CN108148124A - A kind of Human HNRPA 0 polypeptide and its preparation method for antibody - Google Patents

Abstract

The invention discloses the preparation methods of Human HNRPA 0 polypeptide and antibody special in the middle part of a kind of sequence, belong to the biological products of the experiment in vitro characterized by antibody.The amino acid sequence of the special Human HNRPA 0 polypeptide of N-terminal is：PKEDIYSGGGGGGS.Anti-human HNRPA0 polypeptide antibodies are prepared as follows：(1) Human HNRPA 0 Characterization of antigenic epitopes；(2) Human HNRPA 0 epitope polypeptide synthesizes；(3) synthesis polypeptide is crosslinked with carrier protein；(4) rabbit-anti Human HNRPA 0 polypeptide antibody is prepared；(5) it collects, the isolated serum containing antibody, antibody purification is to get to anti-human HNRPA0 polypeptide antibodies.Special anti-human HNRPA0 synthesis of polypeptide antibody potency is high in the middle part of sequence prepared by the present invention, affinity is strong, specificity is good, specific binding can occur with natural human HNRPA0 and react；Manufacturing cost is low；Antibody after purification can be used for immunoblotting.Basic research of the antibody for HNRPA0 albumen, such as the characteristic to HNRPA0, function, express spectra and the analysis of content and its research of relevant disease provide an important tool.

Description

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

1. technical field

The present invention relates to a kind of polypeptide and its preparation method for antibody, this antibody is mainly used for the inspection of native protein antigen It surveys.

2. background technology

1) HNRPA0 functions：HNRPA0 albumen belongs to the A/B subfamilies of the hnRNP of wide expression.Core Inhomogenous ribonucleoprotein is rna binding protein, they form compound with core heterogeneous nuclear RNA.In nucleus, these eggs It is closely related with mRNA precursor in vain, take part in mRNA precursor process and mRNA metabolism and transhipment etc. other aspect.Though So all hnRNPs are found in nucleus, but some of which albumen can be but distributed in simultaneously In nucleus and cytoplasm.HnRNP family protein member has different nucleic acid binding properties.HNRPA0 There are two the repetitive sequences for being similar to RNA binding structural domains RRM for the albumen of gene code, and C-terminal is rich in glycine.There is research to send out It is existing, inhibit SAPK2a/p38 compounds HNRPA0 can be blocked by MAPKAP-K2 phosphorylations and cytokines mRNA and HNRPA0 combination (EMBO J.2002Dec 2；21(23)：6505-14).

2) HNRPA0 antibody products information：Through retrieval, there are Anti-HNRPA0 polyclonal antibody products in the market, but phase It is different from antigen polypeptide sequence used in my company to close antigen polypeptide sequence used in antibody product.

3) application of HNRPA0 antibody：

It is found using a proteomic techniques-air gun mass spectrography (shotgun mass spectrometry) analysis, This albumen had occurred in prefrontal lobe cortex on the outside of the back of the body of schizophreniac differential expression (Eur Arch Psychiatry Clin Neurosci.2009Apr；259(3)：151-63).This research prompts us, and HNRPA0 is as one A important potential marker, it is significant to the pathogenesis for illustrating schizophrenia this complex disease.

3. invention content

The present invention provides a kind of HNRPA0 polypeptides, sequence is：PKEDIYSGGGGGGS.It is prepared with this polypeptide anti- HNRPA0 antibody can be in immunoblotting (Western blot) analysis in specific recognition tissue or cell natural HNRPA0 Albumen.

Anti- HNRPA0 antibody through the following steps that obtain：

Step 1：The analysis and design of peptide sequence：Using DNAstar softwares to the amino acid sequence of HNRPA0 albumen into Row Characterization of antigenic epitopes mainly assesses hydrophily, antigenicity, surface possibility, and the indexes such as flex region were prepared anti-in conjunction with the past The practical experience of body, finally determining HNRPA0 protein 17s 5-188 positions 14 amino acid is as synthesis polypeptide amino acid sequence, sequence For PKEDIYSGGGGGGS.

Step 2：Peptide systhesis and crosslinking：Desired polypeptides are synthesized using ACT396 fully-automatic multi-channels Peptide synthesizer, and It is identified using mass spectrum；To enhance the antigenicity of polypeptide, HNRPA0 polypeptides and carrier protein KLH are handed over using Sulfo-SMCC Connection method is crosslinked.

Step 3：It is prepared by polypeptide immune and antiserum：By the HNRPA0-KLH after crosslinking and Freund's adjuvant mixing and emulsifying, New zealand rabbit back carries out intradermal injection immunity, and booster immunization repeatedly, stops until blood examination is taken to survey when antibody titer reaches standard It is immune.

Step 4：Antibody purification：After experimental rabbit antibody titer reaches standard, using heart extracting blood, antiserum is detached, is used After ProteinA purifying whole antibodies, further using peptide affinity purification, target antibody is obtained.

Anti- HNRPA0 antibody through the following steps that identification：

Immunoblotting：Using the HNRPA0 antibody obtained as primary antibody, using the western blotting method of standard, confirm that this is anti- Body can with the natural HNRPA0 protein-interactings after denaturation, available for western blot test.

4. description of the drawings

Fig. 1 schemes for WB, and the purpose band of immunoblotting analysis is 36kDa in figure, basic with the theoretical molecular weight of HNRPA0 albumen Unanimously.

5. specific embodiment

1. the analysis and design of peptide sequence

Characterization of antigenic epitopes is carried out to the amino acid sequence of HNRPA0 albumen using DNAstar softwares, is mainly assessed hydrophilic Property, antigenicity, surface possibility, the indexes such as flex region prepared the practical experience of antibody in conjunction with the past, consider amino acid structure Complexity, oxidizable degree synthesize difficulty, and amino acid classification and distribution etc. finally determine HNRPA0 protein 17s 5-188 positions 14 A amino acid is as synthesis polypeptide amino acid sequence, sequence DSRSEAEAAKNALN.Meanwhile to ensure that the crosslinking of later stage polypeptide carries Body protein and peptide affinity purification increase a cysteine C in N-terminal, and final peptide sequence to be synthesized is C- PKEDIYSGGGGGGS。

2. Peptide systhesis and crosslinking

Using ACT396 fully-automatic multi-channel Peptide synthesizers, desired polypeptides are automatically synthesized according to the program woven, it will Polypeptide after synthesis is dissolved in 50% acetonitrile, is identified using mass spectrograph, and it is purpose polypeptide to confirm obtained polypeptide.Using Carrier protein KLH is crosslinked by Sulfo-SMCC as crosslinking agent with synthesis polypeptide：10mg KLH is taken to be dissolved in 0.5ml ultra-pure waters In；3mg sulfo-SMCC is taken to be dissolved in 0.5ml ultra-pure waters, with 3M NaOH tune pH value 7 or so.In mixing, Sulfo-SMCC solution is slowly added to dropwise in KLH solution, rotates mixing reactant 30min at room temperature.It will be completely reacted Sulfo-SMCC/KLH mixed liquors are loaded in advance with equilibration buffer (0.05M PB, pH6.0) equilibrated 30min's In Sephadex G25 columns, light grey eluent, that is, the sulfo-SMCC/KLH solution activated are collected.With 200ul PBS (pH7.3) 2mg cross linking polypeptides are treated in dissolving, and the sulfo-SMCC/KLH complex solutions of 0.2 volume are added in polypeptide solution, Adjusting pH value, rocked at room temperature 4 hours is spare after being lyophilized 24 hours with freeze dryer after -70 DEG C of freezings to 7.3.It is examined by Ellman methods Polypeptide sulfydryl determines polypeptide cross-linking efficiency before and after test cross connection.

3. prepared by polypeptide immune and antiserum

The 400 μ g of KLH- polypeptides being crosslinked are dissolved in 400 μ l phosphate buffers (0.01M PBS), are added in equal volume not Family name's Freund's complete adjuvant is fully emulsified (to indiffusion in water).Using 3 months rabbit ages, the health of weight 1.75-2.25Kg was new Western blue rabbit is immunized, and is carried out back intradermal injection immunity, at least to be injected 20 points or more.After first immunisation 3 weeks, by 300 μ g Polypeptide is dissolved in 300 μ l phosphate buffers (0.01M PBS), intradermal with the fully emulsified rear progress of the incomplete Freund's adjuvant of equivalent It is immune, as first time booster immunization, it is desirable that back intradermal injection immunity will at least inject 15 points or more.Second 3 weeks immune Afterwards, second of booster immunization is carried out, method and requirement are the same as first time booster immunization.After 1 week, blood is taken using auricular vein is micro, With uncrosslinked synthesis polypeptide coated elisa plate, indirect elisa method detection immune serum potency.It repeats booster immunization and potency is surveyed Fixed, until serum titer reaches more than 1: 10000, using heart extracting blood, standard method obtains antiserum.

4. antibody affinity purification

(1), TIgG is purified：50% Protein-A Sepharose suspensions 10ml is added to 30ml layers with pipettor It analyses in column, removes top lid and bottom cap, the bed volume after liquid outflow is 5ml, is then rinsed 3 times with 25ml deionized waters. Corresponding serum 10ml is taken out, is added in 30ml chromatographic columns after being mixed with 2ml PBS, room temperature (20-25 DEG C) on impeller Mixed 1 hour, allows blood serum sample to flow out.Purify washing lotion with 15ml again and wash chromatographic column 3 times, add in 10ml eluents and eluted.

(2), peptide affinity purification：1ml Sulfo-link gel suspensions (0.5ml gels) are added in chromatographic column, are treated in column Dried liquid stream rinses chromatographic column with 4ml coupling buffers.The HNRPA0 polypeptides synthesized with the dissolving of 1ml coupling buffers, and add in Chromatographic column is added in 1ml coupling buffers to chromatographic column, and room temperature overturns mixing 1 hour.Layer is rinsed with 6ml coupling buffers Column is analysed, then adds in 3ml confining liquids, room temperature mixing 1 hour.It rinses chromatographic column 3 times, 6ml IgG is then added in into chromatographic column And 3ml PBS, room temperature overturn mixing 1 hour.Chromatographic column is rinsed with PBS 3 times, then with 2ml elutions.By what is obtained Antibody purification is packed into 4 DEG C of dialysis in bag filter.Dialysed overnight, then 4000rpm × 35min centrifugations collect supernatant except precipitation.With Indirect elisa method measures antibody titer and measures protein concentration with Bradford methods.

HNRPA0 antibody through the following steps that identification：

Immunoblotting assay

PAGE gel is prepared according to standard method, by the cell or Tissue Lysis that 5 μ l protein concentrations are 5mg/ml Liquid is loaded successively, constant pressure 80V about 30 minutes, when sample ran concentration matrix sheet in straight line, changes 160V voltages, electrophoresis Electrophoresis is terminated when running out of separation gel (about 60 minutes) completely to bromophenol blue indicator, turns 80 using electric transferring film method constant pressure 100V electricity Minute transferring film is to pvdf membrane.Using the HNRPA0 antibody obtained as primary antibody, using a concentration of 0.625 μ g/ml and above-mentioned transferring film Obtained antigen chip hybridizes 1 hour at room temperature, is then hybridized at room temperature 1 hour with the HRP goat anti-rabbit antibodies marked, adopted It is developed the color with ECL development processes, is developed the color and exposed with X pieces in darkroom, obtain immunoblot results.

Sequence table

PKEDIYSGGGGGGS。

Claims (6)

1. a kind of Human HNRPA 0 polypeptide, it is characterised in that the amino acid sequence of polypeptide is：PKEDIYSGGGGGGS.

2. a kind of preparation method for antibody of anti-Human HNRPA 0 polypeptide, it is characterised in that sequent synthesis N-terminal is repaiied as described in claim 1 Peptide is adornd, the N-terminal modified peptides of synthesis and carrier protein are crosslinked, animal is immunized with crosslinked peptide, the blood of immune animal is taken to prepare and is resisted Serum isolates and purifies IgG from serum, wherein the N-terminal is modified to N-terminal one cysteine of increase in amino acid sequence Residue.

3. preparation method for antibody according to claim 2, it is characterised in that carrier protein for keyhole limpet hemocyanin (KLH) or Bovine serum albumin(BSA) (BSA).

4. preparation method for antibody according to claim 2, it is characterised in that middle part modified peptides are by crosslinking agent by its sulfydryl It is crosslinked with carrier protein amino covalence.

5. preparation method for antibody according to claim 2, it is characterised in that after crosslinking peptide and immunologic adjuvant mixing and emulsifying, Rabbit back is subcutaneously injected by multiple spot, and through secondary Yi Shang booster immunization, and the potency of serum is more than 1: 10000.

6. preparation method for antibody according to claim 2, it is characterised in that by ammonium sulfate precipitation, albumin A affinity purification And peptide affinity purification can obtain the IgG of high-purity from antiserum.