CN1490334A - Affinity purifying method for gene specific antibody - Google Patents
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Abstract
An affinity purifying process for the specific antibody generated by genetic immune includes such steps as using the eukaryon expression plasmid with particular gene and 6XHis to immunize animal, extracting the serum or yolk immunoglubulin containing gene specific antibody (IgG), externally transfecting eukaryon, culturing, adding the cell cracked liquid to metallic ion chelating resin column to prepare affinity chromatographic column, affinity chromatographing and eluting. Its advantage is high purity of IgG or IgY.
Description
Goal of the invention: the present invention is a kind of method that relates to the specific antibody that the affinity purification genetic immunization produced, be mainly used in the purifying of the gene specific antibody that is produced behind the genetic immunization, for genetic immunization prepares Mammals IgG or birds IgY, the purity that improves specific antibody provides a kind of practical approach.
Invention field: biomedicine
Technical background
Along with the continuous propelling of proteomics research, protein chip technology has become the important function genome technique platform.Protein chip technology is the high-throughput protein expression technology platform of protein-protein interactions.The protein that is fixed on the upholder surface can be antibody, antigen, acceptor, part, enzyme, substrate and the protein binding factor etc.Wherein antibody chip is used at most.
Antibody chip (Antibody Microarray, antibody microarray) is a kind of of protein chip, is the novel method of protein expression pattern in the detection of biological sample.This new technology makes the researchist once comparing hundreds of proteinic relative abundance in the biological sample in the experiment, thereby can be used to detect a certain specific physiology or the expression pattern of pathologic process associated protein.
The antibody that present antibody chip is adopted all is from commercially available monoclonal antibody.The prior art for preparing monoclonal antibody method is at present, first purifying antigen albumen, and immune mouse prepares monoclonal myelomatosis fusion cell line at last, and wherein antigenic preparation purifying becomes the bottleneck of Monoclonal Antibody.In order to overcome this restriction, released and adopted the method for genetic immunization directly to prepare antibody, because ready-made cDNA expression plasmid is arranged, thereby can directly prepare thousands of antibody for the protein chip needs.
Genetic immunization (also claiming dna immunization) is all by this ultimate principle of its dna encoding according to all antigens of human body, the dna fragmentation of the coded protein in the gene library is inserted in the specific recombinant mammalian expressing vector, then expression plasmid is imported Mammals or birds by intramuscular injection or particle gun method.Can glycosylation correctly in host by expressed protein, thus induce antigenic high-quality immune response.These expression plasmids can increase in intestinal bacteria, but can not duplicate in mammalian cell.Plasmid vector promotor commonly used mostly is and derives from virus genomic cytomegalovirus (CMV) early promoter, has very strong transcriptional activation; In addition, also can comprise some suitable enhansers, terminator, intron, immuno-stimulating sequence and polyadenylic acid signal etc. among the expression plasmid DNA.
Employing as sheep, rabbit, horse, donkey etc., can obtain the polyclonal antibody of anti-this gene product to mammiferous genetic immunization in animal serum, these antibody mostly are the IgG type, and minority can be IgM.Yet, antibody in the serum is the mixture of all immunoglobulin (Ig)s, and what affinity purification methods such as general albumin A, Protein G were collected is these immunoglobulin (Ig) mixtures, and this is for the high-throughput protein chip, can increase cross reaction, obviously undesirable.
Employing is because the specific antibody after the immunity can be present in the yolk of egg in a large number to the immunity of birds such as chicken, is easy to gather and separate, and also the expression amount height, the antibody acidproof heat-proof.Immunoglobulin of Yolk G antibody (IgY) is a kind of 7S immunoglobulin (7SIgG), and is slightly different with Mammals IgG.This protein molecular weight is about 180kDa, contains two subunits, and promptly the light chain of the heavy chain of 67～70kDa and 22～30kDa claims IgY (Yolk Immunoglobulin) again, is present in the yolk of immunity back hen.Though the preparation aspect of IgY is better than the preparation of Mammals IgG, also still there is the problem of antibody mixture in the similar serum, though the fast purifying test kit of the production and sales Igy of many companies special use at present, the issues of purification of specific antibody does not still solve.Therefore, the purifying of gene specific antibody is the gordian technique that genetic immunization prepares protein chip.
The present invention is in order to solve the problem of specific antibody purifying in the gene antibody preparation, adopt the method for vivoexpression band 6 * His recombinant protein to prepare the special antibody method of affinity column purified genes, its principle following (accompanying drawing):
With the recombinant mammalian expressing vector of commercial sources acquisition or the recombinant mammalian expressing vector 1 of oneself preparation; after a large amount of amplifications in prokaryotic cell prokaryocyte; carry out the animal immune of eukaryon expression plasmid; immune animal can be that Mammals 17 is as rabbit, sheep, horse, donkey etc.; also can select birds 16 as chicken, duck, goose etc.; the former collects serum and extracts gene specific IgG (15), and the latter collects egg, extracts gene specific IgY (14) from yolk.Simultaneously with recombinant mammalian expressing vector at in-vitro transfection 293-F cell 2 or other eukaryotic cells, cultivate back harvested cell and lysing cell, cell pyrolysis liquid is added to the chromatography column 6 (Qiagen) of Talon resin column (Clontech) or 5 preparations of Ni-NTA (nickel-nitrilotriacetic acid(NTA)) resin, because recombinant protein has 6 * His fragment, can with Co or the Ni ion chelating 8 in the resin, be adsorbed on resin surface, foreign protein 9 is then washed away.So just made the affinity column 7 that is used to adsorb specific antibody.When carrying out the specific antibody affinity chromatography, the IgG antibody or the IgY antibody of preliminary purification are added to affinity column 12, fully after the combination, specific antibody combines 13 with recombinant protein, wash away nonspecific IgG antibody or IgY antibody 11 then, wash post at last and collect specific IgG antibody or IgY antibody 10.
Concrete operations step of the present invention is as follows:
1. the preliminary purification of genetic immunization process and antibody
1) preparation of cDNA eukaryon expression plasmid
The acquisition approach of recombinant mammalian expressing vector has 2, promptly obtains from commercial channels and oneself preparation.The supply of commodities of at present existing more than 2500 kind of full-length cDNA eukaryon expression plasmid also can be set up eukaryon expression plasmid voluntarily with the cDNA clone.Selected plasmid vector must be to increase on high copy ground in intestinal bacteria, then can efficiently express in zooblast, but not duplicate, and does not also contain the sequence of integrating in the oriented host cell gene group.Adoptable recombinant mammalian expressing vector has: and pcDNA3.1 (InvitroGen, USA), (InvivoGen, USA), wherein pVAC+pBOOST is exclusively used in dna immunization for pVIVO2 and pVAC+pBOOST.
The present invention is a basic framework with the pcDNA3.1/GS plasmid, and these plasmids have bacterium replicon (ori), Eukaryotic promotor and PolyA tailing signal.Promotor derives from viral genome CMV, and PolyA is from bovine growth hormone gene; Screening-gene is selected neomycin resistance gene for use.6 * His fragment is contained in the gene downstream, is used for the purposes such as affinity chromatography, analysis of recombinant protein.
2) dna immunization
The dna immunization mode mainly contains two kinds: the subcutaneous importing of intramuscular injection and particle gun.
The selection of immune animal can be selected Mammals such as rabbit, sheep, horse, donkey etc., also can select birds such as chicken, duck, goose etc., and the former collects serum and extracts specific IgG, and the latter collects egg, extracts specific IgY from yolk.
Intramuscular injection: before injection, carry out plasmid amplification, earlier plasmid is transformed into intestinal bacteria, cultivate amplification, use plasmid DNA extraction agent extracting plasmid then, inject according to the dna immunization program.Particle bombardment: general elder generation is coated on golden micropartical surface with plasmid DNA, will be quickened by the gold grain that DNA is wrapping with particle gun, brings DNA into target tissue effectively.This method efficient height, the DNA of tens nanograms can obtain the intensive immunne response; Shortcoming is that the gold grain operation of preparation DNA bag quilt is complicated, and special equipment requirements is arranged.
The immunity flow process generally adopts the method for multi-section position, gradation booster immunization, begins to occur the rising of antibody titers after one month.
3) serum IgG is collected and preliminary treatment
Mammiferous immunity, general genetic immunization began to produce antibody in 30 days, increased gradually later on.After immunity arrives 3 months, the venous blood that can take a morsel, separation of serum is done immune double diffusion test with recombinant antigen protein, if serum titer＞1: 16, get final product bloodletting.
4) collection of egg and IgY initial gross separation
The immunity of birds, with the source that egg extracts as antibody, more traditional method of from Mammals, producing polyclonal antibody, convenient, simple, cheap.Such as using the genetic immunization chicken, begin to collect egg after one month.IgY antibody is present in the yolk, therefore before purifying, must isolate yolk earlier, removes lipid matter in the yolk then, carries out the affinity purification of following specific antibody on this basis.
2. vivoexpression prepares recombinant protein
1) cell is selected and plasmid transfection
Can select to be exclusively used in the cell strain of eukaryotic expression, as COS-7,293 cells, Chinese hamster ovary celI etc.The FreeStyle293-F cell is the variant of 293 cells, be to make up by human embryo kidney primary cell transfection V-type adenoviral gene to form, sustainablely go down to posterity, suspension culture and serum free medium cultivate, and expression E1A adenoviral gene, participate in the activation of some viral promotors, high level expression allogenic gene albumen.This cell is fit to high-density culture, serum-free large vol suspension culture, has very high transfection efficiency.
Also can select prokaryotic cell prokaryocyte such as e. coli bl21 (DE3) host as expression of recombinant proteins, but eukaryotic expression plasmid expression efficiency in prokaryotic cell prokaryocyte is not high, should select to have procaryotic cell expression plasmid such as the pRSET of the cDNA identical with animal immune.The characteristics of procaryotic cell expression are recombinant protein output height, and shortcoming is that the recombinant protein that is generated lacks corresponding modification, and its surface glycoprotein composition may be distinguished to some extent with the recombinant protein of eukaryotic cell expression.
2) protein urine and quantitative
Adopt ELISA, spot or cell direct staining method to detect the Recombinant Protein Expression situation.Utilize Ni-NTA-HRP (Qiagen Cat.34530) color producing reaction directly to measure Histidine peptide section in the recombinant protein.
3. affinity column preparation
1) Talon resin and Ni-NTA (nickel-nitrilotriacetic acid(NTA)) resin
The Ni-NTA resin is a Qiagen company product, has high affinity with 6 continuous histidine residues in the recombinant protein, good avidity is all arranged under condition natural or sex change, can from liquid that dilutes or the cell pyrolysis liquid of slightly carrying, concentrate recombinant protein by purifying.Purification step divided for 4 steps, i.e. lysis, combination, wash post and wash-out.For cell-membrane receptor, epicyte protein, inclusion body albumen etc., can take the strong denaturant soluble protein.6 * His part of this recombinant protein is very strong with combining of Ni-NTA, only just can dissociate under the powerful competition of 100-250mM imidazoles.
Talon is a Clontech company product, also is the sequestrant that adopts four chelating sites, and metal ion is a cobalt, have the avidity stronger than Ni-NTA, therefore have better resolving power, foreign protein still less, metal ion is not easy to be pulled down when albumen sepn simultaneously, can reuse.The recombinant protein binding capacity of resin is 6-8mg/ml.
2) cytolysis
In order to allow intracellular protein fully discharge, must make lysis, generally use 1%NP40,1%Tween20 or 1%Triton X-100 to make cytoclasis, guarantee that again recombinant protein remains on native state simultaneously.
3) protein expression in vitro is crossed the preparation of post and affinity column
Cell lysates after centrifugal, is added on Talon or the Ni-NTA resin column, makes its gravity filtration chromatography, and perhaps negative pressure-pumping filters, and also can pass through centrifuging, reaches the purpose that allows cell lysates cross post.Because recombinant protein contains 6 * His fragment, therefore can be fixed with Co or the Ni ion chelating in the resin, this chelating is quite firm, is not easy by general elutriant wash-out.The affinity column of specific antibody has just been made like this, can be used for the purifying of following specific antibody.
4. the affinity chromatography of gene specific antibody is separated
With the above-mentioned affinity column adding serum for preparing or the IgY composition of preliminary purification, after suitably hatching, wash post again and remove non-specific antibody, wash post with suitable elutriant at last, collect elutriant, this component is specific absorption antibody.According to elutriant what, select suitable ultrafiltration and concentration method to concentrate specific antibody.Can adopt the immune two-way precipitator method, ELISA method or additive method to measure tiring of specific antibody.
Advantage of the present invention
1. immunity and the shared eukaryon expression plasmid of vivoexpression in the body reduce cost, increase antigen-antibody bonded quality;
2. after the affinity column preparation, can use repeatedly;
3. utilize 6 * His of recombinant protein, can easyly carry out recombinant protein quantitatively, specific antibody quantitatively, easy and simple to handle;
4. specific antibody purity height can reach more than 99%.
Explain implementation process of the present invention in detail below in conjunction with example.
Accompanying drawing: the antibody purification flow process of genetic immunization
The non-specific antibody that 1 recombinant mammalian expressing vector 11 is washed away
But the eukaryotic cell 12 of 2 in-vitro transfection high expression levels is combined with recombinant protein-anti-recombinant protein antibody
3 have the resin of the recombinant protein of 6 * His polypeptide fragment
The foreign protein 13 resins-recombinant protein of 4 cell expressings-anti-recombinant protein antibody
The IgY of resin 14 preliminary purification from the egg of birds of 5 Ni or Co ion chelating
6 are seated in the resin antibody in the post
The preliminary purification from mammiferous serum of 7 resins 15 that are combined with recombinant protein
8 resins and proteic mode of connection IgG antibody
9 cell foreign protein 16 birds that washed away
10 antibody purifications of finally collecting, 17 Mammalss
Example 1 gene specific IgY purifying antibody
1. genetic immunization
1) correlation candidate expressing gene clone's the screening and the preparation of cDNA eukaryon expression plasmid
Any cDNA clone or expression plasmid all can be used for the present invention.This example is selected the cDNA clone of interleukin-(ILs), hemocyte somatomedin.These clones obtain from Invitrogen company, existing more than the 2500 kind of full-length cDNA eukaryon expression plasmid supply of the said firm.The cDNA clone who obtains preferably checks order again.These plasmids are to be basic framework with the pcDNA3.1/GS plasmid, and these plasmids have bacterium replicon (ori), Eukaryotic promotor and PolyA tailing signal.Promotor derives from viral genome CMV, and PolyA is from bovine growth hormone gene; Screening-gene is selected neomycin resistance gene for use.6 * His fragment is contained in the gene downstream, is used for the purposes such as affinity chromatography, analysis of recombinant protein.
2) dna immunization
Adopt the intramuscular injection immunization.Carried out plasmid amplification before injection, earlier plasmid transfection is arrived intestinal bacteria, cultivate amplification, (MidiPrep Kit, Qiagen) extracting plasmid is injected according to the dna immunization program at last to use plasmid DNA extraction agent box then.The dna immunization process is that the recombinant mammalian expressing vector DNA with total amount 50～300 μ g is dissolved in 200 μ l, 0.9% physiological saline, divides 3 multi-section positions to be expelled in the quadriceps muscle of thigh muscle of chicken, and per 2 all booster shots once.30 days begin to collect egg later on, and titre progressively raises, and can keep 1-6 month.For keeping high titre production of antibodies, booster shots in per 1 month once.
3) egg is collected and the IgY initial gross separation
Purifying IgY from egg, (Cat#.G1531) a kind of separator in separates albumen and yolk for EGGstractIgY, Promega at first to use the IgY purification system.With precipitation solution A fat a large amount of in the yolk is removed.Be further purified antibody with solution B.Obtain the IgY antibody of 75% purity with these two kinds of solution precipitations.Carry out the affinity purification of following specific antibody on this basis.
2. vivoexpression prepares recombinant protein
1) plasmid transfection
Select FreeStyle 293-F cell as the eukaryotic expression host cell.Should remove remaining phenol and sodium-chlor before the plasmid transfection, generally should adopt MidiPrep Kit (Cat#.K1910-01) to carry out purifying.Cell culture fluid original volume 30ml during transfection, density 1 * 10
6Cell/ml, promptly total cellular score is 3 * 10
7, plasmid DNA 20-40 μ g, 293fectin 30-45 μ l.The transfection program is as follows:
A), adjust cell density to 1 * 10 in cell index vegetative period
6Cell/ml, the 28ml volume;
B) collecting cell (aseptic), centrifugal 5 minutes of 1000rpm;
C) fresh pre-temperature FreeStyle293 expresses substratum, and re-suspended cell slightly vibrates 10-30 second in the 125ml culturing bottle, guarantees that cell is single suspended state;
D) dilute 30 μ g plasmid DNA, mixing with 1ml Opti-MEM; With 1ml Opti-MEM dilution 33-45 μ g 293fectin, mixing, room temperature was kept somewhere 5 minutes, and both mix, mixing, room temperature was kept somewhere 20-30 minute, allowed the DNA-293fectin complex body form;
E) the DNA-293fectin complex body is added in the Tissue Culture Flask, the negative control list adds the Opti-MEM substratum;
F) 37 ℃, 8%CO
2Cultivate 125rpm rotating and culturing bottle;
G) collecting cell after 48 hours carries out following recombinant protein purification operation.
2) protein urine and quantitative
Adopt ELISA, spot or cell direct staining method to detect the Recombinant Protein Expression situation.(Qiagen, Cat#:34530) color producing reaction is directly measured the Histidine peptide section in the recombinant protein to utilize Ni-NTA-HRP.Its program is as follows:
A) cell culture fluid or cytolysis thing 10 μ l points are dropped on the nitrocellulose filter, 37 ℃ of dryings 2 hours are washed film 2 times, 10 minutes with TBS (10mM Tris.Cl, pH7.5,150mM NaCl);
B) 3%BSA of TBS configuration soaked film 1 hour, TBS-Tween (20mM Tris.Cl, pH7.5; 500mM NaCl; 0.05% (v/v) Tween20;＜Sigma, Cat.No.P1379＞) wash film 3 times;
C) TBS-Tween 1/1000 dilution Ni-NTA-HRP soaked film 1 hour;
D) TBS-Tween washed film 3 times, with HRP staining fluid dyeing 1-5 minute;
E) with distillation washing film termination reaction, how much judge Recombinant Protein Expression according to the colour developing degree.
3. affinity column preparation
1) select the Talon resin as the chromatography column weighting material
Talon is a Clontech company product, is the sequestrant that adopts four chelating sites, and metal ion is cobalt (Co
++), have the avidity stronger than Ni-NTA, therefore have better resolving power, foreign protein still less, metal ion is not easy to be pulled down when albumen sepn simultaneously, can reuse.The recombinant protein binding capacity of resin is 6-8mg/ml.This example employing Talon recombinant protein purification test kit (Clontech, Cat#:K1253-1).
2) cytolysis
In order to allow intracellular protein fully discharge, must make lysis, generally use 1%NP40,1%Tween 20 or 0.2%Triton X-100 (20 mM Tris.Cl, pH7.5; 500 mM NaCl; 0.05% (v/v) Tween 20; 0.2% (v/v) Triton X-100＜Sigma, Cat.No.X-100＞) make cytoclasis, guarantee that again recombinant protein remains on native state simultaneously.
3) protein expression in vitro is crossed the preparation of post and affinity column
Cell lysates after centrifugal, is added on the Talon resin column, makes its gravity filtration chromatography, reaches the purpose that allows cell lysates cross post.Because recombinant protein contains 6 * His fragment, therefore can be fixed with the Co ion chelating in the resin, this chelating is quite firm, is not easy by general elutriant wash-out.The affinity column of specific antibody has just been made like this, can be used for the purifying of following specific antibody.Its step is as follows:
A) add 1ml extraction buffer balance Talon post, 700 * g washed post in centrifugal 2 minutes, repeated 2 times;
B) add above-mentioned cell pyrolysis liquid, shake gently and make the resin suspension, left standstill 2 minutes;
C) 700 * g is centrifugal 2 minutes, adds washing lotion 2ml, centrifugal 2 minutes;
D) add the TBS that contains 0.02% sodium azide, 4 ℃ of preservations are ready to use in affinity chromatography antibody and separate.
4. the chromatographic separation of gene specific antibody
1) purifying of the specific antibody of birds IgY
With the IgY crude extract 5ml of the above-mentioned affinity column adding preliminary purification for preparing, 4 ℃ of overnight incubation.Being washed till 280nm OD value with 0.05mol/L pH7.0 PBS is 0, changes 3mol/L pH6.0 potassium sulfocyanate and washes post, collects elutriant, and this component is the IgY antibody of gene specific.
2) ultrafiltration and concentration
According to elutriant what, select Centricon Plus-20 or Plus-80 (Millipore) centrifuging ultrafiltration and concentration.Plus-80 can be concentrated to 300 μ l with maximum 80ml elutriants, and CentriconPlus-80 is through handling and can reusing.
3) double immunodiffusion is measured antibody titer
Measure antibody titer and adopt the antigen-antibody double dilution, promptly antibody adopts two-fold dilution such as 1/2,1/4,1/8,1/16,1/32, carries out the double diffusion test with the recombinant protein of two-fold dilution's different concns.Occurring in antigen group or antibody group, the concentration of weak precipitation line is best antigen titre or optimum antibody titre.
The purifying of example 2. gene specific antibody IgG
1. genetic immunization
1) selection of the screening of correlation candidate expressing gene and cDNA eukaryon expression plasmid
Any cDNA clone or expression plasmid all can be used for this example.When preparing expression plasmid voluntarily, can be according to general molecular biology operation, cloned DNA.The cDNA clone who obtains preferably checks order again.When selecting plasmid, plasmid vector must be to increase on high copy ground in intestinal bacteria, then can efficiently express in zooblast, but not duplicate, and does not also contain the sequence of integrating in the oriented host cell gene group.Adoptable recombinant mammalian expressing vector has: and pcDNA3.1 (InvitroGen, USA), (InvivoGen, USA), wherein pVAC+pBOOST is exclusively used in dna immunization for pVIVO2 and pVAC+pBOOST.Selected plasmid must have 6 * His tailing part.
2) dna immunization--the subcutaneous importing of particle gun
Plasmid DNA can be imported in the animal tissues by the particle bombardment technology and to go, this is at present as the most effective the most frequently used transfer method of genetic immunization.Its base program is that elder generation is coated on golden micropartical surface with plasmid DNA, will be quickened by the gold grain that DNA is wrapping with particle gun, brings DNA into target tissue effectively.Operate by following program:
A) in the 1.5ml centrifuge tube, add 10 μ l DNA (1mg/ml), 25 μ l microparticles (about 10 μ g), 50 μ l CaCl
2(2.5mol/L) and 50 μ l spermidines (0.1mol/L), left standstill behind the mixing 10 minutes, be beneficial to DNA and be wrapped in the microparticle surface;
B) centrifugal (10000rpm) 5 minutes removes supernatant, washes one time with 70% ethanol;
C) centrifugal (10000rpm) 5 minutes removes supernatant, washes one time with dehydrated alcohol;
D) the centrifugal supernatant that goes is resuspended in precipitation in the 100ml dehydrated alcohol;
E) will be surrounded by DNA plasmid microparticle suspension and be added on the particle gun carrier with micropipet, and dry under room temperature, vacuum is preserved;
Carrier with microparticle when f) using is pasted on particle gun Kapton dish down with glycerine, is close to position to be imported, and the promotor gene rifle drives microparticle and enters epithelium, answers the injection of multiple spot particle gun.Can be used for the subcutaneous importing of DNA of Mammals and birds.
G) same intramuscular injection of time (referring to example 1).
3) serum IgG is collected and preliminary treatment
General genetic immunization began to produce antibody in 90 days, increased gradually later on.After immunity arrives 3 months, the venous blood that can take a morsel, separation of serum is done immune double diffusion test with recombinant antigen protein, if serum titer＞1: 16, get final product bloodletting.As it is not high to tire, continue escalated dose and strengthen 1～2 time, and after meeting the requirements, the carotid artery bloodletting, separation of serum, packing stores for future use.
2. vivoexpression prepares recombinant protein
1) cell is selected and the plasmid transfection cell
Host cell is selected FreeStyle 293-F cell.Should remove remaining phenol and sodium-chlor before the plasmid transfection, generally should adopt MidiPrep Kit (Cat.K1910-01) to carry out purifying.Cell culture fluid original volume 30ml during transfection, density 1 * 10
6Cell/ml, promptly total cellular score is 3 * 10
7, plasmid DNA 20-40 μ g, 293fectin30-45 μ l.The transfection program is referring to example 1.
2) protein urine and quantitative
Adopt ELISA, spot or cell direct staining method to detect the Recombinant Protein Expression situation.Utilize Ni-NTA-HRP (Qiagen Cat.34530) color producing reaction directly to measure Histidine peptide section in the recombinant protein.Its trace routine is referring to example 1.
3. affinity column preparation
1) Ni-NTA (nickel-nitrilotriacetic acid(NTA)) resin
The Ni-NTA resin is a Qiagen company product, has high affinity with 6 continuous histidine residues in the recombinant protein, good avidity is all arranged under condition natural or sex change, can from liquid that dilutes or the cell pyrolysis liquid of slightly carrying, concentrate recombinant protein by purifying.This recombinant protein is very strong with combining of Ni-NTA, only just can dissociate under the powerful competition of 100-250mM imidazoles.
2) cytolysis
Purification step is divided into lysis, combination, washes 4 steps such as post and wash-out.For cell-membrane receptor, epicyte protein, inclusion body albumen etc., general solubleness is poor, can take the strong denaturant soluble protein.In order to allow intracellular protein fully discharge, must make lysis, generally use 1%NP40,1%Tween 20 or 1%Triton X-100 to make cytoclasis, guarantee that again recombinant protein remains on native state simultaneously.
3) protein expression in vitro is crossed the preparation of post and affinity column
Cell lysates, after centrifugal, be added to the Ni-NTA resin column (Qiagen, Cat#:30622) on, its negative pressure-pumping is filtered, reach the purpose that allows cell lysates cross post.This product has the ability of parallel processing, cooperates BioRobot 3000 Workstation of the said firm, can a parallel processing 24 parts, and the protein binding capacity of each post reaches as high as 15mg, enough as the usefulness of affinity chromatography.Because recombinant protein contains 6 * His fragment, therefore can be fixed with the Ni ion chelating in the resin, this chelating is quite firm, is not easy by general elutriant wash-out.The affinity column of specific antibody has just been made like this, can be used for the purifying of following specific antibody.
4. the chromatographic separation of gene specific antibody
1) purifying of mammalian blood serum IgG specific antibody
A) saltout
Mammalian immune serum is added equivalent physiological saline mixing, the saturated ammonium sulphate of slow adding and dilute serum equivalent under induction stirring, 4 ℃ of placements are spent the night, centrifugal (3000rpm/min) 30 minutes, abandon supernatant, precipitation is used for following affinity chromatography with physiological saline 5ml dissolving.
B) affinity chromatography
The above-mentioned affinity column for preparing is added the serum composition 5ml that saltouts, 4 ℃ of overnight incubation.Being washed till 280nm OD value with 0.05mol/L pH7.0 PBS is 0, changes 3mol/L pH6.0 potassium sulfocyanate and washes post, collects elutriant, and this component is gene specific IgG antibody.
2) ultrafiltration and concentration
According to elutriant what, select Centricon Plus-20 or Plus-80 (Millipore) centrifuging ultrafiltration and concentration.Plus-80 can be concentrated to 300 μ l with maximum 80ml elutriants, through handling and can reusing.
3) double immunodiffusion is measured antibody titer
Measure antibody titer and adopt the antigen-antibody double dilution, promptly antibody adopts two-fold dilution such as 1/2,1/4,1/8,1/16,1/32, carries out the double diffusion test with the recombinant protein of two-fold dilution's different concns.Occurring in antigen group or antibody group, the concentration of weak precipitation line is best antigen titre or optimum antibody titre.
Claims (7)
1. the method for the specific antibody that produced of an affinity purification genetic immunization, this method comprises:
A) the recombinant mammalian expressing vector immune animal of specific gene and 6 * His be will have, the serum or the Yolk immunoglobulin crude extract that contain gene specific antibody produced;
B) this plasmid is transformed prokaryotic cell prokaryocyte at in-vitro transfection eukaryotic cell or preparation prokaryotic expression plasmid, after the cultivation cell pyrolysis liquid is added to the metal ion-chelant resin column, because recombinant protein has 6 * His fragment, can with the metal ion-chelant in the resin, be adsorbed on resin surface, make affinity column;
C) when carrying out the specific antibody affinity chromatography, the serum or the Yolk immunoglobulin crude extract that will contain gene specific antibody are added to affinity column, specific antibody is with after the corresponding antigen recombinant protein fully combines, wash nonspecific antibody and foreign protein earlier off, wash the antibody that post is collected gene specific at last.
2. according to the described recombinant mammalian expressing vector of claim 1, it is characterized in that these plasmids can be in vivo with external eukaryotic cell in high level expression, plasmid has specific for expressing gene and 6 * His sequence, and these plasmids comprise pcDNA3.1/GS, pVIVO2 and pVAC+pBOOST etc.
3. according to the described immune animal of claim 1, it is characterized in that immune animal is a mammal, as rabbit, sheep, horse, donkey etc., the immunity back is collected animal serum and is extracted gene specific immunoglobulin (Ig) (IgG).
4. according to the described immune animal of claim 1, it is characterized in that immune animal is birds, as chicken, duck, goose etc., egg is collected in the immunity back, extracts gene specific Yolk immunoglobulin (IgY) from yolk.
5. according to the described eukaryotic cell of claim 1, it is characterized in that these eukaryotic cells can high level expression allogenic gene, for example FreeStyle-293F, COS-7, Chinese hamster ovary celI etc.
6. according to the described prokaryotic cell prokaryocyte of claim 1, it is characterized in that these prokaryotic cell prokaryocytes can transform prokaryotic expression plasmid such as pRSET and high level expression allogenic gene, these prokaryotic cell prokaryocytes comprise e. coli bl21 (DE3) etc.
7. according to the described metal ion-chelant resin of claim 1, it is characterized in that being chelated with divalent-metal ion in these resins, comprise cobalt (Co), nickel (Ni), zinc (Zn), copper (Cu) etc., wherein Co
++Resin (Talon, Clontech) or Ni
++-NTA (nickel-nitrilotriacetic acid(NTA)) resin (Qiagen) has supply of commodities.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899110A (en) * | 2010-07-16 | 2010-12-01 | 浙江大学 | A method for isolating immunoglobulin IgY (ΔFc) from goose blood |
| CN101180317B (en) * | 2005-05-25 | 2013-06-19 | 弗·哈夫曼-拉罗切有限公司 | Methods used to purify antibodies |
| CN114235990A (en) * | 2021-11-29 | 2022-03-25 | 宁夏医科大学 | A screening method and application of tuberculosis serum markers |
-
2002
- 2002-10-18 CN CNA021375097A patent/CN1490334A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101180317B (en) * | 2005-05-25 | 2013-06-19 | 弗·哈夫曼-拉罗切有限公司 | Methods used to purify antibodies |
| CN101899110A (en) * | 2010-07-16 | 2010-12-01 | 浙江大学 | A method for isolating immunoglobulin IgY (ΔFc) from goose blood |
| CN101899110B (en) * | 2010-07-16 | 2012-08-08 | 浙江大学 | Method for separating immune globulin IgY(delta Fc) from goose blood |
| CN114235990A (en) * | 2021-11-29 | 2022-03-25 | 宁夏医科大学 | A screening method and application of tuberculosis serum markers |
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