CN102192941B - Method for quickly estimating specificity of influenza A virus host - Google Patents
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Abstract
The invention relates to a method for quickly estimating specificity of an influenza A virus host. The method comprises the following steps of: primarily purifying the hemagglutinin of influenza A virus and preparing a sugar chain magnetic particle complex; combining the primarily purified hemagglutinin of the influenza A virus with the sugar chain magnetic particle complex; eluting the hemagglutinin of the influenza A virus, which is combined with the sugar chain magnetic particle complex; and identifying the eluted hemagglutinin of the influenza A virus so as to acquire the result of the specificity of the influenza A virus host and judge whether the influenza virus infects human bodies. The technical problem that the mutation of the pathogen of the influenza A virus and the specificityof the host of the influenza A virus cannot be completely estimated by the conventional influenza virus detecting means and virus nucleic acid detecting methods is solved. The method has the advantages that the method is quick, simple and convenient, has high efficiency and the like.
Description
Technical field
The present invention relates to the influenza A virus field, be specifically related to a kind of method of assessing the influenza A virus host specificity.
Background technology
Influenza A virus is common influenza virus, and morphs easily.Avian influenza virus is a kind of hypotype of influenza A virus, and bird flu is a kind of acute infectious disease that is caused by avian influenza virus, also can infect the mankind.Avian influenza virus is the production of serious harm animal husbandry not only, and particularly highly pathogenic H5N1 avian influenza virus has been crossed over species barrier and directly caused the human infection and cause the infected's death.
The avian influenza virus antigen numerous types, variation is frequent, causes the highly pathogenic bird flu prevention and control very difficult.Therefore be necessary to develop and can be used for monitoring and assessing the variation of avian influenza virus cause of disease and the new technology of host specificity.There is the kind specificity in interaction between virus and host cell surface sugar chain, mainly identify and in conjunction with being present in the sugar chain acceptor of sialic acid (SA) the α 2-3Gal (galactose) of fowl intestines and stomach surface epithelial cell as avian influenza virus, and the human influenza virus mainly identifies and in conjunction with the sugar chain acceptor that is present in the SA α 2-6Gal on lung and airway epithelial cell surface.Think that at first the difference of this receptor-specific has caused avian influenza virus to propagate in the human world.But recent studies show that, some strains energy direct infection mankind of the H5N1 type of bird flu, and the H9N2 hypotype can directly be infected to the people by bird equally, and its reason is that they can be simultaneously in conjunction with these two kinds of sugar chain acceptors.
The cell receptor of being combined with influenza A virus is the sialic acid (SA) that is positioned at glycoprotein on the cell membrane or glycolipid chain end.The receptor-specific that studies show that influenza virus hemagglutinin (HA) albumen has determined viral host range.Human influenza virus HA mainly identifies and is the sialic acid compound sugar acceptor of SA α 2-6 galactose (Gal) in conjunction with end, it is present in the human respiratory surface epithelial cell, the main identification of avian influenza virus HA and combination end are the sialic acid compound sugar acceptor of SA α 2-3Gal, and it extensively is present in the surface epithelial cell of fowl intestines and stomach.
By the bird flu that influenza A virus causes, its pathological change is because infecting the different of the pathogenic power of virus stain and fowl kind.Its clinical symptoms because of the kind, age in days and the accompanying infection situation that infect fowl and to infect the different symptoms that show such as the pathogenic of strain and other environment very inconsistent.For a long time, the diagnosis of bird flu depends on Pathogen Isolation and evaluation always.In recent years, set up the diffusion of avian influenza virus agar (AGP), hemagglutination-inhibition test (HI) neuraminidase inhibition test (NI), indirect enzyme-linked immunosorbent assay serodiagnosis technology and molecular diagnostic techniques (as RT-PCR) such as (ELISA) both at home and abroad in succession, satisfied the diagnosis of China's avian influenza virus epidemiological surveillance and terrain and anti-pressing for of making.Virus separates cultivation and hemagglutination-inhibition test is frequent as the contrast of estimating new detection avian influenza virus method, but virus is separated cultivation length consuming time, at least need 7 days, be unfavorable for quick diagnosis, the requirement for experiment condition that virus is separated is higher, pathogenic danger is arranged simultaneously, the strict bio-safety measure of considering of wanting is gone up in detection and the management of strain.Avian influenza virus also may morph in chicken embryo culture process.Blood clotting and hemagglutination-inhibition test specificity are good, but its operation is loaded down with trivial details relatively.Serological testing generally can only just can have antibody positive to occur in morbidity 2-3 week even longer time to having made a definite diagnosis retrospective diagnostic value at last.Because avian influenza virus serotype is numerous, new variant constantly occurs, and preparation serotype specificity monoclonal antibody is difficulty relatively, and under the situation of various immunity inoculations, serological diagnostic methods such as hemagglutination-inhibition test also can be ineffective.Nucleic acid amplification technologies (NAT) detects avian influenza virus, chooses target gene and primer and optimizes response parameter, all can obtain high sensitivity and specificity.For the numerous avian influenza virus of serotype, the pathogenic difference of different subtype, its host distributes also different, so fast, somatotype seems particularly important in the avian influenza virus diagnosis specifically.The RT-PCR method can obtain the known HA hypotype of all avian influenza virus.No matter be any detection method in the detection method of above-mentioned influenza virus, all can't be simultaneously the influenza virus of numerous types and hypotype be carried out precise typing.Thereby be necessary to seek a kind of efficient, high flux and fast influenza virus detect and classifying method.The appearing as simultaneously influenza virus to be detected with somatotype of biochip technology provides possible approach.Have more than the ten piece of report of differentiating application about biochip technology in influenza test and hypotype at present both at home and abroad.Method for gene chip only can obtain the known hypotype of all avian influenza virus, can not detect and assess the variation of influenza A virus cause of disease and host's specificity fully.
Summary of the invention
Can not assess the variation of influenza A virus cause of disease and host's specific technical matters fully for the method that solves conventional sense avian influenza virus means and detection viral nucleic acid, the invention provides a kind of method of rapid evaluation influenza A virus host specificity.
Technical scheme of the present invention is as follows:
A kind of method of rapid evaluation influenza A virus host specificity may further comprise the steps:
Step 1: the influenza A virus hemagglutinin is carried out preliminary purification;
Step 2: preparation sugar chain magnetic particle compound;
Step 3: the influenza A virus hemagglutinin behind the preliminary purification and sugar chain magnetic particle compound are carried out combination;
Step 4: the influenza A virus hemagglutinin that will be combined with sugar chain magnetic particle compound carries out wash-out;
Step 5: the influenza A virus hemagglutinin behind the wash-out is identified, according to qualification result, with assessment influenza A virus host specificity.
The method of above-mentioned rapid evaluation influenza A virus host specificity, its special character is:
In the described step 1 the influenza A virus hemagglutinin being carried out preliminary purification may further comprise the steps;
1) gets the influenza A virus suspension;
2) in the influenza A virus suspension, add equal-volume and analyze absolute ether;
3) place 4 ℃ environment 0.5-2 hour again, and fully vibrate once every 10min;
4) the influenza A virus suspension after the step 3) processing is carried out the centrifugal of 10-20min with the speed of 2000r/min;
5) the influenza A virus suspension after centrifugal is carried out static layering, draw lower floor's influenza A virus suspension, and be transferred in another centrifuge tube;
6) centrifuge tube that will fill the lower floor's influenza A virus suspension after the transfer places ventilated environment, and the ether in this influenza A virus suspension thoroughly volatilizees;
7) place the environment that is lower than-18 ℃ to preserve the influenza A virus suspension after the step 6) processing.
Preparation sugar chain magnetic particle compound may further comprise the steps in the above-mentioned steps 2:
1) gets 2 centrifuge tubes, put into the some 1-15mg of equivalent hydroxylation magnetic particle respectively;
2) the hydroxylation magnetic particle is carried out magnetic resolution;
3) the hydroxylation magnetic particle after separating is abandoned supernatant, clean with absolute ethyl alcohol again;
4) the hydroxylation magnetic particle after the absolute ethyl alcohol cleaning is abandoned supernatant again, add coupling buffer again and clean;
5) in 2 centrifuge tubes that the hydroxylation magnetic particle that coupling buffer cleaned is housed, serve as with reference to the coupling buffer that adds 20-40 μ L/mg respectively with the amount of hydroxylation magnetic particle;
Amount with the hydroxylation magnetic particle is reference, again the SA α 2-3Gal sugar chain that adds with 0.3-0.75 μ mol/mg of centrifuge tube therein; The SA α 2-6Gal sugar chain that another centrifuge tube adds with 0.3-0.75 μ mol/mg;
6) hydroxylation magnetic particle, coupling buffer and sugar chain in 2 centrifuge tubes are carried out mixing; And carry out 2-10 hour oscillating reactions; Obtain sugar chain magnetic particle compound;
7) the sugar chain magnetic particle compound that obtains is carried out magnetic resolution, abandon supernatant;
8) also with the sugar chain magnetic particle compound after the binding buffer liquid cleaning magnetic resolution;
9) after cleaning is finished, place the preservation damping fluid to preserve sugar chain magnetic particle compound.
In the above-mentioned steps 3 the influenza A virus hemagglutinin behind the preliminary purification and sugar chain magnetic particle compound being carried out combination may further comprise the steps:
1) gets the above-mentioned two kinds of sugar chain magnetic particle compounds that prepare and put into two other centrifuge tube respectively;
2) be example with the 2mL centrifuge tube, in each centrifuge tube, add 200-1000 μ L binding buffer liquid respectively, the benzyl sulfonephthalein fluorine of 20-500 μ L preliminary purification influenza A virus liquid and 1-10 μ L, and mix;
3) solution behind the mixing is carried out 0.5-2 hour oscillating reactions, obtain being combined with the sugar chain magnetic particle albumen composition of influenza A virus hemagglutinin.
The influenza A virus hemagglutinin that to be combined with sugar chain magnetic particle compound in the above-mentioned steps 4 carries out wash-out and may further comprise the steps:
1) the sugar chain magnetic particle albumen composition that draws is carried out magnetic resolution, abandon supernatant, and clean with cleaning fluid;
2) the sugar chain magnetic particle albumen composition after will cleaning adds eluent 100-1000 μ L;
3) the sugar chain magnetic particle albumen composition that adds eluent is carried out the oscillating reactions of 0.1-2h;
4) reacted sugar chain magnetic particle compound is carried out magnetic resolution, obtain supernatant, the influenza A virus liquid hemagglutinin that namely is purified into;
5) place the environment that is lower than-18 ℃ to preserve to the influenza A virus liquid hemagglutinin that is purified into.
In the above-mentioned steps 5 the influenza A virus hemagglutinin behind the wash-out is identified specifically: the influenza A virus liquid hemagglutinin that two kinds of different sugar chain magnetic particle compound purifying are obtained carries out polyacrylamide gel electrophoresis and silver dyes colour developing respectively, thus assessment influenza A virus host specificity.
Above-mentioned assessment is specifically: if show that in electrophoretogram eluent elutes the albumen of being combined with SA α 2-6Gal tangible band is arranged, molecular weight is about 75KD, can infect the mankind so can judge influenza A virus; If show in the electrophoretogram that eluent elutes the albumen of being combined with SA α 2-3Gal tangible band is arranged, molecular weight is about 75KD, so can judge that influenza A virus can infected poultry.
In the above-mentioned steps 5 the influenza A virus hemagglutinin behind the wash-out identified and may further comprise the steps:
1) suppresses experiment serum with fluorochrome label influenza A virus blood clotting;
2) influenza A virus blood clotting that mark is good suppresses experiment serum and mixes with 1: 9 ratio with phosphate buffer and be made into antibody-solutions;
3) sugar chain magnetic particle compound is combined with the influenza A virus hemagglutinin of preliminary purification;
4) the sugar chain magnetic particle albumen composition in conjunction with the influenza A virus hemagglutinin is cleaned with cleaning fluid;
5) antibody-solutions of adding 100-1500 μ L, the sugared magnetic corpuscular protein compound after obtaining mark after the jolting reaction again in the sugar chain magnetic particle albumen composition after cleaning;
6) the sugar chain magnetic particle albumen composition behind the mark is cleaned with cleaning fluid again;
7) draw the sugar chain magnetic particle albumen composition that 50-200 μ L cleaned and place on the slide, and carry out the drying processing;
8) dried slide is put into the fluorescent scanning instrument and scan, thus assessment influenza A virus host specificity.
Above-mentioned assessment is specifically: if SA α 2-6Gal sugar chain magnetic particle albumen composition shows fluorescence, show that influenza A virus can infect the mankind; If SA α 2-3Gal sugar chain magnetic particle albumen composition shows fluorescence, show that influenza A virus can infected poultry.
Put into the hydroxylation magnetic particle of 3mg in the above-mentioned steps 1 respectively at 2 centrifuge tubes; Wash number in described step 3 and the step 4 is at least 3 times.
The present invention has the following advantages
1, magnetic particle can float on a liquid uniformly, these particulates can be assembled towards certain direction under the effect of externally-applied magnetic field, the direction of magnetic pole is consistent, after removing externally-applied magnetic field, the magnetic pole of particulate again can be very fast revert to original state, still can keep original character, can float on a liquid uniformly, therefore utilize this specific character of magnetic particle, can be the carrier of magnetic particle as the separation and purification biomolecule.The present invention utilizes the similar method for preparing carbohydrate chip covalent coupling sugar chain in purification step, carry out magnetic particle and organic reagent compound, introduce some functional groups at microparticle surfaces, these functional groups carry out in conjunction with the surface that just these materials can be fixed in particulate with the group of introducing on the material.Can high-level efficiency, quick, easy carbohydrate-binding protein (for example influenza A virus hemagglutinin) is carried out separation and purification.
2, the World Health Organization (WHO) judges according to four conditions whether the influenza great outburst arrives at present: the first, and whether human have immunity to this virus; The second, virus can propagate into the people from fowl; The 3rd, can cause infected people's death; The 4th, can be in interpersonal propagation.The method of fast detecting influenza A virus host specificity provided by the invention is applicable to import and export quarantine, health and epidemic prevention and the epidemiology survey of bird and bird product.Can satisfy and realize that quick, high flux detects virus, adapt to and quarantine on a large scale, forecast that prediction is acute, the needs of deadly infectious disease.
Description of drawings
Fig. 1 is the electrophoresis result figure of example for the present invention with H5N2 avian influenza virus Mallard/JX/16/05;
Description of drawings: the 1-8 swimming lane is followed successively by among Fig. 1
1: albumen Marker; 2: not in conjunction with the albumen of SA α 2-3Gal; 3: clean the washing lotion component after finishing; 4: eluent elutes the albumen of being combined with SA α 2-3Gal; 5: not in conjunction with the albumen of SA α 2-6Gal; 6: clean the washing lotion component after finishing; 7: eluent elutes the albumen of being combined with SA α 2-6Gal; 8: viral total protein.
Embodiment:
Realize that experiment material of the present invention is specific as follows:
1), main material and reagent:
Avian influenza virus is provided by the Harbin veterinary institute; Homemade hydroxylation magnetic particle;
3 '-N-Acetylneuraminyl-N-acetyllactosamine sodium salt (α-NeuNAc-(2 → 3)-β-D-Gal-(1 → 4)-D-GlcNAc) or 3 '-Sialyllactose (α-NeuNAc-(2 → 3)-β-D-Gal-(1 → 4)-D-Glc), 6 '-Sialyllactose sodium salt (α-NeuNAc-(2 → 6)-β-D-Gal-(1 → 4)-D-Glc), benzyl sulfonephthalein fluorine (PMSF), monoethanolamine, ProteinMarker, sigma; Other common agents are that homemade analysis is pure.
2), the prescription of main agents:
Coupling buffer: 0.2mol/L pH5.4 acetic acid sodium acetate buffer;
Binding buffer liquid: 20mmol/L Tris-HCl, 0.5mol/L NaCl, 10mmol/L CaCl
2, 6mmol/L MnCl
2, pH7.2;
Cleaning fluid: 20mmol/L Tris-HCl, 0.5mol/L NaCl, 0.05%Tween20, pH7.2;
Elution buffer: 0.5%SDS;
Preserve damping fluid: 10% monoethanolamine.
3), the main instrument that uses:
Magnetic separation rack, Shanxi North America Gene Co., Ltd's product; ZHWY 2102C type constant temperature culture oscillator, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.; The AB204-S electronic balance, Switzerland Mettlertoledo; Liquid-transfering gun, German Epphendorf company; The 5415D hydro-extractor, German Eppendorf company; DK-98-1 type electric-heated thermostatic water bath, Tianjin Tai Site Instr Ltd.; SK-1 high speed vortex mixer, Changzhou Guohua Electric Appliance Co., Ltd.; DYY-8B type voltage stabilization and current stabilization electrophoresis apparatus, Beijing Liuyi Instrument Factory; WD-9406 type film illuminator, Beijing Liuyi Instrument Factory.
Realize that the present invention assesses the method for influenza A virus host specificity, may further comprise the steps:
Step 1: the influenza A virus hemagglutinin is carried out preliminary purification;
1) gets the influenza A virus suspension;
2) in the influenza A virus suspension, add the equal-volume ether;
3) the influenza A virus suspension that will add the equal-volume ether was placed 0.5-2 hour in 4 ℃ environment, and every the 10min jolting once;
4) the influenza A virus suspension after will handling carries out the centrifugal of 10-20min with the speed of 2000r/min;
5) the influenza A virus suspension after centrifugal is carried out static layering, be transferred in another centrifuge tube to draw lower floor's influenza A virus suspension;
6) the lower floor's influenza A virus suspension after the transfer places ventilated environment to arrange ether;
7) the lower floor's influenza A virus suspension behind row's ether being put into the environment that is lower than-18 ℃ preserves.
Step 2: preparation sugar chain magnetic particle compound;
1) gets 2 centrifuge tubes, put into hydroxylation magnetic particle 1-15mg respectively; Be best with 3mg;
2) the hydroxylation magnetic particle is carried out magnetic resolution;
3) the hydroxylation magnetic particle after separating is abandoned supernatant, and the adding absolute ethyl alcohol cleans 3 times.
4) the hydroxylation magnetic particle after the absolute ethyl alcohol cleaning is abandoned supernatant again, add coupling buffer again and clean 3 times;
5) every pipe adds 90 μ L coupling buffers in 2 centrifuge tubes that the hydroxylation magnetic particle that coupling buffer cleaned is housed, and a pipe adds 15mmol/L α-NeuNAc-(2 → 3)-β-D-Gal-(1 → 4)-D-GlcNAc or 3 '-Sialyllactose (α-NeuNAc-(2 → 3)-β-D-Gal-(1 → 4)-D-Glc) 10-100 μ L therein, be good with 10 μ L, another pipe adds 15mmol/L α-NeuNAc-(2 → 6)-β-D-Gal-(1 → 4)-D-Glc 10-100 μ L; Be good with 10 μ L.
6) to the hydroxylation magnetic particle in 2 centrifuge tubes, coupling buffer, α-NeuNAc-(2 → 3)-β-D-Gal-(1 → 4)-D-GlcNAc, α-NeuNAc-(2 → 6)-β-D-Gal-(1 → 4)-D-Glc, carry out mixing; And carry out 2-10 hour oscillating reactions; Obtaining sugar chain magnetic particle compound, was good with 8 hours.
7) the sugar chain magnetic particle compound that draws is carried out magnetic resolution;
8) the sugar chain magnetic particle compound after the magnetic resolution is abandoned supernatant; And clean 3 times with binding buffer liquid;
9) place the preservation damping fluid to preserve to the sugar chain magnetic particle compound after the cleaning of binding buffer liquid.
Step 3: the influenza A virus hemagglutinin behind the preliminary purification and sugar chain magnetic particle compound are carried out combination;
1) gets two kinds of sugar chain magnetic particle compounds that prepare and put into two centrifuge tubes respectively;
2) in each centrifuge tube, add 200-1000 μ L binding buffer liquid respectively, the hemagglutinin of 20-500 μ L preliminary purification influenza A virus liquid and the benzyl sulfonephthalein fluorine of 1-10 μ L, and mix;
3) to mixed binding buffer liquid, influenza virus liquid and benzyl sulfonephthalein fluorine carry out 0.5-2 hour oscillating reactions, obtain being combined with the sugared magnetic corpuscular protein compound of influenza A virus hemagglutinin.
Step 4: the influenza A virus hemagglutinin that will be combined with sugar chain magnetic particle compound carries out wash-out;
1) the sugar chain magnetic particle albumen composition that draws is carried out magnetic resolution, abandon supernatant, and clean 3～5 times with cleaning fluid;
2) the sugar chain magnetic particle albumen composition after will cleaning adds eluent 100-1000 μ L, 200 μ L the bests;
3) the sugar chain magnetic particle albumen composition that adds eluent is carried out the oscillating reactions of 0.1-2h, 2h the best;
4) reacted sugar chain magnetic particle compound is carried out magnetic resolution, obtain supernatant, the influenza A virus liquid hemagglutinin that namely is purified into;
5) place the environment that is lower than-18 ℃ to preserve to the influenza A virus liquid hemagglutinin that is purified into.
Step 5: the influenza A virus hemagglutinin behind the wash-out is identified, to obtain influenza A virus host specificity result, namely the influenza A virus hemagglutinin behind the wash-out is identified it is that the influenza A virus liquid hemagglutinin that respectively two kinds of different sugar chain magnetic particle compound purifying obtained carries out polyacrylamide gel electrophoresis and silver dyes colour developing, thus assessment influenza A virus host specificity.
Referring to Fig. 1, in conjunction with figure this part is further specified.
Step 6: the influenza A virus hemagglutinin behind the wash-out assessed may further comprise the steps:
1) suppresses experiment serum with fluorochrome label influenza A virus blood clotting;
2) influenza A virus blood clotting that mark is good suppresses experiment serum and mixes with 1: 9 ratio with phosphate buffer and be made into antibody-solutions;
3) sugar chain magnetic particle compound is combined with the influenza A virus hemagglutinin of preliminary purification;
4) the sugar chain magnetic particle compound in conjunction with the influenza A virus hemagglutinin is cleaned with cleaning fluid;
5) antibody-solutions of adding 100-1500 μ L, the sugared magnetic corpuscular protein compound after obtaining mark after the jolting reaction again in the sugared magnetic corpuscular protein compound after cleaning;
6) the sugared magnetic corpuscular protein compound behind the mark is cleaned with cleaning fluid again;
7) draw the sugared magnetic corpuscular protein compound suspension that 50-200 μ L cleaned and place on the slide, and carry out the drying processing;
9) if SA α 2-6Gal sugar chain magnetic particle albumen composition shows fluorescence, show that influenza A virus can infect the mankind.
10) if SA α 2-3Gal sugar chain magnetic particle albumen composition shows fluorescence, show that influenza A virus can infected poultry.
Claims (1)
1. the method for a rapid evaluation influenza A virus host specificity may further comprise the steps:
Step 1: the influenza A virus suspension that includes the influenza A virus hemagglutinin is carried out preliminary purification; Specifically may further comprise the steps;
1) gets the influenza A virus suspension;
2) in the influenza A virus suspension, add equal-volume and analyze absolute ether;
3) place 4 ℃ environment 0.5-2 hour again, and fully vibrate once every 10min;
4) the influenza A virus suspension after the step 3) processing is carried out the centrifugal of 10-20min with the speed of 2000r/min;
5) the influenza A virus suspension after centrifugal is carried out static layering, draw lower floor's influenza A virus suspension, and be transferred in another centrifuge tube;
6) centrifuge tube that will fill the lower floor's influenza A virus suspension after the transfer places ventilated environment, and the ether in the influenza A virus suspension thoroughly volatilizees;
7) place the environment that is lower than-18 ℃ to preserve the influenza A virus suspension after the step 6) processing;
Step 2: prepare two kinds of sugar chain magnetic particle compounds; Specifically may further comprise the steps:
1) gets 2 centrifuge tubes, put into the hydroxylation magnetic particle of 3mg respectively;
2) the hydroxylation magnetic particle is carried out magnetic resolution;
3) the hydroxylation magnetic particle after separating is abandoned supernatant, clean with absolute ethyl alcohol again;
4) the hydroxylation magnetic particle after the absolute ethyl alcohol cleaning is abandoned supernatant again, add coupling buffer again and clean;
5) in 2 centrifuge tubes that the hydroxylation magnetic particle that coupling buffer cleaned is housed, serve as with reference to the coupling buffer that adds 20-40 μ L/mg respectively with the amount of hydroxylation magnetic particle;
Amount with the hydroxylation magnetic particle is reference, more therein in centrifuge tube the concentration with 0.3-0.75 μ mol/mg add SA α 2-3Gal sugar chain; Concentration with 0.3-0.75 μ mol/mg in another centrifuge tube adds SA α 2-6Gal sugar chain;
6) hydroxylation magnetic particle, coupling buffer and sugar chain in 2 centrifuge tubes are carried out mixing respectively; And carry out 2-10 hour oscillating reactions; Obtain sugar chain magnetic particle compound;
7) the sugar chain magnetic particle compound that obtains is carried out magnetic resolution, abandon supernatant;
8) also with the sugar chain magnetic particle compound after the binding buffer liquid cleaning magnetic resolution;
9) after cleaning is finished, place the preservation damping fluid to preserve sugar chain magnetic particle compound;
Step 3: the influenza A virus suspension behind the preliminary purification is carried out combination with two kinds of sugar chain magnetic particle compounds respectively, obtain two kinds of sugar chain magnetic particle protein complexes that are combined with the influenza A virus hemagglutinin; Specifically may further comprise the steps:
1) gets two kinds of sugar chain magnetic particle compounds that prepare and put into two other centrifuge tube respectively;
2) adopt the 2mL centrifuge tube, in each centrifuge tube, add 200-1000 μ L binding buffer liquid respectively, the influenza A virus suspension behind the 20-500 μ L preliminary purification and the benzyl sulfonephthalein fluorine of 1-10 μ L, and mix;
3) solution behind the mixing is carried out 0.5-2 hour oscillating reactions, obtain being combined with the sugar chain magnetic particle protein complex of influenza A virus hemagglutinin;
Step 4: the influenza A virus hemagglutinin that will be combined with sugar chain magnetic particle compound carries out wash-out; Specifically may further comprise the steps:
1) the sugar chain magnetic particle protein complex that is combined with the influenza A virus hemagglutinin that obtains is carried out magnetic resolution, abandon supernatant, and clean with cleaning fluid;
2) add eluent 100-1000 μ L in the sugar chain magnetic particle protein complex that is combined with the influenza A virus hemagglutinin after cleaning;
3) the sugar chain magnetic particle protein complex that is combined with the influenza A virus hemagglutinin that adds eluent is carried out the oscillating reactions of 0.1-2h;
4) solution after the oscillating reactions is carried out magnetic resolution, obtain supernatant, the influenza A virus hemagglutinin that namely is purified into;
5) place the environment that is lower than-18 ℃ to preserve the influenza A virus hemagglutinin that is purified into;
Step 5: assessment influenza A virus host specificity;
Method one: the influenza A virus hemagglutinin that will use two kinds of different sugar chain magnetic particle compounds to be purified into carries out polyacrylamide gel electrophoresis and silver respectively and dyes colour developing, thus assessment influenza A virus host specificity;
The assessment of method one is specifically: if show that in electrophoretogram the protein of being combined with SA α 2-6Gal that eluent elutes has tangible band, molecular weight is about 75KD, can infect the mankind so can judge influenza A virus; If show the protein of being combined with SA α 2-3Gal that eluent elutes in the electrophoretogram tangible band is arranged, molecular weight is about 75KD, so can judge that influenza A virus can infected poultry;
Method two: use antibody-solutions that two kinds of sugar chain magnetic particle protein complexes that are combined with the influenza A virus hemagglutinin are carried out mark respectively, carry out fluorescent scanning then, thus assessment influenza A virus host specificity; Specifically may further comprise the steps:
1) suppresses experiment serum with fluorochrome label influenza A virus blood clotting;
2) influenza A virus blood clotting that mark is good suppresses experiment serum and mixes with 1: 9 ratio with phosphate buffer and be made into antibody-solutions;
3) two kinds of sugar chain magnetic particle protein complexes that are combined with the influenza A virus hemagglutinin that step 3 is obtained clean with cleaning fluid respectively;
4) add the antibody-solutions of 100-1500 μ L in two kinds of sugar chain magnetic particle protein complexes after cleaning respectively, again the sugar chain magnetic particle protein complex after obtaining mark after the jolting reaction;
5) two kinds of sugar chain magnetic particle protein complexes behind the mark are cleaned with cleaning fluid respectively again;
6) the sugar chain magnetic particle protein complex of drawing behind the mark that 50-200 μ L cleaned places on the slide, and carries out the drying processing;
7) dried slide is put into the fluorescent scanning instrument and scan, thus assessment influenza A virus host specificity;
The assessment of method two is specifically: if SA α 2-6Gal sugar chain magnetic particle protein complex shows fluorescence, show that influenza A virus can infect the mankind; If SA α 2-3Gal sugar chain magnetic particle protein complex shows fluorescence, show that influenza A virus can infected poultry.
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| CN1599747A (en) * | 2001-10-19 | 2005-03-23 | 纳蒂芒公司 | Isolation of lectins |
| CN102190701A (en) * | 2010-03-05 | 2011-09-21 | 西北大学 | Method for separating and purifying influenza virus hemagglutinin on large scale |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1599747A (en) * | 2001-10-19 | 2005-03-23 | 纳蒂芒公司 | Isolation of lectins |
| CN102190701A (en) * | 2010-03-05 | 2011-09-21 | 西北大学 | Method for separating and purifying influenza virus hemagglutinin on large scale |
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| 孙宇.禽流感病毒血凝素的分离纯化以及快速评估其宿主特异性方法的研究.《西北大学硕士学位论文》.2010, |
| 禽流感病毒血凝素的分离纯化以及快速评估其宿主特异性方法的研究;孙宇;《西北大学硕士学位论文》;20100915;全文 * |
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