CN1594363A - Anti-human phylaxin synthetic polypeptide antibody and its preparation method - Google Patents
Anti-human phylaxin synthetic polypeptide antibody and its preparation method Download PDFInfo
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- CN1594363A CN1594363A CN 200410019932 CN200410019932A CN1594363A CN 1594363 A CN1594363 A CN 1594363A CN 200410019932 CN200410019932 CN 200410019932 CN 200410019932 A CN200410019932 A CN 200410019932A CN 1594363 A CN1594363 A CN 1594363A
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Abstract
The invention discloses an anti-human phylaxin synthetic polypeptide antibody and its preparation method which consists of, synthesizing polypeptides using one segment of the resistin amino acid sequence, using its amino acid sequence CSMEEAINERIQE as semiantigen, preparing artificial immunity antigen through connection with protein carriers, immunizing the animals so as to effectively induce organism immune response, thus obtaining the antibodies.
Description
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 anti-people's phylaxin synthesis of polypeptide antibody and preparation method thereof.
Background technology
(resistin RST) is find calendar year 2001 a kind of by white adipose cell specific expression and excretory neurophysin to phylaxin.Because commercially available gene recombinant human phylaxin costs an arm and a leg, the cost that is used for preparing antibody is too high, not only risky but also uneconomical.
Ab1129:http: //www.abcam.com/? datasheet=1129 " phylaxin antibody (ab1129) data sheet " supplier NovuS-Biologicals (NB200-202) provides rabbit anti-phylaxin polyclonal antibody, its immunogen is synthesized peptide GSWDVRAETTCH, corresponding to the 79-90 position of people's phylaxin aminoacid sequence.
Ab1130:http: //www.abcam.com/? datasheet=1130 " phylaxin antibody (ab1129) data sheet " supplier Novus-Biologicals (NB200-202) provides rabbit anti-phylaxin polyclonal antibody, its immunogen is synthesized peptide AGMDWTGAR, corresponding to the 94-102 position of people's phylaxin aminoacid sequence.
Http:// www.phoenixpeptide.com/catalog%20Files/Risistin/risistin .htm discloses " with the painted rat hypothalamus section of phylaxin (43-65) antiserum(antisera) " product innovation, and personnel selection phylaxin (51-108) enzyme immunoassay medicine box detects healthy people's normal plasma phylaxin level.
Summary of the invention
The present invention is used for immunoprecipitation and Western blot in order to solve the laboratory exactly, setting up the problem of the required test of external immunoassay with biological products, and provides a kind of anti-people's phylaxin synthesis of polypeptide antibody and preparation method thereof.
A kind of anti-people's phylaxin synthesis of polypeptide antibody, this antibody titer be more than 1: 100000, high specificity, and avidity is up to 1.34 * 10
10L/mol as haptenic synthetic amino acid sequence of polypeptide is: CSMEEAINERIQE.
A kind of preparation method of anti-people's phylaxin synthesis of polypeptide antibody, with the CSMEEAINERIQE peptide section sequence of 22-34 position in the phylaxin aminoacid sequence as the synthetic phylaxin 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.
Described preparation method, its carrier proteins is a key hole alkali keyhole limpet hemocyanin.
Described preparation method collects antibody after its link coupled KLH-polypeptide adds the freund adjuvant immune animal.
Described preparation method, its antigen peptide and carrier protein couplet are as immunogen, and immunizing rabbit prepares the anti-people's phylaxin of rabbit synthesis of polypeptide antibody.
Described preparation method, its synthetic polypeptide coupling Sepharos 4B, the preparation affinity column is used for the purifying anti-peptide antibody.
The high-quality phylaxin synthesis of polypeptide antibody of the present invention preparation tire height, avidity is strong, specificity good, can with natural human phylaxin molecule generation specific binding reaction.The cost for preparing antibody with polypeptide antigen has only with about 1/10th of recombinant antigen preparation cost.Antibody can be used for immunoprecipitation and Western blot (Western blot) test fully behind the affinity chromatography purifying, and sets up external immune analysis method.Preparation that should synthetic peptide antibody is for the research of phylaxin molecular studies and relative disease provides a useful instrument.
Embodiment
One. synthesizing of phylaxin antigen peptide: use Antheprot protein analysis software, people's phylaxin aminoacid sequence (1-108) is carried out wetting ability, antigenic type and homology analysis.Selection contains 13 amino acid whose peptide section sequences of 22～34 as antigen peptide, increases progressively synthetic back by fixing carboxyl to aminoterminal and obtain crude product on the 433A full-automatic polypeptide synthetic instrument.After the dissolving of 30% acetonitrile, 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.
Two. the coupling of synthetic polypeptide and carrier: carrier proteins is selected key hole alkali keyhole limpet hemocyanin (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); 3mgMBS (11 μ mol) 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/L NaCl, 0.01mol/L Na 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 solution 0.56ml 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-PAGE (polyacrylamide gel electrophoresis) evaluation coupling efficiency.
Three. 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, back intracutaneous multi-point injection immunizing rabbit.The antigen amount reduces by half (250 μ g) after 4 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.Begin auricular vein after 2 weeks again and take a blood sample on a small quantity, with synthetic polypeptide (1 μ g/ml) coated elisa plate, indirect elisa method monitoring immune serum is tired.When antiserum titre reach the highest after, adopt the carotid artery bloodletting to collect antibody.
Four. the affinitive layer purification anti-peptide antibody:
1. the preparation of the synthetic peptide affinity column of phylaxin: 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 final 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 inserts 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 280nm after diluting 50 times and goes out OD value, and the calculating protein content is also drawn elution curve.
Five. the evaluation of antibody: indirect elisa method is identified antibodies specific, and the Schatchard graphing method is calculated avidity.
Test effect
1. phylaxin synthesizes polypeptide: purity behind the HPLC purifying＞92%, mass spectrum identify that molecular weight is 1551.7, are accredited as aminoacid sequence through protein sequencing to be: CSMEEAINERIQE.
2. the coupling efficiency of polypeptide and carrier: the SDS-PAGE electrophoresis identifies that coupling efficiency roughly conforms to sample recovery rate, is 75%.
3. tiring of anti-peptide antibody: the antiserum titre that obtains with many rabbit is all more than 1:100000.
4. antibodies specific: oneself synthetic peptide cross reacting rate of Resistin polypeptide antibody behind the purifying and people Resistin is near 100%, and with rabbit, mouse, mouse Resistin, insulin human, people's leptin no cross reaction.
5. affinity of antibody: affinity costant is 1.34 * 10
10L/mol.
6. the application of antibody: the realization of above technical indicator guarantees that this antibody can be applied to immunoprecipitation and Western blot fully, and sets up external immunoassay.The above-mentioned test effect that carries out in Institute of Radiation Medicine, Chinese Academy of Medical Sciences test nuclear medicine chamber is good.
Claims (6)
1. anti-people's phylaxin synthesis of polypeptide antibody is characterized in that this antibody titer more than 1: 100000, high specificity, and avidity is up to 1.34 * 10
10L/mol as haptenic synthetic amino acid sequence of polypeptide is: CSMEEAINERIQE.
2. the preparation method of anti-people's phylaxin synthesis of polypeptide antibody is characterized in that synthesizing the phylaxin antigen peptide with the CSMEEAINERIQE peptide section sequence of 22-34 position in the phylaxin aminoacid sequence as 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.
3. preparation method according to claim 2 is characterized in that carrier proteins is a key hole Zhu keyhole limpet hemocyanin.
4. preparation method according to claim 2 is characterized in that collecting antibody after link coupled KLH-polypeptide adds the freund adjuvant immune animal.
5. preparation method according to claim 2 is characterized in that antigen peptide and carrier protein couplet as immunogen, and immunizing rabbit prepares the anti-people's phylaxin of rabbit synthesis of polypeptide antibody.
6. preparation method according to claim 2 is characterized in that synthetic polypeptide coupling Sepharos 4B, and the preparation affinity column is used for the purifying anti-peptide antibody.
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| CN 200410019932 CN1594363A (en) | 2004-07-08 | 2004-07-08 | Anti-human phylaxin synthetic polypeptide antibody and its preparation method |
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| CN 200410019932 CN1594363A (en) | 2004-07-08 | 2004-07-08 | Anti-human phylaxin synthetic polypeptide antibody and its preparation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100423781C (en) * | 2006-08-09 | 2008-10-08 | 郭锡熔 | Application of Resistin binding polypeptide for preparing medicine promoting insulin secretion |
| CN102559694A (en) * | 2012-02-23 | 2012-07-11 | 中国医学科学院医学生物学研究所 | Cloning method for cDNA (complementary Deoxyribonucleic Acid) full-length sequence of macaque resistin gene |
| CN103554249A (en) * | 2013-10-25 | 2014-02-05 | 中国科学院深圳先进技术研究院 | Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof |
| CN106986937A (en) * | 2017-05-11 | 2017-07-28 | 南京谋新生物技术有限公司 | A kind of ETH polypeptide antibodies and preparation method thereof, kit |
| CN108484768A (en) * | 2018-03-20 | 2018-09-04 | 中国人民解放军军事科学院军事医学研究院 | Neutralizing antibody and the application in treating breast cancer is immunized in a kind of anti-phylaxin |
-
2004
- 2004-07-08 CN CN 200410019932 patent/CN1594363A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100423781C (en) * | 2006-08-09 | 2008-10-08 | 郭锡熔 | Application of Resistin binding polypeptide for preparing medicine promoting insulin secretion |
| CN102559694A (en) * | 2012-02-23 | 2012-07-11 | 中国医学科学院医学生物学研究所 | Cloning method for cDNA (complementary Deoxyribonucleic Acid) full-length sequence of macaque resistin gene |
| CN103554249A (en) * | 2013-10-25 | 2014-02-05 | 中国科学院深圳先进技术研究院 | Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof |
| CN106986937A (en) * | 2017-05-11 | 2017-07-28 | 南京谋新生物技术有限公司 | A kind of ETH polypeptide antibodies and preparation method thereof, kit |
| CN106986937B (en) * | 2017-05-11 | 2020-09-08 | 南京羚诺生物医药技术研究院有限公司 | ETH polypeptide antibody composition, preparation method thereof and kit |
| CN108484768A (en) * | 2018-03-20 | 2018-09-04 | 中国人民解放军军事科学院军事医学研究院 | Neutralizing antibody and the application in treating breast cancer is immunized in a kind of anti-phylaxin |
| CN108484768B (en) * | 2018-03-20 | 2021-03-30 | 中国人民解放军军事科学院军事医学研究院 | Anti-resistin immune neutralizing antibody and application thereof in treatment of breast cancer |
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