CN102344490B - Anti-mouse Stk40 polyclonal antibody and preparation thereof - Google Patents
Anti-mouse Stk40 polyclonal antibody and preparation thereof Download PDFInfo
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Abstract
The invention relates to an anti-mouse Stk40 polyclonal antibody and preparation thereof, in particular to an antigen for preparing the anti-mouse Stk40 polyclonal antibody or a fusion protein thereof, a coded sequence thereof and an immune composition containing the antigen or the fusion protein thereof, a method preparing the anti-mouse Stk40 polyclonal antibody, an anti-mouse Stk40 polyclonal antibody prepared with the method and application thereof.
Description
Technical field
The application relates to polyclonal antibody and the preparation thereof of anti-mouse Stk40.
Background technology
Stk40 is an albumen with serine/threonine kinase structural domain, and it is high conservative on evolving, and especially between Mammals, has the homology (seeing the following form) surpassing more than 94%.The expression amount of Stk40 in undifferentiated mouse ES cells is very low, but all risings gradually with the differentiation of mouse ES cells and the formation of embryoid (embryoid bodies, EBs) of its RNA (Figure 1B left) and protein level (Figure 1B is right).Particularly importantly, while crossing expression Stk40 in mouse ES cells, mouse ES cells there will be the form (Fig. 1 C) of noble cells and the expression (Fig. 1 D) of extraembryonic endoderm cell sign gene.
Existing bibliographical information, Stk40 is a downstream gene that is directly subject to Oct4 negative regulation.When the mouse ES cells express Stk40 crossing and to be marked with H2B-GFP fusion rotein is expelled to 3.5 days (embryo day 3.5 of mouse, while E3.5) carrying out the chimeric embryo experiment in body in blastaea, find that these cells overwhelming majority has been distributed to embryo's ExEn region (Fig. 2).This is consistent with the cell of the Oct4 down-regulated expression location in chimeric embryo experiment, further in function, has supported the upstream and downstream regulation relationship of Oct4 and Stk40.
Stk40, as the new target gene that suppressed by Oct4, is bringing into play critical function at ES cell in extraembryonic endoderm cell differentiation procedure.By the method for multi-signal Signal Transduction Pathways inhibitor on probation, find that Stk40 causes that by activating Ras-Erk/MAPK path ES cell breaks up to ExEn direction.Further by the strategy of affinity chromatography, mass spectroscopy, found the calcium binding protein Rcn2 of an energy and Stk40 direct interaction, and studies have reported that recently Rcn2 high expression level is in ExEn cell.What is interesting is, Rcn2 cross to express and also can cause with Stk40 and crosses and express similar phenotypic differentiation in mouse ES cells, and the differentiation of the ExEn direction that causes for Stk40 of Rcn2 is essential.In addition, existing document proof Rcn2 can form protein complexes with an important scaffolding protein Ksr1 in Ras-Erk/MAPK signal path, and prompting Rcn2 likely participates in the regulation and control (Fig. 3) of Ras-Erk/MAPK signal path by activating Ksr1.
In addition, Stk40 gene is expressed in a plurality of important organs of mouse, and this gene and its homologous gene STK40 in the mankind have high conservative, implies that it may and healthyly have important contacting with Human diseases.
Anti-mouse Stk40 polyclonal antibody is provided convenience for studying function and the mechanism of Stk40 in Development Mouse Embryo and in each system (as immunity system), and the Study on Molecular Mechanism relating in the generation evolution for research human diseases provides possibility.Although there are in the market some commercial anti-Stk40 antibody, the effect of these antibody all can not reach the requirement of Western blotting application.For this reason, the invention provides a kind of new anti-mouse Stk40 antibody, this antibody can meet the requirement of Western blotting application, thereby has solved the existing problem in this area.
Summary of the invention
The application's first aspect provides a kind of isolated polypeptide, and the aminoacid sequence of this polypeptide is comprised of long 60-85 between SEQ ID NO:1 340-430 amino acids residue amino acid whose amino acid fragment.
In a specific embodiment, the amino acid of described polypeptide is comprised of the long 63-75 between SEQ ID NO:1 345-425 position amino acid whose amino acid fragment.
In a specific embodiment, the amino acid of described polypeptide is comprised of SEQ ID NO:1 349-420 amino acids.
The application's second aspect provides a kind of isolated polypeptide, and described polypeptide is by long 60-85 between SEQ ID NO:1 340-430 amino acids residue amino acid whose amino acid fragment and for promoting the polypeptide of purifying to form.
In a specific embodiment, described for promoting that the polypeptide of purifying is GST or His.
In a specific embodiment, described amino acid fragment is comprised of the long 63-75 between SEQ ID NO:1 345-425 position amino acid whose amino acid fragment.
In another specific embodiment, described amino acid fragment is comprised of SEQ ID NO:1 349-420 amino acids.
In a specific embodiment, the sequence of described polypeptide is as shown in SEQ ID NO:5.
It is a kind of for immune composition that the application provides on the other hand, and said composition contains the isolated polypeptide described in the application.
In a specific embodiment, described isolated polypeptide is comprised of long 60-85 between SEQ ID NO:1 340-430 amino acids residue amino acid whose amino acid fragment.
In another specific embodiment, described polypeptide is by long 60-85 between SEQ ID NO:1 340-430 amino acids residue amino acid whose amino acid fragment and for promoting the polypeptide of purifying to form.
In a specific embodiment, described amino acid fragment is comprised of the long 63-75 between SEQ ID NO:1 345-425 position amino acid whose amino acid fragment.
In another specific embodiment, described amino acid fragment is comprised of SEQ ID NO:1 349-420 amino acids.
In another specific embodiment, described isolated polypeptide is as shown in SEQ ID NO:5.
The application provides a kind of method of preparing the polyclonal antibody of anti-mouse Stk40 more on the one hand, it is characterized in that, described method comprises isolated polypeptide or its composition immunity Mammals of using the application.
In a specific embodiment, the aminoacid sequence of described polypeptide is as shown in SEQ ID NO:4 or 5.
In a specific embodiment, described method comprises:
(1) build the antigen presentation carrier that contains described polypeptide;
(2) use this expression vector to transform intestinal bacteria;
(3) polypeptide described in purifying;
(4) the composition immunity Mammals of using described polypeptide or containing described polypeptide; With
(5) antibody purification from described mammalian blood serum, thus make described antibody.
The application relates to the anti-mouse Stk40 polyclonal antibody that in employing claim 6-8, the method described in any one claim prepares on the other hand.
The application also relates to the application's isolated polypeptide or the purposes of its composition in the polyclonal antibody of the anti-mouse Stk40 of preparation.
The application also relates to a kind of polynucleotide sequence of separation, the isolated polypeptide described in this polynucleotide sequence coding the application.
Accompanying drawing explanation
Figure 1A shows the structural representation of Stk40; Figure 1B shows that the expression amount of Stk40 in undifferentiated mouse ES cells is very low, but all risings gradually with the differentiation of mouse ES cells and the formation of embryoid of its RNA (Figure 1B left) and protein level (Figure 1B is right); Fig. 1 C demonstration, while crossing expression Stk40 in mouse ES cells, mouse ES cells there will be the form of noble cells; Fig. 1 D shows the expression of extraembryonic endoderm cell sign gene.
Fig. 2 shows when the mouse ES cells of expressing Stk40 crossing and being marked with H2B-GFP fusion rotein is expelled to mouse and within 3.5 days, in blastaea, carries out chimeric embryo in body while testing, and finds that these cells overwhelming majority has been distributed to embryo's ExEn region.
Fig. 3 shows the mode chart of Stk40 mechanism of action.
Fig. 4 shows that WB detects the result of the anti-mouse Stk40 of the application polyclonal antibody.
Fig. 5 shows the result of the antibody (Stk40-C98) that C 98 amino acid of end (C98) of WB detection Stk40 are prepared as antigen section.
Fig. 6 shows the antigenicity index collection of illustrative plates of mouse Stk40.As can be seen from the figure, about Stk40 aminoacid sequence (SEQ ID NO:1) 349-375 amino acids residue and SEQ ID NO:1 386-424 amino acids residue place, there is stronger immunogenicity greatly.
Embodiment
In the application, term " polypeptide " and " protein " refer to the polymkeric substance of amino-acid residue, are not limited to the minimum length of product.Therefore, peptide, oligopeptides, dipolymer, polymer etc. are all included in this definition.The protein of total length and fragment thereof are included in this definition.This term is modified after also comprising the expression of polypeptide, such as glycosylation, acetylize, phosphorylation etc.In addition, for the purposes of the present invention, " polypeptide " refers to comprise the modification of native sequences, for example, lack, add and replace (character is conservative conventionally), as long as protein maintains required activity.These modifications can design by site-directed mutagenesis, can be maybe accidental, for example by producing protedogenous host, suddenly change, or the mistake causing due to pcr amplification.
The application comprises the analogue of polypeptide, and described analogue preferably includes conservative in nature replacement, i.e. these replacements occur in the class of amino acid relevant with their side chain.Particularly, amino acid is generally divided into four classes: (1) acidity---aspartic acid and L-glutamic acid; (2) alkalescence---Methionin, arginine, Histidine; (3) nonpolar---L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane; (4) uncharged polarity---glycine, l-asparagine, glutamine, halfcystine, Serine, Threonine, tyrosine.Sometimes phenylalanine, tryptophane and tyrosine are classified as to aromatic amino acid.For example, have reason to predict: with Isoleucine or α-amino-isovaleric acid, replace leucine, with L-glutamic acid, replace aspartic acid, with Serine, replace Threonine separately, or with the similar conservative amino acid of aminoacid replacement relevant in structure, such replacement will can not have material impact to biological activity.For example, interested polypeptide can comprise or not conservative aminoacid replacement conservative up to about 5-10, for example, has 1-8,1-5, a 1-3 conservative or not conservative aminoacid replacement, as long as the required function of this molecule still remains complete.Those skilled in the art can, in conjunction with Hopp/Woods well known in the art and Kyte-Doolittle graphic representation, easily measure the region that can tolerate change in interested molecule.The application is also included within and in the application's polypeptide, exists 1 or several (for example 1,2,3,4,5 or 6 or more) amino acid lack or insert.
In the application, term " fragment " only refers to the polypeptide that the part by complete full-length polypeptide sequence and structure forms.Therefore, " the amino acid whose amino acid fragment of the long 63-75 between SEQ ID NO:1 345-425 position " as herein described means a part (being " fragment " of this aminoacid sequence) for SEQ ID NO:1 345-425 amino acids sequence, and this partial-length is 63-75 amino acid.
In the application, when relating to a peptide species or polynucleotide, " separation " means that described molecule is separated and separately from find the naturally occurring whole organism of this molecule, or substantially do not have the biomacromolecule of other same type.
In the application, the similar terms such as " containing ", " comprising " comprised " by ... form ", " by ... form ".
In the application, " expression cassette ", " expression constructs " or " expression vector " refer to instruct the assembling of the expression of interested sequence or gene.This expression cassette comprises above-mentioned controlling elements, and as operability is connected in the promotor of interested sequence or gene (transcribing to instruct it), this expression cassette also usually comprises polyadenylation sequence.Construction of expression vector, make concrete encoding sequence be arranged in this and there is the carrier that is applicable to regulating sequence, the position of the encoding sequence relevant with control sequence and orientation make encoding sequence under " control " of control sequence, transcribe (that is this encoding sequence of the rna polymerase transcribe of DNA molecular in, being incorporated into control sequence).May need the sequence of coding molecules of interest to modify to realize this object.For example, may need in some cases to modify this sequence, thereby make it be connected in the control sequence that is applicable to orientation, maintain frame.Before being inserted into carrier, control sequence and other adjusting sequence may be connected in encoding sequence.Or, encoding sequence Direct Cloning can be entered to comprise in the expression vector of control sequence and suitable restriction site.
" conversion " refer in this article by exogenous polynucleotide Insertion Into Host Cell, and do not consider the method inserted: for example, and by the conversion of direct picked-up, transfection, infection etc.Below further describe concrete transfection method.This exogenous polynucleotide can be maintained the carrier of not integrating, episome for example, or can be integrated in host genome.
" host cell " is the cell being converted, or the cell that can be transformed by exogenous DNA array.
The application's first aspect provides a kind of isolated polypeptide, and the aminoacid sequence of this polypeptide is comprised of long 60-85 between SEQ ID NO:1 340-430 amino acids residue amino acid whose amino acid fragment.SEQ IDNO:1 has shown the aminoacid sequence of mouse Stk40 albumen.
This amino acid tablet segment length is 63-80 amino acid, a 63-75 amino acid, a 70-75 amino acid, a 72-75 amino acid.Preferred amino acid fragment is as shown in SEQ ID NO:4.
In addition, preferably, described isolated polypeptide is to contain the aminoacid sequence shown in SEQ ID NO:4 and the amino acid fragment of its length between the SEQ ID NO:1 340-430 amino acids residue between 72-78 amino acid.
In other preferred embodiment, described amino acid fragment preferably contains SEQ ID NO:1 349-375 amino acids residue.In other preferred embodiment, described amino acid fragment preferably contains SEQ ID NO:1 386-423 amino acids residue.
This isolated polypeptide can have 1 or several amino acid mutation, comprises 1,2,3,4,5,6 or more aminoacid insertion, lack and suddenly change, as long as these sudden changes do not change the immunogenicity of this isolated polypeptide.
The application also comprises the fusion rotein of described isolated polypeptide, wherein, described isolated polypeptide with for promoting the polypeptide of purifying to merge.The polypeptide of described promotion purifying comprises GST and His.GST is a label protein, and it can combine with gsh.So by after the application's polypeptide and its fusion, just can be by the GST on fusion rotein and the affine combination of the gsh of immobilization on carrier, the principle of recycling gsh exchange wash-out is carried out purified fusion protein.The sequence of His label protein is 6 continuous Histidines.Conventionally the expression vector of His label protein is PET-30a (this carrier is purchased from Novagen company).Should be understood that the polypeptide merging for immunogenic polypeptide with the application, such as GST and His etc., should not weaken or have influence on the immunogenicity of the application's immunogenic polypeptide.
The application also provides the application's polypeptide, comprises fusion rotein, encoding sequence, and the expression vector that contains described encoding sequence.
It is a kind of for the mammiferous composition of immunity that the application also provides, the polypeptide that said composition contains the application or fusion rotein, pharmaceutically acceptable carrier and/or adjuvant.
Term " adjuvant " refers to any material of auxiliary or regulating drug effect, includes but are not limited to immunological adjuvants, and it makes the immune response of antigen strengthen.Carrier generally includes
" pharmaceutically acceptable " refers to that carrier can give individuality with the application's polypeptide together with fusion rotein, and can in individual body, not cause undesirable biological respinse, or with composition in the interaction that is harmful to of any other composition of containing.Spendable carrier is as water, salt solution, glycerine, polyoxyethylene glycol, hyaluronic acid, ethanol etc.
In composition, the amount of polypeptide or fusion rotein is determined according to actual situation, within this ken of grasping those skilled in the art.For example, in composition the content of polypeptide or fusion rotein be 0.001-50 % by weight, 0.001-20 % by weight, 0.001-10 % by weight, 0.001-1 % by weight not etc.
The application provides a kind of method of preparing the polyclonal antibody of anti-mouse Stk40, and the method comprises polypeptide or its fusion protein immunization Mammals described in use the application.
More specifically, preparation method of the present invention comprises the step that builds the antigen presentation carrier that contains described polypeptide, and conversion, abduction delivering, purifying are to obtain the step of antigen, immune step, and the step of antibody purification.
Can adopt method well known in the art to prepare antigen of the present invention.For example, can build the expression vector of this antigen, then use this expression vector transformed host cell, make this antigen of host cell expression, and separated from this host cell culture, be purified into antigen.
Selection for expression vector there is no particular restriction, as long as it can give expression to antigen of the present invention in host cell.Conventionally, can use according to reality for promoting the polypeptide of purifying to select suitable expression vector.For example, when using GST to merge, can choice for use PGEX-4T-1 carrier.When selecting His to merge, can choice for use PET-30a carrier.For within promoting the selection of polypeptide of purifying and ken that the selection of corresponding expression vector is all grasped in this area thereof.
Host cell is generally intestinal bacteria.Preferably, according to different protein expression vectors, select different bacterial classifications, and be incubated in different substratum.In a preferred embodiment, express the PGEX-4T-1 carrier of gst fusion protein corresponding to BL21 bacterial strain and YTA substratum, and the PET-30a carrier of expressing His fusion rotein is corresponding to BL21-DE3 bacterial strain and LB substratum.
Colibacillary cultivation be there is no to particular restriction, can adopt existing cellar culture method to carry out.
Can be by the antigen immune Mammals of purifying gained.Can adopt conventional method to carry out immunity.In a specific embodiment, use antigen to inject respectively two bull rabbits, carry out at twice immunity, between twice, about interval 1-2 month, altogether use the antigen of 5mg.Immunity for the second time finishes rear tiring of rabbit Serum Antibody to be carried out to ELISA detection, is greater than 2,500,000 and can, by rabbit bloodletting, collects serum, for antibody purification.
Can adopt the method for this area routine to collect serum, antibody purification.
The application also comprises the Stk40 polyclonal antibody that adopts aforesaid method to prepare, the composition that contains this antibody, and this polyclonal antibody or its composition at research Stk40 the function in Development Mouse Embryo and in each system (as immunity system) and the purposes in mechanism.
Below describe the mode with specific embodiment in detail the present invention.Enforcement of the present invention unless otherwise indicated, will be used chemistry well known by persons skilled in the art, biological chemistry, recombinant DNA technology and immunologic ordinary method.These technology have complete explanation in the literature.Referring to, as < < basic immunology > > (Fundamental Virology), second edition, I and II volume (B.N.Fields and D.M.Knipe compile); < < experiment immunization learns to do a > > (Handbook of Experimental Immunology), I-IV volume (D.M.Weir and C.C.Blackwell compile, Blackwell Scientific Publications); T.E.Creighton, < < protein: structure and molecular characterization > > (Proteins:Structures and Molecular properties) (W.H.Freeman and Company, 1993); A.L.Lehninger, < < biological chemistry > > (Biochemistry) (Worth Publishers, Inc. latest edition); Sambrook etc., < < molecular cloning: laboratory manual > > (Molecular Cloning:a Laboratory Manual), second edition, 1989; < < Enzymology method > > (Methods in Engymology) (S.Colowick and N.Kaplan compile, Academic Press, Inc.).
Should be understood that the application is not limited to these concrete embodiments.Those skilled in the art in the situation that do not depart from the application's spirit and scope, can make suitable change to the present invention.In the situation that not expressing, the reagent using and consumption unit thereof are all this area routines.
Embodiment 1: the structure of antigen presentation carrier
Stk40-C72 antigen is that the C of coding Stk40 holds 349-420 position totally 72 amino acid, shown in its sequence SEQ ID NO:4.
Stk40-C72 antigen presentation vector construction flow process is:
1. clone's primer of restriction enzyme site is introduced in design:
C72-F(BamH?I)：5’TTG?GGA?TCC?ATG?CCG?GAC?ATT?GAT?GAC?3’(SEQ?ID?NO：2)
C72-R(Xho?I)：5’TGT?CTC?GAG?ACT?ACA?TTG?GCT?GTG?CGT?3’(SEQ?ID?NO：3)
2. with Flag-LYK4/ppyCAGIP plasmid (Lingjie Li
*, Lei Sun
*, etc., Stk40 Links the Pluripotency Factor Oct4 to the Erk/MAPK Pathway and Controls Extraembryonic Endoderm Differentiation PNAS.2010 Jan; 107 (4): 1402-1407) be template, with C72-F (BamH I) and C72-R (Xho I) primer, carry out PCR and obtain 210bp object fragment; (noting: LYK4 is Stk40)
3. with BamH I and Xho I enzyme, cut PCR product, and carry out glue recovery, with BamH I and Xho I enzyme, cut pGEX-4T-1 plasmid (purchased from Amersham Biosciences) simultaneously, and run glue and reclaim 5Kb carrier segments;
4. connect Insert Fragment and carrier segments, 4 ℃ are spent the night;
5. connection product is converted in bacillus coli DH 5 alpha;
6. choose bacterium, the little enzyme of taking out is cut evaluation, gets correct LYK4-C72/pGEX-4T-1 clone and checks order.
Embodiment 2: antigen purification
1. LYK4-C72/pGEX-4T-1 plasmid embodiment 1 being prepared is converted in BL21 competence;
2. choose bacterium to 5ml YTA (peptone 16g/L; Yeast extract 10g/L; Sodium-chlor 5g/L) medium and small shaking spent the night;
Within 3.1: 30, inoculation 2L YTA shakes 3h; 30 ℃ of 0.2mM (GST) or 1mM (His) IPTG induction 3h;
4.10000g, the centrifugal receipts of 3min bacterium, minute 4 pipe dresses, use respectively 15ml PBS (GST) resuspended, add PMSF (final concentration 1mM) at 1: 100;
5. ultrasonic, intensity: 10-12; Time: 8-10min; Interval: 2s;
6. after ultrasonic, add 1%TritonX-100, centrifugal 10000g, 10min, gets supernatant;
7. during centrifugal, process GST beads: wash 10 times of volume PBS, 500g, 5min 3 times; (storage: 80%)
By the bacterium liquid supernatant after centrifugal with process after GST beads (every ml can in conjunction with 5-10mg albumen) mix, 4 ℃, rotation is in conjunction with 2-4h;
8. washing: wash 3 times with 10 times of volume PBS, each RT rotates 5-10min, centrifugal;
9. wash-out; Elution buffer: 0.1M Tris-cl, 0.2M Nacl, 61.5ng Glutathine, 0.1%TritonX-100; 4 ℃ are rotated 30min-1h, centrifugal; Wash-out 2-3 time;
10. run glue, coomassie brilliant blue staining is quantitative.
This embodiment obtains the fusion rotein of SEQ ID NO:4 and GST, and its sequence is as shown in SEQ ID NO:5.
Embodiment 3: immune rabbit
The work of immune rabbit is to be completed by upper marine Ke Yingmu bio tech ltd.Roughly flow process is as follows: the gst fusion protein antigen that embodiment 2 is prepared is injected respectively two bull rabbits, carries out at twice immunity, between twice about interval 1-2 month, and the gst fusion protein antigen altogether preparing with the embodiment 2 of 5mg.Immunity for the second time finishes rear tiring of rabbit Serum Antibody to be carried out to ELISA detection, is greater than 2,500,000 and can, by rabbit bloodletting, collects serum, for antibody purification.
Embodiment 4: antibody purification
1) get the rabbit anteserum of the rabbit of embodiment 3 immunity: 3 times (approximately 2 months) of the gst fusion protein that this rabbit prepares through embodiment 2 (being dissolved in PBS) immunity, get blood (70-90ml/ is only), 4 ℃ are spent the night and ooze out serum;
2) for the antigen of antibody purification, cross desalting column PD-10 (GE Healthcare Life Sciences), change elution buffer into NaPi damping fluid (100mM NaH
2pO
4/ Na
2hPO
4(pH 8.2); 500mM NaCl; 0.2mM EDTA; 0.1%NP-40; 0.5mM PMSF);
3) 15000g, 10min is centrifugal, gets serum, the every pipe of packing 9-10ml ,-80 ℃ save backup;
4) with CNBr-activated sepharose 4B beads (poisonous) (GE Healthcare Life Sciences) and antigen (gst fusion proteins of embodiment 2 preparations) combination, carry out antibody purification.Beads first will be activated.Rule of thumb, 0.6g beads can purifying 9-10ml serum.Claim, in the centrifuge tube of the 1mM HCl that beads to 1 of 0.6g is equipped with precooling, to mix, until dissolve completely;
5) prepare a large suction pump, a large Erlenmeyer flask that has suction pump interface, an enamel funnel (inside having hole), two 3M filter paper that match with funnel.50ml is poured in funnel containing beads liquid, adds the 1mM HCl of 450ml precooling, slowly suction simultaneously, notes can not allowing beads become dry in aspiration procedure.About 15 minutes of whole aspiration procedure;
6) with 500ml NaPi damping fluid, aspirate again once (continuously pumping completed about 40 seconds).During surplus 5ml left and right bf (attention can not be drained), pull up enamel funnel, with 5ml rifle, beads is collected, then add 5ml NaPi damping fluid and collect remaining beads.5th, 6 steps completed as early as possible in 2 minutes.
7) by 50ml centrifuge tube at room temperature rotation 1-3 hour (beads and antigen in conjunction with), also can 4 ℃ spend the night.In conjunction with rear centrifugal 500g, 4 ℃, 5 minutes, then survey the OD280 of supernatant, relatively, in conjunction with the value of reading of front and back, determine joint efficiency.(method of calculation: Tot Prot=OD * extension rate * albumen volume).The about 2ml of precipitation volume of 0.6g beads, mixes bead with the NaPi damping fluid of 5 times of precipitation volumes (10ml), then abandons supernatant after centrifugal.
8) in precipitation, add 100mM Tris-HCl PH8.0,10 times of precipitation volumes (20ml), mix, and room temperature is rotated and spent the night for 2 hours or 4 ℃, for sealing the upper unnecessary protein binding site of beads;
9) by 20ml, containing the liquid of beads, divide (10ml syringe in two parts of two glass wool posts pouring prior preparation into, the about 1ml of glass wool), stopper is standing 3 minutes beyond the Great Wall, make beads precipitation, then take away stopper and make liquid freely drip (with 500ml plastics bottle graft, porous plastics is put in centre).
10) with following 5 kinds of damping fluids, wash beads, every kind of volume is 10 times of beads precipitation volumes, about 20ml:
A) 100mM Tris-HCl, PH 8.0, contain 0.5M NaCl
B) 10mM Tris-HCl, PH 8.0, contain 0.5M NaCl
C) 200mM glycine, PH 2.5, contain 0.5M NaCl
d)0.5M?NaCl
E) 200mM triethylamine, PH11.5, contains 0.5M NaCl
f)0.5M?NaCl
11) with the about 20ml of NaPi damping fluid of 10 times of precipitation volumes, rinse again beads one time.
12) in every 9ml serum, add 1ml 1M NaPi, PH8.2, mixes.56 ℃, 30min deactivation complement, centrifugal 12500g, 10min is centrifugal, gets supernatant and is added in pillar and filters, and the serum leaching returns in pillar to be filtered 2-3 time again.
13) use 10mM Na
2hPO
4/ NaH
2pO
4, PH8.2, and the about 100ml of 0.5M NaCl drips and washes beads, damping fluid splashes in pillar through 50ml syringe and stilligout;
14) prepare two 15ml centrifuge tubes, wherein respectively add 1ml 1M Na
2hPO
4/ NaH2PO
4, PH8.2 adds 5ml 100mM glycine in pillar, and PH2.5, and 0.5M NaCl collect and contain 1M NaPibuffer, in the 15ml centrifuge tube of PH8.2, mix;
15) repeat the 13rd step;
16) prepare two 15ml centrifuge tubes, wherein add 1ml 1M Na
2hPO
4/ NaH2PO
4, PH8.2 adds 5ml 100mM triethylamine in pillar, PH11.5, and with 0.5M NaCl, collects and contains 1M NaPi damping fluid, in the 15ml centrifuge tube of PH8.2, mixes.The antibody (about 12ml) that merges twice filtration;
17) Protein A pillar is changed the joint (only) that connects syringe, and the in-built 10mlPBS of syringe takes out the stopper of pillar, with PBS, slowly rinses (1ml/min), and liquid will ooze, and can not throw bubble into.(the same antibody of the reusable purifying of Protein A pillar, is stored in 20% ethanol);
18) 12ml antibody is crossed to post, can cross twice.With corresponding bf zeroing, measure the OD280 that spills liquid, to check joint efficiency, guarantee that the effluent liquid value of reading is 0;
19) with 10ml PBS, wash again pillar one time;
20) with 3ml 0.1M citric acid PH3.6 wash-out antibody, elutriant divides 6 ep pipes (500ul/ pipe) to collect, and adds in advance 50ul 1M Tris-HCl PH 9.0 in every pipe, mixes.With corresponding bf zeroing, measure the OD280 of each pipe, determine concentration.Get the 2-6 pipe antibody that concentration is higher, 2.5ml merging mixes altogether;
21) cross desalting column PD-10, change elution buffer into PBS (2.5ml enters, and 3.5ml goes out).Measure final OD280 and the total amount of antibody.The every pipe of packing 500 ,-80 ℃ of preservations.Antibody concentration is that 1ug/ul is comparatively suitable, if antibody concentration is too low, needs to add BSA and prevents degraded, needs to add NP-40 to increase solubleness if too high; Antibody (IgG) total amount=0.75*OD280* volume;
22) following WB (Western Blotting) detects antibody:
(a) collect 9 days formed embryoid (EB) samples of mouse embryo stem cell differentiation of wild-type (WT) and Stk40 disappearance (KO), with 1 * PBS of precooling, wash twice, add the Co-IP lysis buffer (50mM Tris-Cl, pH 7.5,150mM NaCl, 2mM EDTA, 10% (v/v) glycerol, 0.5%NP-40,1mM NaF, 1mM PMSF) of proper volume to carry out cracking.
(b) 4 ℃ vibration 15 minutes, 12,000rpm, 4 ℃ centrifugal 10 minutes, get supernatant.
(c) BCA gets 20 μ g total protein concentrations after quantitatively, adds 1/3 volume 4 * SDS sample-loading buffer (200mM Tris-Cl, pH 7.4,8%SDS, 20% beta-mercaptoethanol, 40% glycerine, 0.04% tetrabromophenol sulfonphthalein), boiling water boiling 5 minutes.
(d) SDS-PAGE electrophoretic separation.Electrophoretic buffer formula is: Tris 0.25M, glycine 1.92M, SDS 1%.
(e) the albumen electricity consumption transfer method in gel is transferred to (200mA, 4 hours) on nitrocellulose filter.Transferring film liquid formula is 0.025M Tris, 0.192M Glycine and 20% methyl alcohol.
(f) with TBS-T damping fluid (0.02M Tris-Cl, pH7.6,0.137M NaCl, 0.1%Tween-20) the room temperature sealing that contains 5% skim-milk or 5%BSA, after 1 hour, discard confining liquid.
(g) add a certain amount of primary antibodie (Stk40-C72 and Stk40-C98 polyclonal antibody) being suitably diluted in containing the anti-mouse Stk40 albumen in the TBS-T damping fluid of 5% skim-milk, 4 ℃ are spent the night.(h) discard primary antibodie, TBS-T buffer solution for cleaning 3 times, each 5 minutes.
(i) two anti-(Ab2 that show in Fig. 4) of the horseradish peroxidase of a certain amount of anti-rabbit are diluted in and are contained in the TBS-T damping fluid of 5% skim-milk, incubated at room 1～2 hour; Discard two and resist, TBS-T buffer solution for cleaning 3 times, each 5 minutes.
(j) on film, drip Pierce chemical luminous substrate, then with X-ray exposure, through the automatic developing fixing of sheet-punching machine.
Fig. 4 has shown that the mouse embryo stem cell that utilizes Stk40-C72 polyclonal antibody to carry out Western blotting detection wild-type (WT) and Stk40 disappearance (KO) breaks up the protein expression situation of 9 days middle Stk40 of formed embryoid (EB).Result is presented at the obvious expression (band at～50kDa place) that Stk40 can be detected in wild-type EB sample, in the EB sample of Stk40 disappearance, can't detect corresponding band, illustrates that this Anti-TNF-α physical efficiency identifies mouse Stk40 albumen specifically.
Embodiment 5: antibody activity detects
By embodiment 4 steps (22), carry out WB detection, only use Stk40-C98 antibody (Lingjie Li
*, Lei Sun
*, etc., Stk40 Links the Pluripotency Factor Oct4 to the Erk/MAPKPathway and Controls Extraembryonic Endoderm Differentiation PNAS.2010 Jan; 107 (4): the Stk40-C72 polyclonal antibody of 1402-1407) replacing the application detects.
Result is presented in Fig. 5.Fig. 5 shows, the antibody (Stk40-C98) that C 98 amino acid of end (C98) of the Stk40 adopting are prepared as antigen section is although can identify Stk40 albumen, but non-specific band (assorted band) is more, and some is assorted, and to take away object band very near, the in the situation that only when running glue, albumen being fully separated, just can see.
Although the mode with specific embodiment has been described the application, it should be understood that, in the situation that do not depart from the application's spirit, can the application is made to suitable change and modification.Within these protection domains that all limit in the application's claim.
Claims (6)
1. an isolated polypeptide, its sequence is as shown in SEQ ID NO:4.
2. an isolated polypeptide, its sequence is as shown in SEQ ID NO:5.
3. for an immune composition, it is characterized in that, said composition contains the isolated polypeptide described in any one in claim 1-2.
4. a method of preparing the polyclonal antibody of anti-mouse Stk40, is characterized in that, described method comprises that right to use requires the composition immunity Mammals described in any one in the isolated polypeptide described in any one in 1-2 or claim 3.
5. method as claimed in claim 4, is characterized in that, described method comprises:
(1) build the antigen presentation carrier that contains described polypeptide;
(2) use this expression vector to transform intestinal bacteria;
(3) polypeptide described in purifying;
(4) the composition immunity Mammals of using described polypeptide or containing described polypeptide; With
(5) antibody purification from described mammalian blood serum, thus make described antibody.
6. a separated polynucleotide sequence, is characterized in that, the polypeptide in this polynucleotide sequence coding claim 1-2 described in any one.
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| CN101501187A (en) * | 2006-06-06 | 2009-08-05 | 田纳西大学研究基金会 | Compositions enriched in neoplastic stem cells and methods comprising same |
| CN101657535A (en) * | 2006-08-01 | 2010-02-24 | 爱丁堡大学管理处 | Come from the multipotential cell of rat and other animal |
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| CN101501187A (en) * | 2006-06-06 | 2009-08-05 | 田纳西大学研究基金会 | Compositions enriched in neoplastic stem cells and methods comprising same |
| CN101657535A (en) * | 2006-08-01 | 2010-02-24 | 爱丁堡大学管理处 | Come from the multipotential cell of rat and other animal |
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