CN101928332A - Preparation method of Human HNRPA0 polypeptide and antibody thereof - Google Patents

Abstract

The invention discloses a preparation method of Human HNRPA0 polypeptide with a sequence of which the intermediate section has specificity and antibody thereof, belonging to a biological product used for in-vitro experiment which takes the antibody as the characteristic. The amino acid sequence of Human HNRPA0 polypeptide with N end being specific is: PKEDIYSGGGGGGS. The anti-human HNRPA0 polypeptide antibody is prepared by the method comprising the following steps: (1) human HNRPA0 epitope analysis; (2) human HNRPA0 epitope polypeptide synthesis; (3) crosslinking of synthesized polypeptide and carrier protein; (4) preparation of anti-human NRPA0 polypeptide antibody; (5) carrying out collection and separation to obtain blood serum containing the antibody, purifying the antibody to obtain the anti-human HNRPA0 polypeptide antibody. The anti-human HNRPA0 polypeptide antibody with a sequence of which the intermediate section has specificity of the invention has high titer, strong affinity and good specificity, can carry out specificity conjugation reaction with natural human HNRPA0; in addition, the preparation cost is low and the purified antibody can be applied to western blot and Immunohistochemistry. The research of the antibody constitutes the basic research of HNRPA0 protein, for instance, analyses of properties, functions, expression profiles and content of the HNRPA0 and provides an important tool for researches of related diseases.

Description

A kind of Human HNRPA 0 polypeptide and preparation method for antibody thereof

1. technical field

The present invention relates to a peptide species and preparation method for antibody thereof, this antibody is mainly used in the native protein detection of antigens.

2. background technology

1) HNRPA0 function: HNRPA0 albumen belongs to the A/B subfamily of the hnRNP of wide expression.HnRNP is a rna binding protein, and they form mixture with the nuclear heterogeneous nuclear RNA.In nucleus, these albumen and mRNA precursor are closely related, have participated in the course of processing of mRNA precursor, and mRNA metabolism and transhipment wait other aspects.Though all hnRNPs all are to find in the nucleus, wherein some albumen but can be distributed in nucleus and the tenuigenin simultaneously.HnRNP family protein member has different nucleic acid binding characteristics.The albumen of HNRPA0 genes encoding has two tumor-necrosis factor glycoproteinss that are similar to RNA binding domains RRM, and C-terminal is rich in glycine.Discover, suppress the SAPK2a/p38 mixture can block HNRPA0 by the combination of MAPKAP-K2 phosphorylation and cytokines mRNA and HNRPA0 ( EMBOJ.2002 Dec 2; 21 (23): 6505-14).

2) HNRPA0 antibody product information: by retrieval, Anti-HNRPA0 polyclonal antibody product is arranged on the market, but relevant this antibody product can not be applied in the immunohistochemical assay detection.

3) application of HNRPA0 antibody:

Utilize a proteomic techniques-air gun mass spectroscopy (shotgun mass spectrometry) analyze to find, this albumen in the prefrontal lobe cortex of schizophreniac's the back of the body outside, taken place differential expression ( Eur Arch Psychiatry Clin Neurosci.2009Apr; 259 (3): 151-63).This research is pointed out us, and HNRPA0 is as an important potential mark, and is significant to the pathogenesis of illustrating this complex disease of schizophrenia.

3. summary of the invention

The invention provides a kind of HNRPA0 polypeptide, its sequence is: PKEDIYSGGGGGGS.With the anti-HNRPA0 antibody of this polypeptide preparation can be in immunoblotting (Western blot) and immunohistochemical methods (IHC) be analyzed natural HNRPA0 albumen in specific recognition tissue or the cell.

Anti-HNRPA0 antibody obtains by following steps:

Step 1: the analysis of peptide sequence and design: utilize DNAstar software that the proteic aminoacid sequence of HNRPA0 is carried out Characterization of antigenic epitopes, main assessment wetting ability, antigenicity, the surface possibility, indexes such as flex region, in conjunction with the practical experience for preparing antibody in the past, finally determine 14 amino acid in HNRPA0 protein 17 5-188 position as synthetic polypeptid acid sequence again, sequence is PKEDIYSGGGGGGS.

Step 2: polypeptide is synthetic and crosslinked: adopt the synthetic desired polypeptides of ACT396 fully-automatic multi-channel Peptide synthesizer, and adopt mass spectrum to identify; For strengthening the antigenicity of polypeptide, adopt the Sulfo-SMCC crosslinking to carry out crosslinked HNRPA0 polypeptide and carrier proteins KLH.

Step 3: polypeptide immune and Antiserum Preparation: with HNRPA0-KLH after crosslinked and freund's adjuvant mixing and emulsifying, carry out intradermal injection immunity, and booster immunization repeatedly, survey and stop immunity when antibody titer reaches standard to getting blood examination at the new zealand rabbit back.

Step 4: antibody purification: after the experimental rabbit antibody titer reaches standard, adopt heart extracting blood, separate antiserum(antisera), behind the employing Protein A purifying whole antibody, further adopt the peptide affinity purification, obtain target antibody.

Anti-HNRPA0 antibody is identified by following steps:

Step 1: immunoblotting: the HNRPA0 antibody that obtained is anti-as one, adopt the western blotting method of standard, confirm this antibody can with the natural HNRPA0 protein-interacting after the sex change, can be used for western blot test.

Step 2: immunohistochemical methods: the HNRPA0 antibody that is obtained is anti-as one, the immunohistochemical methods method of employing standard, be used to detect organization chip (9 kinds of fetal tissues), confirm this antibody can with the HNRPA0 protein-interacting with natural structure, can be used for immunohistochemical methods test.

4. description of drawings

Fig. 1 is WB figure, and the purpose band of the immune marking is 36kDa among the figure, with the proteic theoretical molecular basically identical of HNRPA0.

Fig. 2 is IHC figure, and arrow indication position is the positive signal of antibody response among the figure.

5. embodiment

1. the analysis of peptide sequence and design

Utilize DNAstar software that the proteic aminoacid sequence of HNRPA0 is carried out Characterization of antigenic epitopes, main assessment wetting ability, antigenicity, surperficial possibility, indexes such as flex region, combination prepares the practical experience of antibody in the past again, considered amino acid complex structure degree, easily degree of oxidation, synthetic difficulty, amino acid classification and distribution etc. are finally determined 14 amino acid in HNRPA0 protein 17 5-188 position as synthetic polypeptid acid sequence, and sequence is DSRSEAEAAKNALN.Simultaneously, for guaranteeing crosslinked carrier proteins of later stage polypeptide and peptide affinity purification, increase a halfcystine C at N-terminal, final peptide sequence to be synthesized is C-PKEDIYSGGGGGGS.

2. polypeptide is synthetic and crosslinked

Adopt ACT396 fully-automatic multi-channel Peptide synthesizer,, the polypeptide after synthetic is dissolved in 50% acetonitrile, adopt mass spectrograph to identify, confirm that the polypeptide that is obtained is a desired polypeptides according to the synthetic automatically desired polypeptides of the program that weaves.Adopt Sulfo-SMCC to carry out carrier proteins KLH and synthetic polypeptide crosslinked: to get 10mg KLH and be dissolved in the 0.5ml ultrapure water as linking agent; Get 3mgsulfo-SMCC and be dissolved in the 0.5ml ultrapure water, with 3M NaOH adjust pH about 7.Under the mixing situation, sulfo-SMCC solution is dropwise slowly added in the KLH solution rotation mixing reactant 30min under the room temperature.(0.05M PB, pH6.0) cross in the Sephadex G25 post of 30min, collects light grey elutriant, i.e. activatory sulfo-SMCC/KLH solution by balance to using level pad in advance for sample on the sulfo-SMCC/KLH mixed solution that reaction is good.Treat the sulfo-SMCC/KLH complex solution of 0.2 volume to be joined in the polypeptide solution the crosslinked polypeptide of 2mg with 200ul PBS (pH7.3) dissolving, adjust pH to 7.3, room temperature jolting 4 hours ,-70 ℃ freezing after, the freeze-drying of usefulness Freeze Drying Equipment is standby after 24 hours.Detect crosslinked front and back polypeptide sulfydryl by the Ellman method and determine the polypeptide cross-linking efficiency.

3. polypeptide immune and Antiserum Preparation

Crosslinked good KLH-polypeptide 400 μ g are dissolved in the 400 μ l phosphoric acid buffers (0.01M PBS), add equal-volume Freund's complete adjuvant fully emulsified (extremely indiffusion is as the criterion in water).Adopt 3 months ages of rabbit, the healthy new zealand rabbit of body weight 1.75-2.25Kg carries out immunity, carries out the back intradermal injection immunity, will inject more than 20 at least.First immunisation is after 3 weeks, 300 μ g polypeptide are dissolved in the 300 μ l phosphoric acid buffers (0.01MPBS), carry out intradermal immunization with the Freund's incomplete adjuvant of equivalent after fully emulsified, as the booster immunization first time, require the back intradermal injection immunity, will inject more than 15 at least.After immune 3 weeks for the second time, carry out the booster immunization second time, method and requirement are with the booster immunization first time.After 1 week, adopt the auricular vein trace to get blood, with uncrosslinked synthetic polypeptide coated elisa plate, indirect elisa method detects immune serum and tires.Repeat booster immunization and titration, reach more than 1: 10000 until serum titer, adopt heart extracting blood, standard method obtains antiserum(antisera).

4. antibody affinity purification

(1), TIgG purifying: with pipettor 50% Protein-A Sepharose suspension 10ml is added in the 30ml chromatography column, removes top lid and bottom cap, the column volume after liquid flows out is 5ml, goes from water flushing 3 times with 25ml then.Take out corresponding serum 10ml, mix the back with 2ml PBS and add in the 30ml chromatography column, room temperature on the impeller (20-25 ℃) DL 1 hour allows serum sample flow out.Wash chromatography column 3 times with 15ml purifying washing lotion again, add the 10ml elutriant and carry out wash-out.

(2), peptide affinity purification: in chromatography column, add 1ml Sulfo-link gel suspension (0.5ml gel), treat dried liquid stream in the post, with 4ml coupling buffer flushing chromatography column.With 1ml coupling buffer dissolving synthetic HNRPA0 polypeptide, and add chromatography column, add the 1ml coupling buffer again to chromatography column, room temperature was put upside down mixing 1 hour.With 6ml coupling buffer flushing chromatography column, add the 3ml confining liquid then, room temperature mixing 1 hour.Flushing chromatography column 3 times adds 6ml IgG and 3ml PBS then in chromatography column, room temperature was put upside down mixing 1 hour.Wash chromatography column 3 times with PBS, use 2ml elutriant wash-out then.With the antibody purification that the is obtained 4 ℃ of dialysis in the dialysis tubing of packing into.Dialysed overnight, the centrifugal precipitation of removing of 4000rpm * 35min is collected supernatant then.Measure antibody titer and measure protein concentration with indirect elisa method with the Bradford method.

HNRPA0 antibody is identified by following steps:

1. immunoblotting assay

According to standard method preparation SDS-PAGE gel, be the cell of 5mg/ml with 5 μ l protein concentrations or organize lysate application of sample successively, about 30 minutes of constant voltage 80V, treat that sample ran when concentrating matrix and originally being straight line, change 160V voltage, stop electrophoresis when electrophoresis to tetrabromophenol sulfonphthalein indicator is run out of separation gel (about 60 minutes) fully, adopt electricity to change electric commentaries on classics of membrane method constant voltage 100V and changeed film in 80 minutes to pvdf membrane.The HNRPA0 antibody that is obtained is anti-as one, adopting concentration is that the antigen chip that 0.625 μ g/ml and above-mentioned commentaries on classics film obtain was at room temperature hybridized 1 hour, at room temperature hybridized 1 hour with the goat anti-rabbit antibody of HRP mark then, adopt the ECL development process to develop the color, in the darkroom, develop the color and expose, obtain the immunoblotting result with the X sheet.

2. immunohistochemical analysis

4% formaldehyde solution fixed tissue is cut into the tissue block of 1.5mm * 1.5mm * 2.0-3.0mm, transparent 30 minutes of ethanol gradient dehydration back dimethylbenzene, 62 ℃ of paraffin waxdips are after 2 hours, on tissue embedding machine 9 kinds of tissues are carried out embedding according to 3 * 3 arrangement mode with paraffin.Employing standard paraffin section method is attached at the thick organization chip wax disk(-sc) of 4-5 μ m that cuts on the slide glass that APES handled, 60 ℃ of roasting sheets of roasting sheet machine 1 hour, and 60 ℃ of roasting sheets 6 hours in baking box then.Adopt standard immunoassay group method, cut into slices 30 minutes with the sealing in wet box of 100 μ l, 10% sheep blood serum under the room temperature, adding 100 μ l concentration is the HNRPA0 antibody of 4-8 μ g/ml, 4 ℃ are spent the night in the wet box, PBS clean the back add the biotin labeling goat anti-rabbit igg in wet box 37 ℃ hatched 30 minutes, PBS cleans the strepto-avidin concentrated solution that the back adds the HRP mark then, hatched 30 minutes for 37 ℃ in the wet box, the back 1%DAB colour developing of developing a film, Hematorylin is redyed, multiple blue, dehydration, transparent, mounting, microscopy, check result is also taken pictures.

Sequence table

PKEDIYSGGGGGGS

Claims (7)

1. a Human HNRPA 0 polypeptide is characterized in that amino acid sequence of polypeptide is: PKEDIYSGGGGGGS

2. the preparation method for antibody of an anti-Human HNRPA 0 polypeptide, it is characterized in that by the synthetic middle part of claim 1 described sequence modified peptides, synthetic middle part modified peptides and carrier proteins is crosslinked, with crosslinked peptide immune animal, the blood of getting immune animal prepares antiserum(antisera), separation and purification IgG from serum.

3. according to claim 2 described preparation method for antibody, it is characterized in that N is terminal modified for increasing a cysteine residues at sequence N end.

4. according to claim 2 described preparation method for antibody, it is characterized in that carrier proteins is keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).

5. according to claim 2 described preparation method for antibody, it is characterized in that the middle part modified peptides is crosslinked with its sulfydryl and carrier proteins amino covalence by linking agent.

6. according to claim 2 described preparation method for antibody, it is characterized in that crosslinked peptide and immunological adjuvant mixing and emulsifying after, at rabbit back by the multiple spot subcutaneous injection, and through the above booster immunization of secondary, the tiring of serum greater than 1: 10000.

7. according to claim 2 described preparation method for antibody, it is characterized in that from antiserum(antisera), to obtain highly purified IgG through ammonium sulfate precipitation, albumin A affinity purification and peptide affinity purification.

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