CN101928333A - Human EIF4H polypeptide and preparation method of antibody thereof - Google Patents

Abstract

The invention discloses a middle specific human EIF4H polypeptide and a preparation method of an antibody, belonging to in vitro experiment biological products characterized by the antibody. The amino acid sequence of the middle specific human EIF4H polypeptide is: DSRDDFNSGFRDDF. An anti human EIF4H polypeptide antibody is prepared according to the following method that: (1) human EIF4H epitope is analyzed; (2) a human EIF4H middle polypeptide is synthesized; (3) the synthesized polypeptide is crosslinked with a carrier protein; (4) a rabbit anti human EIF4H polypeptide antibody is prepared; and (5) serum containing the antibody is obtained through collection and separation, and the antibody is purified, and then the anti human EIF4H polypeptide antibody can be obtained. The middle specific anti human EIF4H synthesized polypeptide antibody is high in potency, strong in affinity, good in specificity, is capable of having specific binding reaction with natural human EIF4H and is low in preparation cost; and a purified antibody can be used for immunoblot and immunohistochemistry. The antibody provides an important tool for the basic research on EIF4H protein (such as the analysis of the properties, functions, expression profiles and content of EIF4H) and the research on related diseases (such as Williams syndrome).

Description

A kind of human EIF 4 H 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) EIF4H function: EIF4H is a member of rna binding protein RRM family, and the family member comprises the RRM structural domain of 1-4 copy.RRM structural domain length is 80-100 amino acid, comprises that two are called the conserved sequence of the short stretching, extension of RNP1 and RNP2 and the hydrophobic residue of some high conservatives.EIF4H has a RRM structural domain at N-terminal, and a HHV-1Vhs binding site (J Virol.2008Jul is arranged in the middle of sequence simultaneously; 82 (13): 6600-9).This albumen has two kinds of shear-forms, express comparatively extensive, but as the short shear-form of main existence form only in liver and skeletal muscle expression.Have research to indicate, this albumen can form mixture with EIF4A with EIF4B and EIF4F, has helicase activity, has brought into play vital role (J.Biol.Chem.276:30914-30922 (2001)) in the process by the mRNA synthetic protein.

2) EIF4H antibody product information: by retrieval, Anti-EIF4H 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 EIF4H antibody:

The single allele deficiency of EIF4H gene may be the inducement of the cardiovascular and musculoskeletal abnormality of William Si Shi disease patient.The sequencing results for normal people and William Si Shi disease patient karyomit(e) 7q11.23 comparison shows that the EIF4H gene is positioned at this zone, and shows as single allele in William Si Shi disease patient.(Mamm Genome.2000Oct; 11 (10): 890-8) this just points out us, will help to understand its effect William Si Shi disease for the functional study of this target protein, for the treatment of this disease active effect will be arranged.

3. summary of the invention

The invention provides a kind of EIF4H polypeptide, its sequence is: DSRDDFNSGFRDDF.With the anti-EIF4H antibody of this polypeptide preparation can be in immunoblotting (Western blot) and immunohistochemical methods (IHC) be analyzed natural EIF4H albumen in specific recognition tissue or the cell.

Anti-EIF4H antibody obtains by following steps:

Step 1: the analysis of peptide sequence and design: utilize DNAstar software that the proteic aminoacid sequence of EIF4H 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 EIF4H protein 14 9-162 position as synthetic polypeptid acid sequence again, sequence is DSRDDFNSGFRDDF.

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 EIF4H polypeptide and carrier proteins KLH.

Step 3: polypeptide immune and Antiserum Preparation: with EIF4H-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 ProteinA purifying whole antibody, further adopt the peptide affinity purification, obtain target antibody.

Anti-EIF4H antibody is identified by following steps:

Step 1: immunoblotting: the EIF4H antibody that obtained is anti-as one, adopt the western blotting method of standard, confirm this antibody can with the natural EIF4H protein-interacting after the sex change, can be used for western blot test.

Step 2: immunohistochemical methods: the EIF4H antibody that is obtained is anti-as one, the immunohistochemical methods method of employing standard, be used to detect organization chip (organizing fetal tissue for 9 kinds), confirm this antibody can with the EIF4H protein-interacting with natural structure, can be used for immunohistochemical methods test.

4. description of drawings

Fig. 1 goes immune animal with this invention polypeptide, and the polyclonal antibody that obtains detects the EIF4H native protein in people Jurkat clone, and the band of 28kDa is the proteic signal of EIF4H among the swimming lane B.

Fig. 2 goes immune animal with this invention polypeptide, and the polyclonal antibody that obtains detects the EIF4H native protein in people's renal tissue, and the arrow indication is the proteic positive reaction signal of EIF4H.

5. embodiment

1. the analysis of peptide sequence and design

Utilize DNAstar software that the proteic aminoacid sequence of EIF4H 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 EIF4H protein 14 9-162 position as synthetic polypeptid acid sequence, and sequence is DSRDDFNSGFRDDF.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 CDSRDDFNSGFRDDF.

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: 60000 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, uses the 25ml deionized water rinsing then 3 times.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 EIF4H 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.

Anti-EIF4H 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 EIF4H antibody that is obtained is anti-as one, adopting concentration is that the antigen that 1 μ l/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 EIF4H antibody of 2-4 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.

The aminoacid sequence table

Peptide sequence designed among the present invention is as follows:

Asp?Ser?Arg?Asp?Asp?Phe?Asn?Ser?Gly?Phe?Arg?Asp?Asp?Phe

Claims (7)

1. a people ELF4H polypeptide is characterized in that amino acid sequence of polypeptide is: DSRDDFNSGFRDDF

2. the preparation method for antibody of an anti-people ELF4H polypeptide, it is characterized in that by claim 1 described sequence synthetic mesophase modified peptides, terminal modified peptide of synthetic N 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 terminal modified peptide of N 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 multiple intradermal injections, and through the above booster immunization of secondary, the tiring of serum greater than 1: 60000.

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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