CN101602794A - A kind of human MGF polypeptide and preparation method for antibody thereof - Google Patents

Abstract

The invention discloses a kind of human MGF polypeptide and preparation method for antibody thereof, relating to antibody is the biological products that the in vitro tests of feature is used.The present invention utilizes classical epi-position forecasting software DNAstar to carry out analysis-by-synthesis, determine Characterization of antigenic epitopes and antagonism position, determine that the 76th to the 88th is the target of this antibody, its aminoacid sequence is QRHTDMPKTQKYQ, synthetic through polypeptide, polypeptide and carrier proteins are crosslinked, and immunizing rabbit prepares antibody and antibody purification, obtains target antibody.Tire height, avidity of the high quality polypeptide antibody of the present invention preparation is strong, can with natural human MGF 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.

Description

A kind of human MGF polypeptide and preparation method for antibody thereof

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 special human MGF polypeptide and preparation method for antibody thereof, belongs to field of biomedicine technology.

Background technology

1999, Goldspink etc. placed the tibialis anterior of rabbit and it are carried out electricity irritation after stretching stationary state, find after 7 days that muscle masses have increased 35%.Adopt differential display technique, those be in stretched state and by electricity irritation myofiber in, found a large amount of IGF-1 splicing isomers that can impel muscle to repair, called after Mechano GrowthFactor (MGF).Domestic scholars generally is translated into Mecano growth factor, Mechano growth factor, somatomedin element etc.MGF has the important physical effect at aspects such as promoting damaged muscle reparative regeneration, increase muscle stem cell quantity, neuroprotective fast.

People MGF gene order is compared with IGF-1 from liver, is to have inserted 49 bases on No. 5 exons of its mRNA, because the base of inserting can not divide exactly by 3, so produced the expression product that is different from IGF-1.Express with general and to bring into play the IGF-1 of extensive physiological function different, MGF is many when body is subjected to mechanical stimulus or local muscle/nerve and is damaged, and just can express and performance reparation and defencive function efficiently at the damage happening part.In vivo, confirm that by model animal the action effect of MGF in keeping meromyarian meat quality and process of tissue reparation is also apparently higher than IGF-1.The expression vector that contains MGF cDNA is injected the mouse tibialis anterior muscle, and only muscle quality just can increase by 20% in 15 days, and after carrying the virus vector injection of IGF-1Ea cDNA, then needs 120 days muscle qualities just reach peer-level.Show that MGF is a kind of important function molecule in the muscle repair process, and anathrepsis must exist close inner link between the expression of Substance.By section of preparation muscle and mensuration myofiber size, find that the myofiber ensemble average increases by 25%, but this increase is confined to be subjected to the injection site, shows that the physiological function of MGF had both had the significance of action effect, also has very strong target site specificity.In addition, Olesen etc. discover that MGF also can induce the synthetic of tendon collagen protein, expresses the MGF of rise rapidly and the mRNA synthesis rate positive correlation of collagen protein III after mechanical stimulus.In addition, MGF also has very strong neuroprotective.In the transient cerebral ischemia model of mongolian gerbil, Dluzniewska etc. can give gerbil jird significant neuroprotective by the synthetic peptide of injection MGF; In the hippocampus of opposing brain ischemic, detected and expressed the MGF albumen that obviously raises, and this effect is not rely on the acceptor of IGF-1 and independent performance.Goldspink etc. have studied the reparation damaging action of MGF opposite neural cutting postoperative, find that the neural surface of a wound at a distance of 3 millimeters is only just healed in two weeks fully, obviously are better than IGF-1 Ea and other molecules.Data presentation, MGF has reached more than 80% the protection effect of motor neuron.So far, do not see that introduction is by synthesizing the bibliographical information that polypeptide carries out anti-people MGF polyclonal antibody preparation.

Summary of the invention

The present invention is for solving expression and the natural existence by immunization experiment specific detection people MGF, and set up the problem of the required basic biological products of corresponding external immunoassay, and provide a kind of can specificity at synthesis of polypeptide antibody of people MGF and preparation method thereof.

Anti-people MGF synthesis of polypeptide antibody of the present invention prepares according to following steps:

Characterization of antigenic epitopes and antagonism position are determined: utilize classical epi-position forecasting software DNAstar to carry out analysis-by-synthesis, mainly consider index and comprise hydrophilic structure, antigenic index, surperficial accessibility etc., final definite the 76th to the 88th is the target of this antibody, and its aminoacid sequence is QRHTDMPKTQKYQ.

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 immunogenicity of antigens, 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 human MGF 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, the research of behaviour MGF molecular studies and relative disease thereof provides an important tool, and in biomedical research aspects such as the analysis of the proteic characteristic of respective target, Determination on content, distribution situation and proteic conclusive evidence is all had vital role.

Description of drawings

Fig. 1 is DNASTAR software analysis figure, shows that the selected peptide section of the present invention is positioned at this proteic antigenicity and the strong section of wetting ability.

Fig. 2 is by indirect ELISA method purification Identification antibody relative compatible constant figure as a result, and wherein to detect the how anti-and polypeptide antigen affinity costant of preparation be 10 to ELISA ⁶-10 ⁷L/mol.

Embodiment

1, people MGF antigen peptide is synthetic

With the DNAstar analysis software people MGF aminoacid sequence being carried out hydrophilic structure (Hydrophilicity), antigenic index (Antigenicity), surperficial accessibility (Surface Probability) etc. analyzes and homology analysis (Homology), final definite the 76th to the 88th is target, and its aminoacid sequence is QRHTDMPKTQKYQ.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, by shown in Figure 1.

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 mol) is dissolved in 75 μ L diformamides (DMF).Divide MBS solution 3 times and add KLH solution, rotation mixing, room temperature effect 30 minutes.After centrifugal rapidly, coupling buffer 2 (0.1mol/L phosphoric acid salt, 0.15mol/L NaCL, 0.01mol/LNa are used in reaction mixture (about 0.8mL) adding in advance ₂EDTA, pH=7.0) equilibrated PD-10 post.With coupling buffer 2 wash-outs, collect elutriant (being MBS-KLH solution).The synthetic polypeptide of dissolving 1.5mg is in 0.15mL coupling buffer 2, add MBS-KLH damping fluid 0.56mL, 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, 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.

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 isopyknic complete freund adjuvant.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 isopyknic incomplete freund adjuvant, 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: 10 and stopped immunity at 10000 o'clock to getting blood, adopts the carotid artery bloodletting to collect antibody.

4, affinitive layer purification anti-peptide antibody

1. the preparation of the synthetic polypeptide affinity column of people MGF: 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 again, 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 pH=2.9 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.

5, the evaluation of antibody uses 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, as shown in Figure 2.

Test effect

1. the coupling efficiency of polypeptide and carrier: the SDS-PAGE electrophoresis identifies that coupling efficiency roughly conforms to sample recovery rate, greater than 90%.

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

3. affinity of antibody: relative compatible constant is 10 ⁶-10 ⁷L/mol.

4. 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〉University of Science ﹠ Technology, Beijing

＜120〉a kind of human MGF polypeptide and preparation method for antibody thereof

<160>2

<210>1

<211>110

<212>PRT

＜213〉people

<400>1

GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKYQQRHTDMPKTQKYQKGSTFEEHK

<210>2

<211>13

<212>PRT

＜213〉people

<400>2

QRHTDMPKTQKYQ

Claims (5)

1, a kind of human MGF polypeptide and preparation method for antibody thereof, it is characterized in that, utilize classical epi-position forecasting software DNAstar to carry out analysis-by-synthesis, final definite the 76th to the 88th is the target of this antibody, and its aminoacid sequence is QRHTDMPKTQKYQ, synthetic through polypeptide again, polypeptide and carrier proteins are crosslinked, immune animal, antibody purification obtains target antibody.

2, preparation method as claimed in claim 1 is characterized in that, polypeptide is synthetic to be that the target polypeptide is synthetic in the fully-automatic multi-channel Peptide synthesizer, adopts column chromatography purification, then by HPLC, detects purity with the fine gradient of standard second of per minute 1%.

3, preparation method as claimed in claim 1 is characterized in that, the crosslinked selection key of polypeptide and carrier proteins hole chirp keyhole limpet hemocyanin carries out crosslinked.

4, preparation method as claimed in claim 1 is characterized in that, immune animal is that immunizing rabbit prepares antibody so that antigen peptide-crosslinked carrier proteins is as immunogen.

5, preparation method as claimed in claim 1, it is characterized in that, antibody purification is to meet the requirements of the immune animal bloodletting, antiserum(antisera) is collected in standard method, use sad-ammonium sulfate method and affinity chromatography to cross the column purification method successively, carry out the antiserum(antisera) purifying, by SDS-PAGE and HPLC checking purity.

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