CN101319002A - N terminal specific human MDC1 polypeptide and antibody preparation method - Google Patents

Abstract

The invention discloses N-terminal specific MDC1 polypeptide, as well as an antibody and a preparation method thereof, belonging to in vitro experiment biological products characterized by the antibody. The amino acid sequence of the N-terminal specific MDC1 polypeptide is TQAIDWDVEEEEETE. Anti MDC1 polypeptide antibody is prepared according to the following method that: (1) MDC1 epitope is analyzed; (2) MDC1 N-terminal polypeptide is synthesized; (3) synthesized polypeptide is crosslinked with carrier protein; (4) rabbit anti MDC1 polypeptide antibody is prepared; (5) serum containing the antibody is obtained through collection and separation, and the antibody is purified, and then the anti MDC1 polypeptide antibody can be obtained. The N-terminal specific anti MDC1 synthesized polypeptide antibody high in potency, strong in affinity, good in specificity, capable of having specific binding reaction with natural MDC1 and low in preparation cost; purified antibody can be completely used for immunoblot and enzyme linked immunosorbent assay, as well as the establishment of in vitro immunization analytical method. The antibody provides a useful tool for the research on the biological effects of MDC1 in DNA damage response.

Description

A kind of people MDC1 polypeptide and preparation method for antibody thereof of N terminal specific

Technical field

The present invention relates to antibody is the biological products that the in vitro tests of feature is used, and specifically is a kind of MDC1 polypeptide and preparation method for antibody thereof of N terminal specific, belongs to field of biomedicine technology.

Background technology

The checkpoint mediator MDC1 of DNA plerosis (mediator of DNA damagecheckpoint 1) can participate in the early response of dna damage.It is considered to one of nucleoprotein BRCT (BRCA1C end) superfamily member.It comprises that the N end is with two group (FHA), two C-terminal BRCT groups and 13 repeated fragments in inside that contain 41 aminoacid sequences of being correlated with the arrow of three preceding finger tip heads sometimes.When cultured cells was subjected to the stimulation of dna damage agent, MDC1 promptly transferred to the dna damage position.Changing over to of siRNA (siRNA) will make the MDC1 expression level descend, and the result causes cell extremely sensitive to the dna damage agent, cause that simultaneously the cell cycle check point is activated and the defective of apoptosis.Therefore, MDC1 is a key ingredient of DNA in mammalian cells injury response.

Summary of the invention

The present invention is for solving expression and the natural existence by immunization experiment specific detection MDC1, and set up the problem of the required basic biological products of corresponding external immunoassay, and provide a kind of can specificity at the synthetic polypeptide of the N end of MDC1, corresponding antibodies and preparation method thereof.The present invention also can solve the similar antibody price height of present use simultaneously, and avidity is low, problem such as the not special and antigenicity in the position of antibody binding proteins is relatively poor.Anti-MDC1 of the present invention synthesizes polypeptide, corresponding antibodies, prepares according to following steps:

Characterization of antigenic epitopes and antagonism position are determined: utilize multiple epi-position forecasting software, as DNAstar, OMIGA, UWGCG etc. carry out analysis-by-synthesis, mainly consider index and comprise hydrophilic structure, antigenic index, surperficial accessibility etc., in conjunction with the checking experience of our existing actual fabrication antibody, finally determining the 4th to the 18th is the target of this antibody again, and its aminoacid sequence is TQAIDWDVEEEEETE.

Polypeptide synthetic: the target polypeptide adopts the fully-automatic multi-channel Peptide synthesizer synthetic, and column chromatography purification then by HPLC, detects purity with the fine gradient of standard second of per minute 1%.

Polypeptide and carrier proteins are crosslinked: because this synthetic polypeptide molecular weight is too little, can not induce the generation immune response in animal body, therefore polypeptide and carrier proteins just can be carried out immunity after crosslinked, select key hole chirp keyhole limpet hemocyanin to carry out crosslinked, can significantly increase antigenic size and immunogenicity, thereby prepare artificial immunogen.

Immune animal: described preparation method, its antigen peptide-crosslinked carrier proteins is as immunogen, and immunizing rabbit prepares antibody.Choose of the right age immunity new zealand rabbit, carry out the multi-point injection immunity after raising through adaptability.Booster immunization stops immunity to getting when blood survey antibody titer reaches standard repeatedly.

Antibody purification: meet the requirements of the immune animal bloodletting, antiserum(antisera) is collected in standard method, uses sad-ammonium sulfate method and affinity chromatography to cross the column purification method successively, carries out the antiserum(antisera) purifying, by SDS-PAGE and HPLC checking purity, obtains target antibody.

Tire height, avidity of the high quality polypeptide antibody of the present invention preparation is strong, can with natural MDC1 molecule generation specificity association reaction.It is low that the conventional recombinant antigen of cost for preparing antibody with polypeptide prepares antibody method, and antibodies specific is good, can be used for inspection-free survey of various enzymes and WB test requirements document behind affinitive layer purification, can be used for setting up external immunoassay.The preparation that should synthesize peptide antibody, for the research of MDC1 molecular studies and relative disease thereof (as the formulation of diagnosis policy, epidemiology survey, prevention and control) an important instrument is provided, and in biomedical research, aspects such as the analysis of the proteic characteristic of respective target, assay, distribution situation and proteic conclusive evidence are all had vital role.

Description of drawings

Fig. 1 is that liquid chromatography is identified synthetic polypeptide purity figure as a result.

Fig. 2 is that mass spectrum is identified synthetic polypeptide molecular weight figure as a result.

Fig. 3 is by indirect ELISA method purification Identification antibody relative compatible constant figure as a result.

After Fig. 1 showed that process HPLC carries out purifying to the synthetic polypeptide, liquid chromatography identified that purity is 82.4%.

Fig. 2 shows that the polypeptide molecular weight theoretical value is 1925, and mass spectrum is identified measured value [M+H ⁺] be 1926.1.

The how anti-and polypeptide antigen affinity costant of ELISA detection preparation is 10 among Fig. 3 ⁵-10 ⁶L/mol.

Embodiment

Synthesizing of embodiment 1:MDC1 antigen peptide.

Use DNAstar, OMIGA, analysis of protein softwares such as UWGCG, the MDC1 aminoacid sequence is carried out hydrophilic structure (hydrophilicity), antigenic index (antigenicity), surperficial accessibility (surface probability) and homology analyses such as (homology), have the checking experience of actual fabrication antibody again in conjunction with us, final definite the 4th to the 18th is target, and its aminoacid sequence is TQAI DWDVEEEEETE.On full-automatic polypeptide synthetic instrument, obtain crude product to the aminoterminal synthetic back of successively decreasing by fixing carboxyl.After the dissolving of 30% second eyeball, carry out high pressure liquid chromatography (HPLC) analysis, calculate main peak area and collection, the synthetic peptide of purifying after freezing vacuum is drained, mass spectrum is identified.

Embodiment 2: the coupling of synthetic polypeptide and carrier.

Carrier proteins is selected KLH (Keyhole limpet hemocyanin), with bifunctional reagent maleic acid amides phenylformic acid-N-succinate (MBS) connection method KLH and synthetic peptide are carried out coupling: get KLH 5mg (0.11 μ mol, contain Methionin 2.2 μ mol), be dissolved in 0.75mL coupling buffer 1 (the 50mmol/L borate buffer solution, pH8.5); 3mg MBS (11 μ mo) is dissolved in 75 μ L diformamides (DMF).Divide MBS solution 3 times and add KLH solution, the rotation mixing, effect is 30 minutes under the room temperature.After centrifugal rapidly, coupling buffer 2 (0.1mol/L phosphoric acid salt, 0.15mol/LNaCL, 0.01mol/LNa are used in reaction mixture (about 0.8mL) adding in advance ₂EDTA, pH7.0) equilibrated PD-10 post.With coupling buffer 2 wash-outs, collect elutriant (being MBS-KLH solution).The synthetic polypeptide of dissolving 1.5mg adds MBS-KLH damping fluid 0.56 in 0.15mL coupling buffer 2, and rotation mixes under the room temperature, acting on 2 hours postposition spends the night for 4 ℃, phosphoric acid buffer equilibrated PD-10 post is used in reaction mixture (about 0.7mL) adding in advance, and the phosphoric acid buffer wash-out is collected effluent liquid.KLH, MBS-KLH and conjugate are carried out SDS-KLH and conjugate to carry out SDS-PAGE (polyacrylamide gel electrophoresis) and identifies coupling efficiency.

Embodiment 3: the preparation of anti-peptide antibody.

Get link coupled KLH-polypeptide 500 μ g, be dissolved in 500 μ L phosphoric acid buffers, add the complete freund adjuvant of equal-volume.Immunity is chosen of the right age immune with new zealand rabbit (weight standard is the 2-2.5 kilogram), and after raising through adaptability, the back intracutaneous is no less than 15 injections.The antigen amount reduces by half (250 μ g) after 2 weeks, adds the incomplete freund adjuvant of equal-volume, for the first time booster immunization.Carry out the booster immunization second time after 2 weeks, method is the same.Auricular vein is taken a blood sample on a small quantity after 2 weeks again, and with synthetic polypeptide (1 μ g/mL) coated elisa plate, indirect elisa method detects immune serum and tires.Booster immunization is repeatedly surveyed antibody titer and is reached 1: 16 and stopped immunity at 10000 o'clock to getting blood, adopts the carotid artery bloodletting to collect antibody.

Embodiment 4: the affinitive layer purification anti-peptide antibody.

1. the preparation of the synthetic polypeptide affinity column of MDC1: take by weighing 1.5g CNBr activation Sepharose 4B, after fully embathing the expansion gel with 1mmol/L hydrochloric acid, wash 2 times with coupling buffer, mix with the synthetic polypeptide 500 μ g that are dissolved in the 5mL coupling buffer rapidly, rotation mixed 2 hours under the room temperature.Add 0.2mol/L glycine 4mL termination reaction, rotation mixed 1 hour under the room temperature.Alternately wash gel each 2 times with coupling buffer and glycine buffer, wash gel with the 50mL phosphate buffered saline buffer again, add sodium azide (NaN by stopping concentration 0.02% ₃), put 4 ℃ of preservations.

2. affinitive layer purification anti-peptide antibody: anti-polypeptide serum of 20mL rabbit and 1.6mL polypeptide link coupled Sepharose 4B are added the 50mL centrifuge tube jointly, and 4 ℃ of rotations mixed 6 hours.Gel is added chromatography column, discard effluent liquid, with 15mL phosphate buffered saline buffer flushing gel.Add 0.8mL pH2.9 at every turn, 0.1mol/L glycine buffer wash-out, totally 8 times, elutriant adds 8 respectively and adds 80 μ L 2mol/L Tris-HCL damping fluids (pH7.5) in advance, and the 20mL phosphate buffered saline buffer is washed gel.Above-mentioned whole operating process is finished under 4 ℃.Every pipe is got elutriant 2 μ L, surveys the OD value of 280nm after diluting 50 times, and the calculating protein content is also drawn elution curve.

Embodiment 5: the evaluation of antibody, and use indirect ELISA method to detect the antibody relative compatible constant.

With the crosslinked BSA of synthetic peptide, respectively with the concentration of 5 μ g/mL and 10 μ g/mL, under 4 degree, wrap and spent the night by the ELISA check-out console, antibody behind the purifying of lowlenthal serum room temperature sealing known protein concentration of adding doubling dilution after 2 hours of 10%, the goat anti-rabbit igg two anti-0.1mL that add the HRP mark again, TMB chromogenic assay A ₄₅₀Logarithm with antibody concentration is an X-coordinate, the A when being up to the standard with curve ₄₅₀Value is 100%, is the ordinate zou mapping with other percentage ratios with respect to this concentration, obtains A ₅₀It is the corresponding antibody concentration of 50%A value.According to Kaff=(n-1)/2 (n[Ab '] t-2[Ab] t) calculate the antibody relative compatible constant, wherein [Ab '] t, [Ab] t refers to A ₅₀The time envelope antigen volumetric molar concentration of antibody during the antibody semi-saturation when being respectively 5 μ g/mL and 10 μ g/mL, [Ag] t, [Ag '] t refer in this experiment of antigen concentration of bag quilt and promptly refer to 10 μ g and 5 μ g, n=[Ag] t/[Ag '] t, n=2 in this experiment.

Test effect

1.MDC1 synthetic polypeptide: behind the HPLC purifying, use liquid chromatography to identify that purity is 82.4%.The polypeptide molecular weight theoretical value is 1925, and mass spectrum is identified measured value [M+H ⁺] be 1926.1

2. the coupling efficiency of polypeptide and carrier: the SDS-PAGE electrophoresis identifies that coupling efficiency roughly conforms to sample recovery rate, greater than 80%.

3. antibody titer: the antiserum titre that many rabbit obtain is all at 1: 10 more than ten thousand.

4. affinity of antibody: relative compatible constant is 10 ⁵-10 ⁶L/mol.

5. the application of antibody: the realization of above technical indicator guarantees that this antibody can be applied to Western blot fully and various enzyme is exempted from experiment, and sets up corresponding external immunological assay reagents.

Sequence table

＜110〉ImmunoHunt Corporation

＜120〉a kind of people MDC1 polypeptide and preparation method for antibody thereof of N terminal specific

<160>2

<210>1

<211>2089

<212>PRT

＜213〉people

<400>1

MEDTQAIDWDVEEEEETEQSSESLRCNVEPVGRLHIFSGAHGPEKDFPLHLGKNVVGRMPDCSVALPFPSISKQHAEIEILAWDKAPILRDCGSLNGTQILRPPKVLSPGVSHRLRDQELILFADLLCQYHRLDVSLPFVSRGPLTVEETPRVQGETQPQRLLLAEDSEEEVDFLSERRMVKKSRTTSSSVIVPESDEEGHSPVLGGLGPPFAFNLNSDTDVEEGQQPATEEASSAARRGATVEAKQSEAEVVTEIQLEKDQPLVKERDNDTKVKRGAGNGVVPAGVILERSQPPGEDSDTDVDDDSRPPGRPAEVHLERAQPFGFIDSDTDAEEERIPATPVVIPMKKRKIFHGVGTRGPGAPGLAHLQESQAGSDTDVEEGKAPQAVPLEKSQASMVINSDTDDEEEVSAALTLAHLKESQPAIWNRDAEEDMPQRVVLLQRSQTTTERDSDTDVEEEELPVENREAVLKDHTKIRALVRAHSEKDQPPFGDSDDSVEADKSSPGIHLERSQASTTVDINTQVEKEVPPGSAIMHIKKHQVSVEGTNQTDVKAVGGPAKLLVVSLEEAWPLHGDCETDAEEGTSLTASVVADVRKSQLPAEGDAGAEWAAAVLKQERAHEVGAQGGPPVAQVEQDLPISRENLTDLVVDTDTLGESTQPQREGAQVPTGREREQHVGGTKDSEDNYGDSEDLDLQATQCFLENQGLEAVQSMEDEPTQAFMLTPPQELGPSHCSFQTTGTLDEPWEVLATQPFCLRESEDSETQPFDTHLEAYGPCLSPPRAIPGDQHPESPVHTEPMGIQGRGRQTVDKVMGIPKETAERVGPERGPLERETEKLLPERQTDVTGEEELTKGKQDREQKQLLARDTQRQESDKNGESASPERDRESLKVEIETSEEIQEKQVQKQTLPSKAFEREVERPVANRECDPAELEEKVPKVILERDTQRGEPEGGSQDQKGQASSPTPEPGVGAGDLPGPTSAPVPSGSQSGGRGSPVSPRRHQKGLLNCKMPPAEKASRIRAAEKVSRGDQESPDACLPPAVPEAPAPPQKPLNSQSQKHLAPPPLLSPLLPSIKPTVRKTRQDGSQEAPEAPLSSELEPFHPKPKIRTRKSSRMTPFPATSAAPEPHPSTSTAQPVTPKPTSQATRSRTNRSSVKTPEPVVPTAPELQPSTSTDQPVTSEPTSQVTRGRKSRSSVKTPETVVPTALELQPSTSTDRPVTSEPTSQATRGRKNRSSVKTPEPVVPTAPELQPSTSTDQPVTSEPTYQATRGRKNRSSVKTPEPVVPTAPELRPSTSTDRPVTPKPTSRTTRSRTNMSSVKTPETVVPTAPELQISTSTDQPVTPKPTSRTTRSRTNMSSVKNPESTVPIAPELPPSTSTEQPVTPEPTSRATRGRKNRSSGKTPETLVPTAPKLEPSTSTDQPVTPEPTSQATRGRTNRSSVKTPETVVPTAPELQPSTSTDQPVTPEPTSQATRGRTDRSSVKTPETVVPTAPELQASASTDQPVTSEPTSRTTRGRKNRSSVKTPETVVPAAPELQPPTSTDRPVTPEPTSRATRGRTNRSSVKTPESIVPIAPELQPSTSRNQLVTPEPTSRATRCRTNRSSVKTPEPVVPTAPEPHPTTSTDQPVTPKLTSRATRRKTNRSSVKTPKPVEPAASDLEPFTPTDQSVTPEAIAQGGQSKTLRSSTVRAMPVPTTPEFQSPVTTDQPISPEPITQPSCIKRQRAAGNPGSLAAPIDHKPCSAPLEPKSQASRNQRWGAVRAAESLTAIPEPASPQLLETPIHASQIQKVEPAGRSRFTPELQPKASQSRKRSLATMDSPPHQKQPQRGEVSQKTVIIKEEEEDTAEKPGKEEDVVTPKPGKRKRDQAEEEPNRIPSRSLRRTKLNQESTAPKVLFTGVVDARGERAVLALGGSLAGSAAEASHLVTDRIRRTVKFLCALGRGIPILSLDWLHQSRKAGFFLPPDEYVVTDPEQEKNFGFSLQDALSRARERRLLEGYEIYVTPGVQPPPPQMGEIISCCGGTYLPSMPRSYKPQRVVITCPQDFPHCSIPLRVGLPLLSPEFLLTGVLKQEAKPEAFVLSPLEMSST

<210>2

<211>15

<212>PRT

＜213〉people

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TQAIDWDVEEEEETE

Claims (7)

1. the MDC1 polypeptide of a N terminal specific is characterized in that amino acid sequence of polypeptide is: TQAIDWDVEEEEETE.

2. a special anti-MDC1N end synthesis of polypeptide antibody is characterized in that this antibody titer more than 1: 100000, high specificity, and avidity is up to 10 ⁵-10 ⁶L/mol.

3. the preparation method of a special anti-MDC1N end synthesis of polypeptide antibody is characterized in that with the 4th to the 18th TQAIDWDVEEEEETE peptide section sequence in the MDC1 aminoacid sequence as the synthetic MDC1 antigen peptide of antigen peptide; The antigen peptide of purifying and protein carrier coupling are behind column chromatography purification; Prepare artificial immunogen, immune animal prepares polypeptide antibody, and forms through affinitive layer purification.

4. preparation method according to claim 3 is characterized in that carrier proteins is a key hole chirp keyhole limpet hemocyanin.

5. preparation method according to claim 3 is characterized in that collecting antibody after link coupled KLH-polypeptide adds the freund adjuvant immunity.

6. preparation method according to claim 3 is characterized in that antigen peptide and carrier protein couplet as immunogen, and immunizing rabbit prepares the anti-MDC1 synthesis of polypeptide antibody of rabbit.

7. preparation method according to claim 3 is characterized in that synthetic polypeptide coupling Sepharose 4B, and the preparation affinity column is used for the purifying anti-peptide antibody.

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