CN108148126A

CN108148126A - A kind of people C21ORF13 polypeptides and its preparation method for antibody

Info

Publication number: CN108148126A
Application number: CN201611143852.1A
Authority: CN
Inventors: 江虹
Original assignee: Tianjin Aoweiya Biotechnology Co Ltd
Current assignee: Tianjin Aoweiya Biotechnology Co Ltd
Priority date: 2016-12-05
Filing date: 2016-12-05
Publication date: 2018-06-12

Abstract

The invention discloses the preparation methods of a kind of special people C21ORF13 polypeptides of N-terminal and antibody, belong to the biological products of the experiment in vitro characterized by antibody.The amino acid sequence of the special people's C21ORF13 polypeptides of N-terminal is：KRSPGTGDFSRNSN.Anti-human C21ORF13 polypeptide antibodies are prepared as follows：(1) people C21ORF13 Characterization of antigenic epitopes；(2) people C21ORF13 N-terminals Peptide systhesis；(3) synthesis polypeptide is crosslinked with carrier protein；(4) rabbit-anti people's C21ORF13 polypeptide antibodies are prepared；(5) it collects, the isolated serum containing antibody, antibody purification is to get to anti-human C21ORF13 polypeptide antibodies.The anti-human C21ORF13 synthesis of polypeptide antibody potency of N-terminal prepared by the present invention specifically is high, affinity is strong, specificity is good, can occur to specifically bind with natural human C21ORF13 and react；Manufacturing cost is low；Antibody after purification can be used for immunoblotting and immunohistochemistry.Basic research of the antibody for C21ORF13 albumen, such as the characteristic to C21ORF13, function, express spectra and the analysis of content and its research of relevant disease provide an important tool.

Description

A kind of people C21ORF13 polypeptides 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) C21ORF13 functions：Down syndromes (Down Syndrome), also known as 21- Trisomies (21trisomy Syndrome) or mongolism, it is a kind of most common clinical hereditary metabolic disorders, the incidence in life birth baby is 0.12-0.16%.Cytogenetic Features are in three signs for No. 21 chromosome, are occurred mainly since reproduction cell is subtracting With the period of the day from 11 p.m. to 1 a.m or fertilized eggs, No. 21 chromosomes generations in mitosis do not detach for speed division formation, and embryoid body is made to exist into the cell One No. 21 additional chromosome.Primary Study finds that the overexpression of C21ORF13 albumen is considered additional with this 21 Number chromosome is related, and therefore, the further investigation to C21ORF13 albumen, to the pathogenesis of the disease, clinical classification is precisely treated And prevention has important impetus.(PLoS One.2016Apr21；11(4)).

2) C21ORF13 antibody products information：Through retrieval, there is the polyclonal antibody of C21ORF13 albumen in the market, but this The epitope of a little antibody is different with the epitope of my company.

3) application of C21ORF13 antibody：Primary Study discovery, excessive table of the C21ORF13 albumen in Down syndromes Up to the exercising result for being this additional No. 21 chromosome.This suggests that us, which is possible to the generation in the disease, develops Cheng Zhong plays an important role, be likely to become soon in the future one it is potential with basic research and clinical value Therefore disease marker, researches and develops polypeptide corresponding with its target protein and its specific antibody, will be to studying the albumen and its at this Disease occurs, the effect in evolution, and epigenetics and clinic precisely treatment and prevention provide accurate reliably tool and material Material, is of great significance.

3. invention content

The present invention provides a kind of C21ORF13 polypeptides, sequence is：KRSPGTGDFSRNSN.It is prepared with this polypeptide anti- C21ORF13 antibody can be natural in specific recognition tissue or cell in immunoblotting (Western blot) analysis C21ORF13 albumen.

Anti- C21ORF13 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 C21ORF13 albumen Progress Characterization of antigenic epitopes, mainly assesses hydrophily, antigenicity, surface possibility, and the indexes such as flex region were prepared in conjunction with the past The practical experience of antibody, finally determining C21ORF13-43 14 amino acid of protein 29 are as synthesis polypeptide amino acid sequence, sequence It is classified as KRSPGTGDFSRNSN.

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, KRSPGTGDFSRNSN polypeptides and carrier protein KLH are used Sulfo-SMCC cross-linking methods are crosslinked.

Step 3：It is prepared by polypeptide immune and antiserum：By the C21ORF13-KLH after crosslinking and Freund's adjuvant mixing and emulsifying, Intradermal injection immunity, and booster immunization repeatedly are carried out at new zealand rabbit back, is stopped until blood examination is taken to survey when antibody titer reaches standard Only it is immunized.

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

Anti- C21ORF13 antibody through the following steps that identification：

Step 1：Immunoblotting：Using the C21ORF13 antibody obtained as primary antibody, using the immunoblotting side of standard Method, confirm the antibody can with the natural C21ORF13 protein-interactings after denaturation, available for western blot test.

4. description of the drawings

Fig. 1 is western blot figure (Western blot)

5. specific embodiment

1. the analysis and design of peptide sequence

Characterization of antigenic epitopes, main assessment parent are carried out to the amino acid sequence of C21ORF13 albumen using DNAstar softwares Aqueous, antigenicity, surface possibility, the indexes such as flex region prepared the practical experience of antibody in conjunction with the past, consider amino acid knot Structure complexity, oxidizable degree synthesize difficulty, and amino acid classification and distribution etc. finally determine C21ORF13 protein 29s -43 14 amino acid are as synthesis polypeptide amino acid sequence, sequence KRSPGTGDFSRNSN.Meanwhile to ensure the crosslinking of later stage polypeptide Carrier protein and peptide affinity purification increase a cysteine C in N-terminal, and final peptide sequence to be synthesized is C- KRSPGTGDFSRNSN。

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 3MNaOH 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: 60000, 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 C21ORF13 polypeptides synthesized with the dissolving of 1ml coupling buffers, and add Enter chromatographic column, add in 1ml coupling buffers to chromatographic column, room temperature overturns mixing 1 hour.It is rinsed with 6ml coupling buffers Then chromatographic column adds in 3ml confining liquids, room temperature mixing 1 hour.It rinses chromatographic column 3 times, 6ml [gG 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.

Anti- C21ORF13 antibody through the following steps that identification：

1. 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 C21ORF13 antibody obtained as primary antibody, the antigen core obtained using a concentration of 1 μ l/ml and above-mentioned transferring film Piece hybridizes 1 hour at room temperature, is then hybridized at room temperature 1 hour with the HRP goat anti-rabbit antibodies marked, using ECL development processes It develops the color, is developed the color and exposed with X pieces in darkroom, obtain immunoblot results.

Polypeptid acid sequence table

Lys Arg Ser Pro Gly Thr Gly Asp Phe Ser Arg Asn Ser Asn.

Claims

1. a kind of people C21ORF13 polypeptides, it is characterised in that the amino acid sequence of polypeptide is：KRSPGTGDFSRNSN.

A kind of 2. preparation method for antibody of anti-human C21ORF13 polypeptides, it is characterised in that the sequent synthesis N-terminal as described in claim 1 The N-terminal modified peptides of synthesis and carrier protein are crosslinked, animal are immunized with crosslinked peptide, the blood of immune animal is taken to prepare by modified peptides Antiserum isolates and purifies IgG from serum, wherein the N-terminal is modified to the N-terminal one and half Guang ammonia of increase in amino acid sequence Sour 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 N-terminal modified peptides by crosslinking agent by its sulfydryl with Carrier protein amino covalence is crosslinked.

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.