CN108101976A - A kind of N-terminal special human DNA mismatch repair protein MLH1 polypeptides and its preparation method for antibody - Google Patents
A kind of N-terminal special human DNA mismatch repair protein MLH1 polypeptides and its preparation method for antibody Download PDFInfo
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- CN108101976A CN108101976A CN201611076181.1A CN201611076181A CN108101976A CN 108101976 A CN108101976 A CN 108101976A CN 201611076181 A CN201611076181 A CN 201611076181A CN 108101976 A CN108101976 A CN 108101976A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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Abstract
The invention discloses the preparation method of a kind of special human DNA mismatch repair protein MLH1 polypeptides of N-terminal and its antibody, belong to the biological products of the in vitro test characterized by antibody.The amino acid sequence of the special human MutL protein homolog 1. polypeptide of N-terminal is：EVIQRPANAIKEMIE.Anti-human MLH1 polypeptide antibodies are prepared as follows：(1) analysis of human MutL protein homolog 1. epitope；(2) synthesis of human MutL protein homolog 1. N-terminal polypeptide；(3) synthesis polypeptide is crosslinked with carrier protein；(4) rabbit-anti human MutL protein homolog 1. polypeptide antibody is prepared；(5) collect, the isolated serum containing antibody, antibody purification is to get to anti-human MLH1 polypeptide antibodies.The anti-human MLH1 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 MLH1；Manufacturing cost is low；Antibody after purification can be entirely used for Western blotting and enzyme-linked immunosorbent assay and establish ion vitro immunization analysis method.The antibody provides more useful instrument for the biological function of research human MutL protein homolog 1. and its with the relation of relevant disease.
Description
Technical field
The present invention relates to the biological products of the in vitro test characterized by antibody, are specifically a kind of special people DNA of N-terminal
Mismatch repair protein MLH1 polypeptides and its preparation method for antibody, belong to field of biomedicine technology.
Background technology
MLH1 (MutL protein homolog 1) is that a kind of DNA mismatch repairs albumen.The reparation of mismatched dna is to maintain
The necessary link of different phase hereditary information integralities.Interchain mispairing during DNA replication dna and sliding can cause one small heavy
Multiple change, this is referred to as microsatellite instability.Existing research will be the defects of this DNA repair functions and the cause of the mankind at present
Cancer effect links together.With illustrating for the hereditary nonpolyposis carcinoma of the rectum (HNPCC) hereditary basis, the weight of mismatch repair gene
The property wanted increasingly is come apparent.MSHS2 participates in replicating the starting identification process of mismatched nucleotide in mispairing repair process, studies table
It is bright, after MSH2 is combined with the DNA dimers of mispairing, i.e., it is connected with the MLH1 and PMSH heterodimers formed, three is together
Aid in the later steps of mispairing reparation.
The content of the invention
The present invention be for solve by the expression of immunization experiment specific detection human DNA mismatch repair protein MLH1 with it is natural
In the presence of, and the problem of establish basic biological products needed for the analysis of corresponding ion vitro immunization, and provide it is a kind of can specificity be directed to people
The synthesis polypeptide of the N-terminal of MLH1, corresponding antibodies and preparation method thereof.The present invention can also solve the similar antibody used at present simultaneously
The problems such as price is high, and affinity is low, and the position of antibody binding proteins is not special and antigenic poor.It is of the present invention anti-human
MLH1 synthesis polypeptides, corresponding antibodies, prepare according to following steps：
Characterization of antigenic epitopes and antagonism location determination：Utilize a variety of Antigen Epitope Prediction softwares, such as DNAstar, OMIGA, UWGCG
Deng progress comprehensive analysis, primary concern index includes hydrophilic structure, antigenic index, surface accessibility etc., in conjunction with us
There is the verification experience for actually preparing antibody, final to determine the 23rd to the 37th target for the antibody, amino acid sequence is
EVIQRPANAIKEMIE。
The synthesis of polypeptide：Target polypeptide is synthesized using fully-automatic multi-channel Peptide synthesizer, then column chromatography purifying passes through
HPLC detects purity with the per minute 1% fine gradient of standard second.
Polypeptide is crosslinked with carrier protein：Since this synthesis polypeptide molecular weight is too small, it not can induce generation in animal body and exempt from
Epidemic disease is reacted, therefore could be immunized after polypeptide is crosslinked with carrier protein, and keyhole chirp keyhole limpet hemocyanin is selected to be crosslinked, can be significantly
Increase the size and immunogenicity of antigen, so as to prepare artificial immunogen.
Immune animal：The preparation method, Antigenic Peptide-crosslinking carrier protein are prepared as immunogene, immunizing rabbit
Antibody.Of the right age immune new zealand rabbit is chosen, carrying out multi-point injection after adaptability is raised is immunized.Booster immunization repeatedly, until
Blood is taken to survey when antibody titer reaches standard to stop being immunized.
Antibody purification：Reach the immune animal bloodletting of requirement, antiserum is collected in standard method, successively using octanoic acid-sulfuric acid
Ammonium method and affinity chromatography cross Column methods, carry out antiserum purifying, verify purity by SDS-PAGE and HPLC, obtain mesh
Labeling antibody.
High quality polypeptide antibody potency prepared by the present invention is high, affinity is strong, can occur with natural human MLH1 molecules special
Property association reaction.The more conventional recombinant antigen of cost that antibody is prepared with polypeptide prepares that antibody method is low, and antibody specificity is good, warp
It can be used for the inspection-free survey of various enzymes and WB test requirements documents after affinitive layer purification, analyzed available for ion vitro immunization is established.
Description of the drawings
Fig. 1 is liquid chromatogram identification synthesis polypeptide purity result figure.
Fig. 2 is Mass Spectrometric Identification synthesis polypeptide molecular weight results figure.
Fig. 3 is by indirect ELISA method purification Identification antibody relative compatible constant result figure.
Fig. 1 shows to carry out after purification the polypeptide of synthesis by HPLC, and liquid chromatogram identifies that purity is 98.45%.
Fig. 2 shows polypeptide molecular weight theoretical value 1741.04, Mass Spectrometric Identification measured value【M+H+】For 1742.92.
More anti-and polypeptide antigen affinity costants that in Fig. 3 prepared by ELISA detections are 109-1010L/mol。
Specific embodiment
Embodiment 1：The synthesis of human MutL protein homolog 1. Antigenic Peptide.
With Protein analysis softwares such as DNAstar, OMIGA, UWGCG, hydrophilic structure is carried out to MLH1 amino acid sequences
(hydrophilicity), antigenic index (antigenicity), surface accessibility (surface probability) and same
The analyses such as source property (homology) have the verification experience for actually preparing antibody in conjunction with us, final to determine the 23rd to the 37th
Target is in position, amino acid sequence EVIQRPANAIKEMIE.From fixing carboxyl to aminoterminal on full-automatic polypeptide synthetic instrument
Successively decrease and obtain crude product after synthesizing.After the dissolving of 30% second eyeball, carry out high pressure liquid chromatography (HPLC) and analyze, calculate main peak area simultaneously
It collects, chilled vacuum purifies synthetic peptide, Mass Spectrometric Identification after draining.
Embodiment 2：The coupling of synthesis polypeptide and carrier.
Carrier protein selection KLH (Keyhole limpet hemocyanin), with bifunctional reagent maleic amide benzene first
KLH and synthetic peptide are coupled by acid-N- succinates (MBS) connection method：Take KLH 5mg (0.11 μm of ol, containing 2.2 μ of lysine
Mol), it is dissolved in 0.75ml coupling buffers 1 (50mmol/L borate buffer solutions, pH8.5)；3mg MBS (11 μm of o) are dissolved in 75 μ l
Diformamide (DMF).MBS solution is added three times KLH solution, rotates mixing, is acted on 30 minutes at room temperature.After Quick spin,
By reaction mixture (about 0.8ml) add in advance with coupling buffer 2 (0.1mol/L phosphate, 0.15mol/LNaCL,
0.01mol/LNa2EDTA, pH7.0) balance PD-10 columns.It is eluted with coupling buffer 2, collecting eluent, (i.e. MBS-KLH is molten
Liquid).1.5mg synthesis polypeptides are dissolved in 0.15ml coupling buffers 2, add in MBS-KLH buffer solutions 0.56, rotation is mixed at room temperature
It closes, reaction mixture (about 0.7ml) overnight, is added in the PD-10 balanced in advance with phosphate buffer by 4 DEG C of postposition when effect 2 is small
Column, phosphate buffer elution, collects efflux.KLH, MBS-KLH and conjugate are subjected to SDS-KLH and conjugate carries out SDS-
PAGE (polyacrylamide gel electrophoresis) identifies coupling efficiency.
Embodiment 3：The preparation of anti-peptide antibody.
The 500 μ g of KLH- polypeptides of coupling are taken, 500 μ l phosphate buffers is dissolved in, adds isometric Split completely.It is immune
It chooses of the right age immune with new zealand rabbit (weight standard is 2-2.5 kilograms), after adaptability is raised, back intracutaneous no less than 15
Point injection.Amount of antigen halves (250 μ g) after 2 weeks, adds isometric incomplete freund adjuvant, first time booster immunization.2 weeks laggard
Second of booster immunization of row, method are the same.Start auricular vein after 2 weeks again to take a blood sample on a small quantity, be coated with synthesis polypeptide (1 μ g/ml)
ELISA Plate, indirect elisa method detection immune serum potency.Booster immunization repeatedly, until blood is taken to survey antibody titer when reaching 1: 16 ten thousand
Stop being immunized, antibody is collected using arteria carotis bloodletting.
Embodiment 4：Affinitive layer purification anti-peptide antibody.
1. the preparation of MLH1 synthesis polypeptide affinity columns：1.5g CNBr activation Sepharose 4B are weighed, use 1mmol/
After L hydrochloric acid fully embathes expanded gel, then with coupling buffer 2 times are washed, rapidly with being dissolved in the synthesis polypeptides of 5ml coupling buffers
500 μ g are mixed, when rotation mixing 2 is small at room temperature.It adds in 0.2mol/L glycine 4ml and terminates reaction, rotation mixing 1 is small at room temperature
When.Alternately gel is washed with coupling buffer and glycine buffer each 2 times, then washes gel with 50ml phosphate buffers, by end
Only concentration 0.02% plus Sodium azide (NaN3), put 4 DEG C of preservations.
2. affinitive layer purification anti-peptide antibody：By 20ml rabbit-anti polypeptide serum and the Sepharose of 1.6ml polypeptides coupling
4B adds in 50ml centrifuge tubes jointly, when 4 DEG C of rotation mixing 6 are small.Gel is added in into chromatographic column, discards efflux, with 15ml phosphoric acid
Salt buffer rinses gel.Every time plus 0.8ml pH=2.9,0.1mol/L glycine buffer elute, totally 8 times, eluent point
Not Jia Ru 8 be previously added 80 μ l 2mol/L Tris-HCL buffer solutions (pH7.5), 20ml phosphate buffers wash gel.On
All operationss process is stated to complete at 4 DEG C.Often pipe takes 2 μ l of eluent, and the OD values of 280nm are surveyed after 50 times of dilution, calculate protein
Content simultaneously draws elution curve.
Embodiment 5：Identification of the antibodies detects antibody relative compatible constant using indirect ELISA method.
BSA is crosslinked with synthetic peptide, respectively with the concentration of 5 μ g/mL and 10 μ g/mL, ELISA detection plate mistakes are coated under 4 degree
Night, 10% lowlenthal serum room temperature closing 2 it is small when after add in doubling dilution known protein concentration antibody after purification, then add
Enter goat anti-rabbit igg secondary antibody 0.1mL, the TMB chromogenic assay A of HRP marks450.Using the logarithm of antibody concentration as abscissa, with curve
Reach A during level450It is worth for 100%, is mapped using other compared with the percentage of the concentration as ordinate, A50 i.e. 50% is obtained
The corresponding antibody concentration of A values.Antibody relative compatible constant is calculated according to Kaff=(n-1)/2 (n [Ab '] t-2 [Ab] t), wherein
[Ab '] t, [Ab] t refer to rubbing for antibody during antibody semi-saturation when i.e. envelope antigen is respectively 5 μ g/mL and 10 μ g/mL during A50
That concentration, [Ag] t, [Ag '] t refer to i.e. finger 10 μ g and 5 μ g, n=[Ag] t/ [Ag '] t in coated this experiment of antigen concentration,
N=2 in this experiment.
Test effect
1.MLH1 synthesis polypeptides：HPLC identifies that purity is 98.45% after purification, using liquid chromatogram.Polypeptide molecular weight is managed
It is 1741.04 by value, Mass Spectrometric Identification measured value【M+H+】For 1742.92.
2. the coupling efficiency of polypeptide and carrier：SDS-PAGE electroresis appraisals coupling efficiency is substantially consistent with sample recovery rate,
More than 80%.
3. antibody titer：The antiserum titre that more rabbit obtain is 1: 100 more than ten thousand.
4. affinity of antibody：Relative compatible constant is 109-1010L/mol。
5. the application of antibody：The realization of more than technical indicator ensures that the antibody can be completely applied to western blot and various
Enzyme exempts to test and establishes corresponding ion vitro immunization analytical reagent.
Claims (7)
1. the special human DNA mismatch repair protein MLH1 polypeptides of a kind of N-terminal, it is characterised in that the amino acid sequence of polypeptide is：
EVIQRPANAIKEMIE。
A 2. species specific anti-human DNA mismatch repair protein MLH1N ends synthesis of polypeptide antibody, it is characterised in that the antibody titer exists
1: 100 more than ten thousand, high specificity, affinity is up to 109-1010L/mol。
3. the preparation method of a species specific anti-human DNA mismatch repair protein MLH1N ends synthesis of polypeptide antibody, it is characterised in that with
The EVIQRPANAIKEMIE peptide section sequences of the 23rd to the 37th resist as antigen peptide synthesis human MutL protein homolog 1. in human MutL protein homolog 1. amino acid sequence
Former peptide；The Antigenic Peptide of purifying is coupled with protein carrier, through column chromatography after purification；Artificial immunogen is prepared, animal is immunized, is prepared
Polypeptide antibody, and formed through affinitive layer purification.
4. preparation method according to claim 3, it is characterised in that carrier protein is keyhole chirp keyhole limpet hemocyanin.
5. preparation method according to claim 3, it is characterised in that received after the KLH- polypeptide Freund adjuvant immunities of coupling
Collect antibody.
6. preparation method according to claim 3, it is characterised in that Antigenic Peptide, as immunogene, is exempted from carrier protein couplet
Epidemic disease rabbit prepares rabbit-anti human MutL protein homolog 1. synthesis of polypeptide antibody.
7. preparation method according to claim 3, it is characterised in that synthesis polypeptide is coupled Sepharose4B, prepares affine
Chromatographic column, for purifying anti-peptide antibody.
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| CN201611076181.1A CN108101976A (en) | 2016-11-24 | 2016-11-24 | A kind of N-terminal special human DNA mismatch repair protein MLH1 polypeptides and its preparation method for antibody |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110845612A (en) * | 2019-08-09 | 2020-02-28 | 无锡傲锐东源生物科技有限公司 | Anti-mismatch repair protein MLH1 monoclonal antibody and immunodetection application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319006A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | N terminal specific human Dbf4 polypeptide and antibody preparation method |
| CN101318996A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | C terminal specific human BTRC polypeptide and antibody preparation method |
| CN101318997A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | C terminal specific human DNA mismatch repair protein MLH1 polypeptide and antibody preparation method |
| CN101319004A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | N terminal specific human YWHAE polypeptide and antibody preparation method |
| CN101319001A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | N terminal specific human DBF4B polypeptide and antibody preparation method |
-
2016
- 2016-11-24 CN CN201611076181.1A patent/CN108101976A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319006A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | N terminal specific human Dbf4 polypeptide and antibody preparation method |
| CN101318996A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | C terminal specific human BTRC polypeptide and antibody preparation method |
| CN101318997A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | C terminal specific human DNA mismatch repair protein MLH1 polypeptide and antibody preparation method |
| CN101319004A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | N terminal specific human YWHAE polypeptide and antibody preparation method |
| CN101319001A (en) * | 2007-06-06 | 2008-12-10 | 北京意宏安生物科技有限公司 | N terminal specific human DBF4B polypeptide and antibody preparation method |
Non-Patent Citations (1)
| Title |
|---|
| YACHIE N ET AL.: ""DNA mismatch repair protein Mlh1 isoform 1 [Homo sapiens]"，Accession Number：NP_000240.1", 《GENBANK》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110845612A (en) * | 2019-08-09 | 2020-02-28 | 无锡傲锐东源生物科技有限公司 | Anti-mismatch repair protein MLH1 monoclonal antibody and immunodetection application thereof |
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Application publication date: 20180601 |