CN108117592A - A kind of people ZNF294 polypeptides and its preparation method for antibody - Google Patents

Abstract

The invention discloses the preparation method of a kind of special people ZNF294 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 ZNF294 polypeptides of N-terminal is：GKNKQRTKGNLRPS.Anti-human ZNF294 polypeptide antibodies are prepared as follows：(1) people ZNF294 Characterization of antigenic epitopes；(2) people ZNF294 N-terminals Peptide systhesis；(3) synthesis polypeptide is crosslinked with carrier protein；(4) rabbit-anti people's ZNF294 polypeptide antibodies are prepared；(5) collect, the isolated serum containing antibody, antibody purification is to get to anti-human ZNF294 polypeptide antibodies.The anti-human ZNF294 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 reaction with natural human ZNF294；Manufacturing cost is low；Antibody after purification can be used for Western blotting and immunohistochemistry.The antibody is the basic research of ZNF294 albumen, for example the characteristic to ZNF294, function, express spectra and the analysis of content and its research of relevant disease provides an important instrument.

Description

A kind of people ZNF294 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) ZNF294 functions：Down syndromes (Down Syndrome), also known as 21- Trisomies (21 trisomy 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 separate 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 ZNF294 albumen is considered No. 21 additional with this Chromosome is related, therefore, the further investigation to ZNF294 albumen, the pathogenesis sick to this, clinical classification, precisely treatment and pre- It is anti-to have important impetus.(Gene.2013 Jan；10：125(2)-25).

2) ZNF294 antibody products information：Through retrieving, there is the polyclonal antibody of ZNF294 albumen in the market, but these are anti- The epitope of body is different with the epitope of my company.

3) application of ZNF294 antibody：Primary Study finds that overexpression of the ZNF294 albumen in Down syndromes is The exercising result of this additional No. 21 chromosome.This suggests that us, which is possible in the sick generation, evolution In, it plays an important role, is likely to become a potential disease with basic research and clinical value soon in the future Therefore sick marker, researches and develops polypeptide corresponding with its target protein and its specific antibody, will be to studying the albumen and its in the disease Occur, effect and clinical precisely treatment and prevention in evolution provide accurate reliable instrument and material, have important Meaning.

3. the content of the invention

The present invention provides a kind of ZNF294 polypeptides, sequence is：GKNKQRTKGNLRPS.It is prepared with this polypeptide anti- ZNF294 antibody can be in Western blotting (Western blot) analysis in specific recognition tissue or cell natural ZNF294 Albumen.

Anti- ZNF294 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 ZNF294 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 as synthesis polypeptide amino acid sequence, sequence is definite ZNF294 albumen 49-62 positions 14 amino acid GKNKQRTKGNLRPS。

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, ZNF294 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 ZNF294-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 separated, is used After Protein A purifying whole antibodies, further using peptide affinity purification, target antibody is obtained.

Anti- ZNF294 antibody through the following steps that identification：

Step 1：Western blotting：Using the ZNF295 antibody obtained as primary antibody, using the western blotting method of standard, Confirm the antibody can with the natural ZNF295 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 is carried out to the amino acid sequence of ZNF295 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 ZNF295 albumen 56-69 positions 14 Amino acid is as synthesis polypeptide amino acid sequence, sequence QSLFTNKENESQTV.Meanwhile to ensure that later stage polypeptide is crosslinked carrier Albumen and peptide affinity purification increase a cysteine C in N-terminal, and final peptide sequence to be synthesized is C- QSLFTNKENESQTV。

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 level pad (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, PH value is adjusted to 7.3, when rocked at room temperature 4 is small, after -70 DEG C of freezings, with freeze dryer freeze 24 it is small when standby use.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), intracutaneous 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, then with 25ml deionized water rinsings 3 times. Corresponding serum 10ml is taken out, is added in after being mixed with 2ml PBS in 30ml chromatographic columns, room temperature (20-25 DEG C) on impeller When mixed 1 is small, blood serum sample is allowed 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 ZNF294 polypeptides synthesized with the dissolving of 1ml coupling buffers, and add in Chromatographic column adds 1ml coupling buffers into chromatographic column, room temperature overturn mixing 1 it is small when.Layer is rinsed with 6ml coupling buffers Column is analysed, 3ml confining liquids are then added in, when room temperature mixing 1 is small.It rinses chromatographic column 3 times, 6ml IgG is then added in into chromatographic column And 3ml PBS, room temperature overturn mixing 1 it is small when.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- ZNF294 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 ZNF294 antibody obtained as primary antibody, concentration is used to be obtained for 1 μ l/ml with above-mentioned transferring film Antigen chip hybridize at room temperature 1 it is small when, then with HRP mark goat anti-rabbit antibody hybridize at room temperature 1 it is small when, use ECL development processes develop the color, and are developed the color and are exposed with X pieces in darkroom, obtain immunoblot results.

Polypeptid acid sequence table

Gly Lys Asn Lys Gln Arg Thr Lys Gly Asn Leu Arg Pro Ser。

Claims (6)

1. a kind of people ZNF294 polypeptides, it is characterised in that the amino acid sequence of polypeptide is：GKNKQRTKGNLRPS.

2. a kind of preparation method for antibody of anti-human ZNF294 polypeptides, 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 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, 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.