influenza virus) - Google Patents

Method for separating and purifying neuraminidase (avian influenza virus) Download PDF

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CN103525785A
CN103525785A CN201310438024.0A CN201310438024A CN103525785A CN 103525785 A CN103525785 A CN 103525785A CN 201310438024 A CN201310438024 A CN 201310438024A CN 103525785 A CN103525785 A CN 103525785A
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李铮
马恬然
钟耀刚
胡圆
杨刚龙
赵菲
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Northwest University
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Abstract

The invention relates to a method for separating and purifying neuraminidase (avian influenza virus), which comprises the following steps: fixing a neuraminidase (NA)-specific competitive inhibitor 4-aminophenyl oxamic acid to the surface of epoxy magnetic particles (MPs) as an affinity ligand to prepare a pair of NA-specific affinity functional magnetic particles, combining the particles with an NA-containing influenza virus strain in a buffer system with the pH value of 5.5, and cleaning; and eluting with a buffer system with the pH value of 9.1 to separate and purify the NA. The method overcomes the defect that the NA acquired by the gene engineering method can not satisfy the NA glycosylation research, and solves the technical problems of complex operating procedure for separating the NA with a commercial oxalic acid agarose affinity column, high price, large elution volume, difficulty in implementing trace amounts, and the like. The method is simple and quick to operate, has high specificity, and has favorable purification effect on the NA.

Description

The method of a kind of separation and purification of nerve propylhomoserin enzyme (avian influenza virus)
technical field
The present invention relates to the method for a kind of separation and purification of nerve propylhomoserin enzyme (avian influenza virus), be specifically related to a kind of method based on magnetic particle separation and purification of nerve propylhomoserin enzyme (avian influenza virus).
Background technology
Magnetic particle (Magnetic particle, MP) is called for short magnetic grain, as a kind of carrier that has superparamagnetism and be easy to be modified by various functional groups, is widely applied to biological chemistry, cytobiology, microbiology, and the various fields such as clinical medicine.The reason that magnetic particle is widely used is that it has following characteristics: magnetic responsiveness is good, and coercive force is zero substantially, possesses superparamagnetism; Particle diameter can reach nano level; There is good chemical stability; Production technique is relatively simple, and reasonable price.So in protein separation, often by immobilization, there is bioactive enzyme, or the antibody that can be combined with antigen-specific and lectin sugar chain to specific recognition ability, be prepared into and can be used for separated specific proteins, antigen and glycoprotein/sugared lipid material.These application all magnetic particle based in solution can be separated rapidly under the action of a magnetic field, and when removing magnetic field, can well be suspended in solution, and still maintain property is stable, is conducive to the wash-out of purifying protein, and this process operation is simple, is easy to widespread use.
4-amino-benzene oxaminic acid, claim again the acid of p-aminophenyl oxime, neuraminidase (Neuraminidase, NA) special competitive inhibitor, can with the specific combination of NA, in this experiment, using it as affinity ligand, modify in epoxidation magnetic particle surface, make this 4-amino-benzene oxaminic acid functional Fe_3 O_4 magnetic particles there is special avidity to neuraminidase.And this tests homemade epoxidation magnetic grain, be by Fe 4o 3magnetic particle is modified through 3-Racemic glycidol propyl ether base Trimethoxy silane (GPTS), structurally there is suitable extension brachium (being the length between affinity ligand and support magnetic particle), the epoxidation group of its end has active chemical reactivity, meets the requirement that connects affinity ligand 4-amino-benzene oxaminic acid in experiment.
Compare with other virus components, in the sepn process of NA the most difficult realization to be NA with HA separated.Both are all positioned at viral Nang film surface, and are all glycoprotein, and the NA tetramer is close with HA tripolymer molecular weight, and therefore general separation means is difficult to be carried out separation.Once used traditionally salting-out process, density gradient ultracentrifugation etc., not only operated complicated and extraction effect is barely satisfactory, and from virus, obtained neuraminidase and there is no very effective means.In recent years, obtained influenza neuraminidase both at home and abroad generally based on two kinds of thinkings.Be to use engineered means, use that known avian influenza virus NA gene carries out that sequences Design, primer are synthetic, gene clone construction of expression vector, and express in the cell strains such as clostridium, insect cell, thereby obtain the NA albumen of expression.Although this kind of method can be obtained purer NA albumen, but this kind of albumen or do not have the posttranslational modifications such as glycosylation, even if or exist, what well imagine is that the state of its posttranslational modification also will mostly be different from naturally occurring state in virus, can not be applied to the research of the aspects such as glycosylation of neuraminidase.Also have one to be that method is mainly based on a kind of commercial oxaminic acid agarose affinity column, N-(p-aminophenyl) oxamic acid agarose(U.S. sigma company), use this method from virus, to extract NA, effect is better than traditional method, also can be applicable to the research of the aspects such as glycosylation of NA, but exist expensively, the nonspecific absorption of agarose is larger, and elution volume is difficult to greatly the technical problems such as traceization.
Summary of the invention
The object of the present invention is to provide the method for a kind of separation and purification of nerve propylhomoserin enzyme (avian influenza virus), the method is using the special competitive inhibitor 4-amino-benzene oxaminic acid of NA as affinity ligand, be fixed on epoxidation magnetic particle surface, be prepared into functional Fe_3 O_4 magnetic particles NA to special avidity, and carry out combination, clean with neuraminidase under the buffer system that is 5.5 at pH with this particulate, use the buffer system that pH is 9.1 to carry out the neuraminidase that wash-out obtains separation and purification.
Technical scheme of the present invention is:
The method of a kind of separation and purification of nerve propylhomoserin enzyme (avian influenza virus), its special character is: the method is using the special competitive inhibitor 4-amino-benzene oxaminic acid of neuraminidase as affinity ligand, be fixed on epoxidation magnetic particle surface, be prepared into functional Fe_3 O_4 magnetic particles neuraminidase to special avidity, and with this particulate, take the buffer system that pH is 5.5 with the strains of influenza viruses that contains neuraminidase and be combined, clean; The buffer system wash-out separation and purification of nerve propylhomoserin enzyme that the pH of take is 9.1.
Above-mentioned being prepared into has the functional Fe_3 O_4 magnetic particles of special avidity to comprise to neuraminidase: extract the suspension containing 200 mg epoxidation magnetic particles, this epoxidation magnetic particle is modified to 4-amino-benzene oxaminic acid functional Fe_3 O_4 magnetic particles;
Described modification comprises:
The first step is modified: above-mentioned suspension is placed in reactor, with dehydrated alcohol, cleans; Reactor is placed on magnetic separator and carries out magnetic resolution again, discard the clear liquid that does not contain magnetic particle in upper strata;
Take 0.1081g Ursol D and be dissolved in 50mL dehydrated alcohol, after fully dissolving, proceed in reactor; Make epoxy magnetic grain and Ursol D fully mix, react, epoxidation magnetic grain is modified into Ursol D and modifies magnetic grain; After having reacted, magnetic resolution, and abandoning supernatant;
Second step is modified: the Ursol D obtaining after above-mentioned reaction is completed is modified magnetic grain and cleaned with dehydrated alcohol, then cleans with ultrapure water, changes to new reactor, after magnetic resolution, discards the clear liquid that does not contain magnetic particle in upper strata;
To the 0.1～0.2M MES damping fluid that adds 50 mL pH 5.0～6.0 in flask, add again 0.1260g oxalic acid dihydrate, stirring is fully dissolved oxalic acid and is mixed with magnetic grain, add EDC 0.1970 g, NHS 0.1151 g, in 4 ℃ of reactions, more than 8 hours, Ursol D is modified to magnetic grain and make 4-amino-benzene oxaminic acid functionalized magnetic particles;
After having reacted, magnetic resolution, abandoning supernatant; With ultrapure water, clean magnetic grain, be settled to 20 mL, proceed to reagent bottle, difference assay weighs the solid contents that calculates this 4-amino-benzene oxaminic acid functionalized magnetic particles suspension, labeling, and 4 ℃ save backup.
In this modification, the consumption of described reactant, reaction system and proportioning are optimum regime, in actually operating, can according to circumstances suitably adjust, and in the scope of the following stated, also can reach modification object:
Using the total mass number of initial starting material Ursol D as reference, every gram of Ursol D can corresponding dehydrated alcohol 30-100 mL, MES damping fluid 30-100 mL, EDC 0.12-2.7 g, NHS 0.06-1.5 g, and meet EDC:NHS ratio and remain between 1:1-1:5.
This ratio of Ursol D: oxalic acid dihydrate=1:1.166 preferably remains unchanged, and wherein oxalic acid dihydrate can replace with oxalic acid, and after replacing, ratio is Ursol D: oxalic acid=1:0.833
By different proportionings, add attentive response system pH after EDC, NHS, if exceed pH 5-6, should be adjusted to this scope.
The strains of influenza viruses of above-mentioned neuraminidase is from 10 age in days chick embryo allantoic liquids of inoculation; The particle of this virus strain is suspended in allantoic fluid.
Before being used, the above-mentioned strains of influenza viruses that contains neuraminidase needs the particle of this virus strain to carry out cracking and albumen is slightly carried; Described cracking and albumen are slightly carried employing ether extraction.
The method of above-mentioned separation and purification of nerve propylhomoserin enzyme (avian influenza virus), its special character is, the method specifically,
(1) sealing of magnetic grain: get a 2mL centrifuge tube, in add wherein 3 mg4-amino-benzene oxaminic acid magnetic grains, magnetic resolution, abandons supernatant; Then in centrifuge tube, add sealing damping fluid 500
Figure 222252DEST_PATH_IMAGE001
, mix, be placed in gas bath vibrator, 37 ℃ of 180 rpm reacts 30 min;
(2) balance of magnetic grain and activation: upper step is placed in centrifuge tube on magnetic separator and carries out magnetic resolution after having reacted, and abandoning supernatant adds 1 mL level pad at every turn, cleans: add again 1 mL binding buffer liquid to clean magnetic grain;
(3) combination of sample and magnetic grain: get a new centrifuge tube, the thick leach protein 5mg of virus that adds ether to process, adds 2 * binding buffer liquid and ultrapure water in proportion, make system binding buffer liquid concentration be 1 *, volume is 1mL; Be labeled as " former state " stand-by; Get this solution 950 add and be equipped with in the centrifuge tube that step activated magnetic grain, jiggle to mixing, be placed in 37 ℃ of 180 rpm reaction 2h of gas bath vibrator; Reacted rear magnetic resolution;
(4) clean: in centrifuge tube, add 1 mL cleaning buffer solution, be positioned in gas bath vibrator, temperature is adjusted to 37 ℃, rotating speed 180 rpm jolting 10 min, magnetic resolution;
(5) wash-out: add 500 in centrifuge tube
Figure 265656DEST_PATH_IMAGE001
elution buffer, 25 ℃ of 180 rpm jolting 1 h, after magnetic resolution, collecting supernatant liquor, to be labeled as " wash-out " stand-by; Be the suspension that contains target protein neuraminidase.
In this separation and purification process, consumption, proportioning and the reaction conditions of described magnetic grain, the thick leach protein former state of virus, buffer system can reach best effect, in actually operating, can adjust as one sees fit, in the scope of the following stated, also can reach preferably separated object:
Every milligram of magnetic grain can correspondence walk damping fluid volume 70-400 mL, albumen former state 1-4 mg;
Temperature of reaction 20-37 ℃;
Rotating speed: 120-240 rpm
Reaction times: sealing 0.5-3 h, in conjunction with 1.5-3 h, cleans 8-15 min, wash-out 0.5-3 h
In addition, the selection of attentive response container, reaction system maximum volume should not surpass 2/3 of reaction vessel maximum capacity, and minimum volume is advisable to be no less than 1/4 of reaction vessel maximum capacity.
Above-mentioned steps (4) is cleaned the supernatant liquor that need to repeat this step 3 time last magnetic resolution of collection.
Above-mentioned reactor is flask or beaker or test tube.
Advantage applies of the present invention exists:
The binding ability of 4-amino-benzene oxaminic acid and neuraminidase changes with the variation of combining environmental pH, and the appropriate pH that it is combined with neuraminidase is 5.5, and raises gradually while reaching 6.5 as pH, and binding substances starts to decompose.
Utilize this characteristic, in the present invention, choose pH and be 5.5 buffer system and carry out the combination of magnetic grain and NA and the cleaning of impurity albumen, choose pH and be 9.1 buffer system and carry out the wash-out of NA, set up the method based on this kind of magnetic grain separation and purification influenza neuraminidase.
Extraction conditions (magnetic grain consumption, binding time, elution time) is optimized, verification method specificity, and identify by SDS-PAGE and MALDI-TOF-MS technology.
The separation and purification that the present invention is influenza neuraminidase provides a kind of effective means, both avoided neuraminidase that gene engineering method is obtained cannot meet the defect of neuraminidase glycosylation research, solved again the separated neuraminidase schedule of operation of commercialization oxalic acid agarose affinity column complicated, expensive, elution volume is large, is difficult to the technical problems such as traceization.The method is simple and efficient to handle, specificity is high, and neuraminidase is had to good refining effect, extraction that also can expanded application NA in other complex samples.The present invention finally applies the method the neuraminidase of the existing 8 kinds of strains in laboratory is carried out to separation and purification, for experiment material is extracted in this research of testing follow-up NA sugar chain spectrum.
Accompanying drawing explanation
Fig. 1 is schematic flow sheet of the present invention;
Fig. 2 is that schematic diagram is modified in the first step reaction;
Fig. 3 is that schematic diagram is modified in second step reaction;
Fig. 4 is magnetic grain consumption and wash-out concentration relationship schematic diagram;
Fig. 5 is binding time and wash-out concentration relationship schematic diagram;
Fig. 6 is elution time and wash-out concentration relationship schematic diagram;
Fig. 7 is specificity checking electrophoresis result figure;
Fig. 8 is electrophoresis qualification result;
Fig. 9 is Mass Spectrometric Identification result;
Figure 10 is Bradford method protein quantification typical curve.
Embodiment
, instrument and material
, main agents and material
Table 2-1 main agents and material and supplier thereof
Figure 789041DEST_PATH_IMAGE002
Note: experiment is processed through Milli-Q 50 pure water systems (U.S. Millipore company) with ultrapure water.
Table 2-2 virus strain information gathers
Figure 218886DEST_PATH_IMAGE003
Note: in experiment, 8 kinds of bird flu strains used are granted by Harbin veterinary institute
, main laboratory apparatus
The table main laboratory apparatus of 2-3 and production firm thereof
Figure 522828DEST_PATH_IMAGE004
, main solution preparation
The preparation of table 2-4 purification NA related solution
Figure 797952DEST_PATH_IMAGE005
Note: the octyl group glycopyranoside in above-mentioned damping fluid can replace with Triton X-100, sodium-acetate, sodium bicarbonate buffer liquid also available other common damping fluids, as replacements such as borate buffer solution, phosphate buffered saline buffers, but must be noted that to keep pH consistent.
 
Table 2-5 SDS-PAGE and silver dye related solution preparation
Figure 441423DEST_PATH_IMAGE006
, 4-amino-benzene oxaminic acid functional Fe_3 O_4 magnetic particles preparation, referring to Fig. 1;
(1) preparation of epoxidation magnetic grain;
Get Fe 3o 4magnetic particle 0.8g in beaker, magnetic resolution, supernatant discarded;
With approximately 100 mL dehydrated alcohols, clean magnetic grain, totally 5 times; Every less important the magnetic grain that accumulates in beaker bottom is washed down completely, and make it to be uniformly dispersed;
This step can complete by means of shaking table;
Measure dehydrated alcohol 200 mL, add in beaker magnetic particle is finally all proceeded to three neck round-bottomed flasks, and install electric stirring reaction unit;
Fully stir and pass into nitrogen protection; Progressively accelerate whipping appts is moved, wait for that rotor is stable.Because GPTS is to the extremely responsive facile hydrolysis of moisture, pass into nitrogen protection;
After 5min, slowly add successively 5 mL GPTS, 0.4 mL Glacial acetic acid, turns speed to 500 rpm, and 25 ℃ are reacted 2 hours; Wherein after sample introduction approximately 20 min, being about to nitrogen passes into speed and is adjusted to lower level;
After reaction finishes, magnetic grain is proceeded to beaker, each 100 mL dehydrated alcohols clean at least five times, are settled to 50 mL, proceed to reagent bottle, and difference assay weighs and calculates this epoxidation magnetic particle suspension solid contents, labeling, and 4 ℃ save backup;
(2) the 4-amino-benzene oxaminic acid of epoxidation magnetic grain is modified
Modify and carry out in two steps altogether;
The first step is modified: get 200 mg epoxidation magnetic particles and be placed in round-bottomed flask, with dehydrated alcohol, clean once; After cleaning, the round-bottomed flask that fills magnetic grain suspension is positioned on magnetic separator and carries out magnetic resolution, discard the clear liquid that does not contain magnetic particle in upper strata, referring to Fig. 2;
Take 0.1081g Ursol D and be dissolved in 50mL dehydrated alcohol, after fully dissolving, proceed in round-bottomed flask; Round-bottomed flask is placed on water-bath and assembles fixing with electric mixer; Turn on agitator stirs epoxy magnetic grain and Ursol D is fully mixed, and progressively agitator speed is risen to after 500 rpm, and 45 ℃ are reacted 4 hours, epoxidation magnetic grain is modified into Ursol D and modifies magnetic grain; After having reacted, magnetic resolution, and abandoning supernatant;
Second step is modified:
Above-mentioned Ursol D is modified to magnetic grain and with dehydrated alcohol, clean 5 times, then clean 5 times with ultrapure water, change to new flask, after magnetic resolution, discard the clear liquid that does not contain magnetic particle in upper strata, referring to Fig. 3;
Assembling fixes flask and agitator, to the 0.2M MES damping fluid that adds 50 mL pH5.0 in flask, add again 0.1260g oxalic acid dihydrate, turn on agitator stirs and oxalic acid is fully dissolved and mix with magnetic grain, add EDC 0.1970 g, NHS 0.1151 g, 4 ℃ of reactions more than 8 hours, are modified magnetic grain by Ursol D and are made 4-amino-benzene oxaminic acid functionalized magnetic particles;
After having reacted, magnetic resolution, abandoning supernatant; With ultrapure water, clean magnetic grain five times, be settled to 20 mL, proceed to reagent bottle, difference assay weighs the solid contents that calculates this 4-amino-benzene oxaminic acid functionalized magnetic particles suspension, labeling, and 4 ℃ save backup;
In this modification, the consumption of described reactant, reaction system and proportioning are optimum regime, in actually operating, can according to circumstances suitably adjust, and in the scope of the following stated, also can reach modification object:
Using the total mass number of initial starting material Ursol D as reference, every gram of Ursol D can corresponding dehydrated alcohol 30-100 mL, MES damping fluid 30-100 mL, EDC 0.12-2.7 g, NHS 0.06-1.5 g, and meet EDC:NHS ratio and remain between 1:1-1:5.
This ratio of Ursol D: oxalic acid dihydrate=1:1.166 preferably remains unchanged, and wherein oxalic acid dihydrate can replace with oxalic acid, and after replacing, ratio is Ursol D: oxalic acid=1:0.833
By different proportionings, add attentive response system pH after EDC, NHS, if exceed pH 5-6, should be adjusted to this scope.
, functionalized magnetic particles separation and purification of nerve propylhomoserin enzyme method foundation
(1) in chick embryo allantoic liquid, the cracking of virion and albumen are slightly carried
This experiment all virus strain used are all from this kind of 10 viral age in days chick embryo allantoic liquids of inoculation; Virion is suspended in allantoic fluid; Because neuraminidase is virus envelope surface glycoprotein, therefore, before the separation and purification of neuraminidase, need first lytic virus particle, dissolve and also slightly carry viral protein.Influenza virus surface has cyst membrane, and lipoid is the primary structure composition of formation virion cyst membrane, and major part is phosphatide, and containing a small amount of glycolipid and cholesterol, existence form is for forming lipid bilayer.Generally can choose tensio-active agent as Triton X-100, organic chemical reagent breaks and dissolves virion as ether, sucrose-acetone etc.Wherein ether can well lipin dissolving layer, discharges envelope protein, protein be soluble in the aqueous phase and with the layering of ether phase.Therefore this experiment select simple and easy to do and on follow-up test system substantially without the ether extraction affecting.
Ether extraction: get and divide in advance the viral chick embryo allantoic liquid that installs-80 degrees Celsius of preservations, add equal-volume ether to mix, be placed in 4 ℃ of 2 h, make to mix once every 10 min joltings;
After so processing, this mixed solution is placed in to whizzer, centrifugal 20 min of 2,000 4 ℃ of rpm, ether phase and water layering, discard upper strata ether phase, collects lower floor's water protein liquid in centrifuge tube;
With sealed membrane, centrifuge tube is sealed, and prick out some apertures with disposable syringe syringe needle, be vertically placed on test-tube stand, put into Cool Room 4℃ and spend the night, remaining ether can be got rid of totally;
After processing through this this, use Nano Photometer trace dna protein analyzer to measure protein concentration in viral crude extract, 4 ℃ save backup;
(2) 4-amino-benzene oxaminic acid functionalized magnetic particles separation and purification avian influenza virus neuraminidase
Below in experiment, using H5N1 (CK/GX/4/09) virus as example sample.
The sealing of magnetic grain: get in 2mL centrifuge tube and add 3 mg 4-amino-benzene oxaminic acid magnetic grains, magnetic resolution, abandons supernatant; Then in centrifuge tube, add sealing damping fluid 500
Figure 776589DEST_PATH_IMAGE001
, mix, be placed in gas bath vibrator, 37 ℃ of 180 rpm reacts 30 min;
The balance of magnetic grain and activation: upper step is placed in centrifuge tube on magnetic separator and carries out magnetic resolution after having reacted, and abandoning supernatant adds 1 mL level pad at every turn, cleans 3 times: add again 1 mL binding buffer liquid to clean magnetic grain, repeat 3 times;
The combination of sample and magnetic grain: get a new centrifuge tube, the thick leach protein 5mg of virus that adds ether to process, adds 2 * binding buffer liquid and ultrapure water in proportion, make system binding buffer liquid concentration be 1 *, volume is 1mL; Be labeled as " former state " stand-by; Get this solution 950
Figure 567828DEST_PATH_IMAGE001
add and be equipped with in the centrifuge tube that step activated magnetic grain, jiggle to mixing, be placed in 37 ℃ of 180 rpm reaction 2h of gas bath vibrator; Reacted rear magnetic resolution, it is stand-by that collection supernatant liquor is labeled as " supernatant ".
Clean: in centrifuge tube, add 1 mL cleaning buffer solution, be positioned in gas bath vibrator, temperature is adjusted to 37 ℃, rotating speed 180 rpm jolting 10 min, magnetic resolution; Repeating this step 3 time collects the supernatant liquor of last magnetic resolution to be labeled as " cleaning 3 " stand-by.
Wash-out: add 500 in centrifuge tube
Figure 646642DEST_PATH_IMAGE001
elution buffer, 25 ℃ of 180 rpm jolting 1 h, after magnetic resolution, collecting supernatant liquor, to be labeled as " wash-out " stand-by;
By the sample of label collection in each step respectively loading carry out SDS-PAGE experiment and silver dyes;
(3) optimization for extracting condition and specificity checking
The selection of magnetic grain consumption: get 1mg, 2mg, 3mg, 4mg, 5 mg4-amino-benzene oxaminic acid magnetic grains add respectively in 5 centrifuge tubes, by step in (2), through sealing, balance activation, in conjunction with, clean final wash-out and carry out after parallel test, use Nano Photometer trace dna protein analyzer to measure protein concentration in elutriant, and by wash-out concentration, magnetic grain consumption mapped; Choose suitable magnetic grain consumption;
Determining of binding time: respectively add 3mg4-amino-benzene oxaminic acid magnetic grain in 5 centrifuge tubes, by step in (2), through sealing, balance activation, in 1-5 centrifuge tube, control respectively as 1h 1.5h with viral protein crude extract binding time, 2h, 2.5h, 3h, cleans, collects respectively elutriant after wash-out, use Nano Photometer trace dna protein analyzer to measure protein concentration in elutriant, and by wash-out concentration, binding time is mapped.Choose optimum reacting time.
Determining of elution time: respectively add 3mg4-amino-benzene oxaminic acid magnetic grain in 5 centrifuge tubes, by step in (2), after sealing, balance activation, cleaning, in 1-5 centrifuge tube, the elution time of target protein is controlled respectively as 1h, 1.5h, 2h, 2.5h, 3h, collects elutriant, use Nano Photometer trace dna protein analyzer to measure protein concentration in elutriant, and by wash-out concentration, elution time is mapped.Choose suitable time of eluent.
The specific checking of method: compare with Viral experiment sample, the negative chick embryo allantoic liquid of the not virus inoculation of processing through same procedure, except not containing each protein ingredient of virus, consistent with virus other compositions that slightly get sample in this, all contain the protein ingredient of chick embryo allantoic liquid own.Therefore be extremely suitable for the material as the checking of this extracting method specificity and separated neuraminidase negative control group.Through ether method, process sample equally, after processing, protein content in sample is carried out quantitatively.By step in (2), test, collect extract magnetic resolution in each step clear liquid respectively loading carry out SDS-PAGE experiment and silver dyes.According to the power of albumen wash-out band, have or not to judge the specific quality of this experimental technique.
, the SDS-PAGE that extracts NA sample identifies
Electrophoresis utensil used (as electrophoresis chamber, seal strip, comb, glass offset plate etc.) is cleaned up and dry for standby in advance.Between two blocks of glass offset plates, properly sandwich seal strip, after assembling, be loaded on electrophoresis chamber and compacting plastic dam is fixed.Preparation separation gel, measures 4.85 mL with 5 mL pipettors and injects two blocks of glass offset plates, then adds immediately 1 mL dehydrated alcohol to flatten glue face.Under light, wait to coagulate, observe when obvious line of delimitation appears in liquid level intersection and coagulate.The concentrated glue of preparation, outwells upper strata dehydrated alcohol, treats that its remnants also volatilize totally, injects the concentrated glue of 1 mL in separation gel upper strata, then carefully inserts comb, must guarantee and coagulant liquid between there is not bubble.Under light, wait for and solidifying.Take away after plastic dam is taken out seal strip the upset of glass offset plate is commutated, make fluted one facing to swimming groove inside, compacting plastic dam again.Gently sample comb is extracted straight up.Assembling has been sealed electrophoresis apparatus and has been added 1 * electrode buffer.Extracting the protein sample of collecting in each step of NA, be mixed in proportion with 5 * sample buffer respectively, will dye in advance and in Marker and individual step, collect sample and in water-bath, boil 3-5 min.Cooling, loading 15 μ L successively in every hole, dye Marker2-3 μ L in advance.Connect electrophoresis apparatus and form loop line, this experimental selection current stabilization pattern is carried out electrophoresis.First adjust electric current to 10 mA, after approximately 15 min (when sample starts to enter separation gel by concentrated glue), then electric current is adjusted to 20 mA.When tetrabromophenol sulfonphthalein band runs to apart from gel below approximately 0.5 centimeters, deenergization.
After electrophoresis finishes, take electrophoresis apparatus apart, carefully take out gel, cut away and discard concentrated glue and carry out silver and dye.It is as follows that silver dyes process:
(1) fixing: in the glass dish of stationary liquid is housed, separation gel tiling to be immersed, more than fixing 1 h; (2) sensitization: discard stationary liquid and change to soak solution, soak 30 min; (3) rinsing: discard soak solution and change to ultrapure water rinsing 60 min, change during this period ultrapure water 4-5 time; (4) silver dyes: discard rinsing liquid and change to silver-colored dye liquor, 20 min dye; (5) colour developing: appropriate ultrapure water Rapid Cleaning silver dyes rear separation gel 2 times, adds nitrite ion; (6) stop: observe band colour-change, wait protein band clear manifesting comparatively, at once discard nitrite ion and change to stop buffer; (7) take a picture: finally film is transferred to ultraviolet according to Jiao Tai, takes a picture and retain result.
, purifying NA MALDI-TOF-MS one-level Mass Spectrometric Identification
Carry out before Mass Spectrometric Identification, first utilize 3 KD filtration unit Amicon Ultra-0.5 to carry out desalination processing to sample.To extract NA and be added in 3 KD centrifugal columns 4 ℃, centrifugal 15 min of 14,000 * g.Add the ultrapure water of 450 μ L in post, 4 ℃, centrifugal 15 min of 14,000 * g, repeat twice.The centrifuge tube renewing is inverted on it 4 ℃ by 3 KD posts, centrifugal 5 min of 3,000 * g.Collect the liquid in centrifuge tube, freeze-drying is standby.
The mass spectrograph of this experiment use is the Microflex MALDI-TOF instrument of German Bruker Daltonics company.The albumen extracting dissolves with 50% ACN, dissolves with the ratio of 1:1 with matrix CHCA, gets 1 μ L point sample on 96 target plates of Anchorchip, and vacuum is drained.With angiotonin (angiotensin, m/z 1046.54) and Sigma I8405 (bovine insulin, m/z 5730.61), as external standard, proofread and correct mass spectrograph.With the positive ion mode of straight line, identify albumen, concrete model selection LP 66 kD.Excitation light source is N2 laser (337 nm), and acceleration voltage is 20 KV.While detecting sample, get multipoint acquisition collection of illustrative plates, each collection of illustrative plates scanning 100 times, all spectrograms automatically stack obtain last protein mass collection of illustrative plates.
, 8 kinds of avian influenza virus NA separation and purification and quantitatively
According to best magnetic grain consumption, binding time and the elution time of carrying out choosing after condition optimizing, experimental procedure is adjusted, other conditions are constant, respectively separation and purification after ether is processed 8 kinds of avian influenza virus in neuraminidase.Using the negative allantoic fluid of virus inoculation not as negative control, carry out parallel running processing.
Protein sample after extracting is carried out to Bradford quantitative: take the BSA of 1.0mg, be dissolved in the BSA mother liquor of preparing 1.0mg/mL in 1mL ultrapure water, dilution preparation BSA standard protein concentration gradient is followed successively by 0,0.2,0.4,0.6,0.8,1.0 mg/mL; In the every hole of enzyme plate, add 200
Figure 144619DEST_PATH_IMAGE001
rise again in advance to the Bradford reagent of room temperature 30 min, then give successively in corresponding pipe, add different concns gradient BSA standard substance 10
Figure 916266DEST_PATH_IMAGE001
, NA sample 10 to be measured
Figure 929222DEST_PATH_IMAGE001
, fully piping and druming mixes, and before noting measuring, does not have bubble; After reaction, enzyme mark pipe is placed on 96 hole enzyme plates, microplate reader light absorption wavelength is set to 595nm and carries out single wavelength mensuration, reads (OD) value after standard protein and NA sample to be measured and Bradford reagent react; Standard protein concentration BSA concentration is as X-coordinate, and optical density value is ordinate zou mapping, obtains typical curve; And calculate the protein concentration in sample to be tested according to typical curve.
, experimental result and analysis
2.1, magnetic grain consumption determines
By wash-out concentration, magnetic grain consumption is mapped, referring to Fig. 4,
By finding out on figure, for H5N1 CK strain, slightly carry the applied sample amount of viral protein 5mg, when magnetic grain consumption is progressively increased to 3mg from 1mg, the extracted amount of albumen (replacing with wash-out concentration for simplicity because elution volume is identical) increases with the increase of magnetic grain consumption, and increasing degree is larger; From 3mg, though increase magnetic grain consumption protein extraction amount, slightly to increase amplitude minimum.The magnetic grain consumption of visible 3mg can meet extraction object substantially, and has alleviated the problem of the excessive non-specific adsorption of magnetic grain, therefore still chooses 3mg as the suitableeest magnetic grain consumption.
By wash-out concentration, binding time is mapped, referring to Fig. 5,
As can be seen from Figure, when binding time was increased to 1.5 hours from 1 hour, extract protein content and have more obviously increase, and As time goes on, extract protein content and do not continue to increase, even occur situation about reducing, consider the prolongation along with extraction time simultaneously, protein occurs that the possibility of degraded can progressively raise, therefore choose can reach optimum extraction efficiency 1.5 hours shortest times as the best combination time.Combination in former extracting method is shortened to 1.5 hours in 2 hours.
By wash-out concentration, elution time is mapped, referring to Fig. 6
As can be seen from Figure, along with the prolongation of elution time, extract protein content and increasing, but this increase tendency was mainly reflected in 2 hours, substantially reached maximum elution concentration in the time of 2 hours, though there is afterwards slightly increase not obvious.When guaranteeing extraction efficiency, shorten as much as possible elution time, therefore should choose can reach optimum extraction efficiency 2 hours shortest times as suitable time of eluent.And elution time in former extracting method 1 hour is obviously too short, do not reach the basic object of wash-out completely, elution time extended to 2 hours by 1 hour.
, method specificity checking
Referring to Fig. 7, electrophoresis result can be found out, former state and supernatant be indifference almost, and wash-out band silver dyes result and with the naked eye cannot differentiate, can prove thus: method that should be based on 4-amino-benzene oxaminic acid magnetic grain separation and purification NA is minimum to other components in test sample (albumen containing as chick embryo allantoic liquid itself) non-specific adsorption, and specificity is good.
, extract the qualification result of NA sample
In order to determine more accurately method reliability, in separation and purification H5N1 (CK) subtype virus, in the experiment of neuraminidase, the albumen that wash-out obtains is identified through SDS-PAGE and two kinds of methods of MALDI-TOF-MS, corroborates each other.Qualification result is referring to Fig. 8, Fig. 9.In electrophoresis result, can find out, although the albumen abundance itself existing in chick embryo allantoic liquid is very high, the protein band of virus own is covered to some extent, but what still can find is, extraction through magnetic particle, at～58 kD places, there is notable difference in the band in upper cleer and peaceful former state, and this～protein band at 58 kD places is just in time one of two master tapes in wash-out object band, illustrates that the albumen of this molecular weight has obtained separation and enrichment after leaching process.Appear at～30 kD places of another wash-out band.Simultaneously from swimming lane 4 almost blank band can find out, through three times, clean and reached good cleaning performance.From mass spectrum result, can find out, eluted protein qualification result and electrophoresis result are coincide, and obtain molecular weight and be two peaks of 28.7 and 57.3, and in having highly sensitive MALDI-TOF one-level mass spectrum result, assorted peak is also not obvious, substantially can ignore.
By with reference in additive method (as commercial oxaminic acid agarose affinity column etc.) extract relatively can finding of H5N1 virus neuraminidase experimental result, the molecular weight that extracts albumen in the present invention is consistent with each reference, the monomer that 57 kD components are neuraminidase, and the Partial Fragment that 28 kD components are monomer.Eluted protein is proved this kind of viral neuraminidase through identifying, has proved the reliability of present method, and in addition, present method is extracted result Za Feng, assorted band still less, is better than present means in specificity, can obtain more highly purified NA protein sample.
Neuraminidase concentration determination
Use Bradford method to carry out quantitatively.Referring to Figure 10, obtain typical curve.
According to above typical curve, calculate the protein concentration of wash-out NA in each sample.In Table 2-6:
Each sample wash-out of table 2-6 NA concentration
Figure 2013104380240100002DEST_PATH_IMAGE007
Note: CK: chicken; DK: duck; M: wild duck; O: ostrich; KP information is not clear.
From measurement result, can find out, the neuraminidase amount of extracting in each virus roughly maintains an equal level, and reflects the good consistence of Virus Sample; In negative control, elute the albumen of minute quantity, show that non-specific adsorption is less, specificity is good.
The separation and purification that the present invention is influenza neuraminidase provides a kind of effective means, both avoided neuraminidase that gene engineering method is obtained cannot meet the defect of neuraminidase glycosylation research, solved again the separated neuraminidase schedule of operation of commercialization oxalic acid agarose affinity column complicated, expensive, elution volume is large, is difficult to the technical problems such as traceization.The method is simple and efficient to handle, specificity is high, and neuraminidase is had to good refining effect, extraction that also can expanded application neuraminidase in other complex samples.

Claims (7)

1. the method for a separation and purification of nerve propylhomoserin enzyme (avian influenza virus), it is characterized in that: the method is using the special competitive inhibitor 4-amino-benzene oxaminic acid of neuraminidase as affinity ligand, be fixed on epoxidation magnetic particle surface, be prepared into functional Fe_3 O_4 magnetic particles neuraminidase to special avidity, and with this particulate, take the buffer system that pH is 5.5 with the strains of influenza viruses that contains neuraminidase and be combined, clean; The buffer system wash-out separation and purification of nerve propylhomoserin enzyme that the pH of take is 9.1.
2. the method for separation and purification of nerve propylhomoserin enzyme (avian influenza virus) according to claim 1, it is characterized in that, described being prepared into has the functional Fe_3 O_4 magnetic particles of special avidity to comprise to neuraminidase: extract the suspension containing 200 mg epoxidation magnetic particles, this epoxidation magnetic particle is modified to 4-amino-benzene oxaminic acid functional Fe_3 O_4 magnetic particles;
Described modification comprises:
The first step is modified: above-mentioned suspension is placed in reactor, with dehydrated alcohol, cleans; Reactor is placed on magnetic separator and carries out magnetic resolution again, discard the clear liquid that does not contain magnetic particle in upper strata;
Take 0.1081g Ursol D and be dissolved in 50mL dehydrated alcohol, after fully dissolving, proceed in reactor; Make epoxy magnetic grain and Ursol D fully mix, react, epoxidation magnetic grain is modified into Ursol D and modifies magnetic grain; After having reacted, magnetic resolution, and abandoning supernatant;
Second step is modified: the Ursol D obtaining after above-mentioned reaction is completed is modified magnetic grain and cleaned with dehydrated alcohol, then cleans with ultrapure water, changes to new reactor, after magnetic resolution, discards the clear liquid that does not contain magnetic particle in upper strata;
To the 0.1～0.2M MES damping fluid that adds 50 mL pH 5.0～6.0 in flask, add again 0.1260g oxalic acid dihydrate, stirring is fully dissolved oxalic acid and is mixed with magnetic grain, add EDC 0.1970 g, NHS 0.1151 g, in 4 ℃ of reactions, more than 8 hours, Ursol D is modified to magnetic grain and make 4-amino-benzene oxaminic acid functionalized magnetic particles;
After having reacted, magnetic resolution, abandoning supernatant; With ultrapure water, clean magnetic grain, be settled to 20 mL, proceed to reagent bottle, difference assay weighs the solid contents that calculates this 4-amino-benzene oxaminic acid functionalized magnetic particles suspension, labeling, and 4 ℃ save backup.
3. the method for separation and purification of nerve propylhomoserin enzyme (avian influenza virus) according to claim 1, is characterized in that: the strains of influenza viruses of described neuraminidase is from 10 age in days chick embryo allantoic liquids of inoculation; The particle of this virus strain is suspended in allantoic fluid.
4. the method for separation and purification of nerve propylhomoserin enzyme (avian influenza virus) according to claim 3, is characterized in that, described in contain neuraminidase strains of influenza viruses need before using the particle of this virus strain to carry out cracking and albumen is slightly carried; Described cracking and albumen are slightly carried employing ether extraction.
5. the method for separation and purification of nerve propylhomoserin enzyme (avian influenza virus) according to claim 1, is characterized in that, the method specifically,
(1) sealing of magnetic grain: get a 2mL centrifuge tube, in add wherein 3 mg4-amino-benzene oxaminic acid magnetic grains, magnetic resolution, abandons supernatant; Then in centrifuge tube, add sealing damping fluid 500 L, mix, be placed in gas bath vibrator, 37 ℃ of 180 rpm reacts 30 min;
(2) balance of magnetic grain and activation: upper step is placed in centrifuge tube on magnetic separator and carries out magnetic resolution after having reacted, and abandoning supernatant adds 1 mL level pad at every turn, cleans: add again 1 mL binding buffer liquid to clean magnetic grain;
(3) combination of sample and magnetic grain: get a new centrifuge tube, the thick leach protein 5mg of virus that adds ether to process, adds 2 * binding buffer liquid and ultrapure water in proportion, make system binding buffer liquid concentration be 1 *, volume is 1mL; Be labeled as " former state " stand-by; Get this solution 950 L and add and be equipped with in the centrifuge tube that step activated magnetic grain, jiggle to mixing, be placed in 37 ℃ of 180 rpm reaction 2h of gas bath vibrator; Reacted rear magnetic resolution;
(4) clean: in centrifuge tube, add 1 mL cleaning buffer solution, be positioned in gas bath vibrator, temperature is adjusted to 37 ℃, rotating speed 180 rpm jolting 10 min, magnetic resolution;
(5) wash-out: in centrifuge tube, add 500 L elution buffers, 25 ℃ of 180 rpm jolting 1 h, after magnetic resolution, collecting supernatant liquor, to be labeled as " wash-out " stand-by; Be the suspension that contains target protein neuraminidase.
6. the method for separation and purification of nerve propylhomoserin enzyme (avian influenza virus) according to claim 5, is characterized in that: described step (4) is cleaned and need to be repeated this step 3 time and collect the supernatant liquor of last magnetic resolution.
7. the method for separation and purification of nerve propylhomoserin enzyme (avian influenza virus) according to claim 6, is characterized in that: described reactor is flask or beaker or test tube.
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