CN106810606A - A kind of preparation and application of Apolipoprotein C-III antigen polypeptide and its polyclonal antibody - Google Patents
A kind of preparation and application of Apolipoprotein C-III antigen polypeptide and its polyclonal antibody Download PDFInfo
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- CN106810606A CN106810606A CN201510867041.5A CN201510867041A CN106810606A CN 106810606 A CN106810606 A CN 106810606A CN 201510867041 A CN201510867041 A CN 201510867041A CN 106810606 A CN106810606 A CN 106810606A
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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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Abstract
The invention discloses a kind of apoC-III antigen polypeptides, the preparation and application of anti-apoC-III polyclonal antibodies belong to biological technical field.Anti- apoC-III polyclonal antibodies of the present invention are with amino acid sequence such as SEQ ID NO：Polypeptide shown in 1 is immunogene, antiserum is prepared with new zealand rabbit is immunized after ovalbumin OVA couplings, and prepare antibody affinity purification filler with apoC-III antigen polypeptide couplings of the invention with the agarose resin that NHS is activated, antibody affinity purification is carried out, ELISA, Western blot identifications are carried out to antibody after purification.Qualification result shows the apoC-III that the polyclonal antibody can be in specific recognition human plasma, can be used for the detection of apoC-III in primary hypertriglyceridemiapatients patients blood plasma, for hypertriglyceridemia diagnosis provides help, and its clinical prognosis can be instructed to judge.
Description
Technical field
The present invention relates to a kind of apoC-III antigen polypeptides, the preparation and application of anti-apoC-III polyclonal antibodies belong to biological technical field.
Background technology
The micro-molecule glucoprotein that apoC-III is made up of 79 amino acid, it is the most abundant class of content in apoC race, main to synthesize in liver, only fraction synthesizes in small intestine, and apoC-III is distributed mainly in chylomicron, VLDL and HDL.ApoC-III genes are located at No. 11 chromosome long arm q23 areas, and between apolipoprotein A-1 and Apolipoprotein A-IV, three collectively constitutes a gene family.The transcription of apoC-III is by the regulation and control of many factors such as insulin and peroxisome proliferation-activated receptors.
ApoC-III has the function of suppressing lipoprotein lipase and hepatic lipase activity, can suppress lipoprotein and lipidolysis of the liver intake rich in triglycerides, is the main regulatory factors of plasma triglyceride level.ApoC-III is mutually shifted between plasma high density lipoprotein level and VLDL, in the serum of blood fat normal individual, apoC-III major parts are combined with HDL, and in primary hypertriglyceridemiapatients patients serum, then major part is combined with VLDL.Hypertriglyceridemia is the most common abnormalities of sugar/lipid metabolism disease of China, is one of the independent hazard factor for causing coronary heart disease.With the increase of China's obese people, the quantity of primary hypertriglyceridemiapatients patients is also increasing year by year, its morbidity causes mainly due to the dyslipoproteinemia rich in triglycerides, apoC-III contents are significantly higher than normal population in patients blood plasma, the promising tumor marker that the hypertriglyceridemia that apoC-III turned into develops, the diagnostic reagent that apoC-III polyclonal antibodies can be used to develop hypertriglyceridemia risk is prepared, the pathogenesis to inquiring into hypertriglyceridemia is significant.
The content of the invention
It is an object of the invention to provide a kind of hypertriglyceridemia mark apoC-III antigen polypeptides.
It is a further object of the present invention to provide a kind of polyclonal antibody of anti-apoC-III.
It is yet another object of the invention to provide the application of the polyclonal antibody of above-mentioned anti-apoC-III.
The present invention is achieved through the following technical solutions above-mentioned purpose：A kind of apoC-III antigen polypeptides, amino acid sequence such as SEQ ID NO：Shown in 1.
A kind of polyclonal antibody of anti-apoC-III, is that immune animal is prepared from apoC-III antigen polypeptides of the invention as immunogene.
The preparation method of the polyclonal antibody of above-mentioned anti-apoC-III, step is as follows：ApoC-III antigen polypeptides are coupled with carrier protein ovalbumin OVA, as immunogene and adjuvant mixed immunity new zealand rabbit, period carries out five times and is immunized, detect serum antibody titer up to after 1: 16000 after ELISA, collection rabbit anteserum, affinity purification is carried out, by after ELISA and Western blot identifications, obtaining the polyclonal antibody of anti-apoC-III.
The polypeptide antigen of chemical synthesis is small molecule, and itself is difficult the antigenicity that has had, can only the very weak immune response of induced animal generation, thus be critically important with carrier protein couplet.Carrier protein contains many epitopes, can stimulate t helper cell, and then induce B cell reaction.Carrier protein for being coupled with polypeptide has various, wherein more conventional hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin(BSA) (bovine serum albumin, BSA), ovalbumin (ovalubumin,) and bovine thyroglobulin (bovine thyroglobulin, THY) OVA.Immunogenicity is most strong after being coupled with apoC-III antigen polypeptides of the invention due to OVA, therefore the present invention selects OVA as coupling carrier albumen.BSA is also often used as peptide carrier, but the antibody that the method is produced above is had some limitations in application because BSA is usually used in the blocking agent of detection experiment.
Compared with prior art, the present invention has following beneficial outcomes：
The present invention is analyzed to physicochemical property, immunogenicity, hydrophilic and hydrophobic, surface accessibility of apoC-III amino acid sequences etc., it is determined that suitable polypeptide sequence carry out it is artificial synthesized；New zealand rabbit is immunized after the polypeptide that will synthesize and ovalbumin OVA couplings；Detected with ELISA method antagonist potency by five immune rabbit anteserums, potency collects immune rabbit anteserum after reaching ideal value, and prepare antibody affinity purification filler with apoC-III antigen polypeptide couplings of the invention with the agarose resin that NHS is activated, the affinity purification of antibody will be carried out after filler dress post, ELISA, Western blot identifications are carried out to antibody after purification.Qualification result shows that the polyclonal antibody can specific recognition apoC-III, with specificity it is good, purity is high the characteristics of, can be used to detect the apoC-III expressed in human plasma, for primary hypertriglyceridemiapatients patients diagnosis provides help, and its clinical prognosis can be instructed to judge.
Brief description of the drawings
Fig. 1 are shown the polypeptide sequence fragment from synthesis through DNAstar software analysis apoC-III immunogenicities, hydrophilic and hydrophobic and surface accessibility in square frame.
SDS-PAGE electrophoresis (12%) testing result of the polyclonal antibody of the anti-apoC-III of Fig. 2 through antigen affinity purification post after purification.Swimming lane 1：Marker；Swimming lane 2：Loading；Swimming lane 3：Flow through；Swimming lane 4：Balance；Swimming lane 5：Wash-out superiors；Swimming lane 6：The lower peak of wash-out.
The Western blot qualification results of the anti-apoC-III polyclonal antibodies of Fig. 3.Swimming lane 1：Human plasma；Swimming lane 2：HepG2 cells.
Fig. 4 experiment flows of the invention.
Specific embodiment
The present invention should be expanded on further in conjunction with specific embodiments below.It should be understood that these embodiments are only used for explaining the present invention, rather than limitation the scope of the present invention.On the premise of without departing substantially from technical scheme, any change that those of ordinary skill in the art made for the present invention easily realize is fallen within scope of the presently claimed invention.
Embodiment 1：ApoC-III antigen polypeptides are designed
1. according to apoC-IIIGenBank accession number GI：4557323, obtain the protein sequence (UniProt of human apolipoprotein C-III：P02656), 79 amino acid are contained.
2. with ProtParam (http：//web.expasy.org/protparam/) on-line analysis apoC-III albumen basic physical and chemical.Analysis result is shown in Table 1, and apoC-III molecular weight is 8.7646KD, and isoelectric point is 4.72, is acidic protein.
ApoC-III basic physical and chemical the analysis results of table 1
3., with DNAstar software analysis apoC-III immunogenicities, hydrophilic and hydrophobic and surface accessibility, the analysis result nearly C-terminal of such as Fig. 1, apoC-III has 16 amino acid antigenicities, hydrophily and surface accessibility stronger.
4. apoC-III antigen polypeptides screening
16 amino acid sequences that will be filtered out are analyzed through Blastp, and final screening polypeptide sequence is：KFSEFWDLDPEVRPTS (60aa-75aa, SEQ ID NO：1).
Embodiment 2：ApoC-III antigen polypeptides synthesize and and carrier protein couplet
1. polypeptide is synthesized by Shanghai gill biochemical corp, and purity is 93.85%.
2. polypeptide and carrier protein couplet
5mg polypeptides are dissolved with 50 μ l DMSO, it is to be dissolved completely after, coupling buffer is added to be diluted to final volume for 1ml, 200 μ l are taken out to be detected for ELISA, after adding the OVA solution that 400 μ l concentration are 10mg/ml to mix in remaining 800 μ l polypeptide solutions, the EDC that 200 μ l concentration are 10mg/ml is added, the polypeptide solution containing EDC, OVA, room temperature reaction 2h are mixed immediately.
3. the ultrafiltration of coupled product and packing
Coupled product is diluted to 4ml with PBS, ultrafiltration is carried out to coupled product with 10KD Milipore ultra-filtration centrifuge tubes, filtrate in down tube is discarded after each ultrafiltration, when remaining liq about 1ml in upper pipe, continue to carry out ultrafiltration centrifugation after being diluted with PBS, after loop ultrafiltration 4 times, remaining liq 1ml adds PBS to be diluted to 3ml mixings in taking pipe, protein concentration is 1.272mg/ml after using NanoDrop spectrophotometric determinations to be coupled, after 0.22 μm of filter filtration sterilization, the μ l of albumen 500, -80 DEG C of preservations after often pipe packing coupling.
Embodiment 3：Anti- apoC-III polyclonal antibodies are prepared and purified
1. animal immune
Blood is taken in rabbit ear vein before immune, -80 DEG C of preservations of centrifuging and taking serum, make negative control sera after standing.After taking coupling protein and Freund's complete adjuvant after packing or the isometric mixing of incomplete Freund's adjuvant, emulsified with injection emulsifier, emulsification immune new zealand rabbit (2~2.5kg of body weight) after terminating, specific immune programme for children is shown in Table 2.
The antigen immune program list of table 2
2.ELISA detection antibody potency
Respectively antibody titer ELISA detections are carried out immune within 29th, 43,57 days.Polypeptide antigen coating buffer (0.1M carbonate buffer solutions) is diluted into final concentration of 5 μ g/ml, is added in 96 orifice plates with the μ l of every hole 100, after 4 DEG C are coated with overnight, washing lotion (phosphate buffer PBST：NaCl 8g, KCl 0.2g, Na2HPO43.58g, KH2PO40.27g, NaOH 0.085g, μ l of tween 500) wash 3 times, after patting dry, 200 μ l confining liquids (hyclone mixes with PBST with 1: 1000) are added per hole, after 37 DEG C of closing 2h, wash 3 times, pat dry.Separate serum after rabbit ear vein blood sampling, with PBS doubling dilutions after, be loaded onto in ELISA Plate be coated with per the μ l of hole 100, meanwhile, choose respectively it is immune before rabbit anteserum as negative control, used as blank, 37 DEG C are not incubated 90min to increase serum, and washing 3 times is patted dry.The secondary antibody of 1: 5000 dilution is added, per hole 100 μ l, 37 DEG C of incubation 30min, is washed 3 times, patted dry.50 μ l developers A, 50 μ l developer B are added per hole, gently concussion is mixed, after 37 DEG C of lucifuges are incubated 10min, 50 μ l terminate liquids (2M H are added per hole2SO4).Returned to zero with blank well, the absorbance in each hole is measured at 450nm wavelength, be limited more than 2.5 with the ratio with negative control hole OD values, as the critical point for being judged as the positive or determination potency.Result such as table 3, after the 5th time immune, antiserum titre reaches 1: 16000, and can gather serum carries out the purifying of polyclonal antibody.
The antibody titer ELISA testing results of table 3
3. the preparation of antigen affinity purification post
Sieve plate is placed in bottom of the pillar compacting.Rock uniform after taking NHS-Activated Sepharose4 Fast Flow fillers (purchased from GE companies) balance to room temperature, take during 1ml adds pillar.Pillar is balanced with PBS, to remove storing liquid.Xiang Zhuzhong adds the proteantigen of 20mg couplings.After mixing, it is placed in room temperature shaker and is incubated 3h or 4 DEG C overnight.Pillar is balanced with PBS, confining liquid (0.5M Tris-HCl, pH 6.8) is added, room temperature shaker is placed in and is incubated 30min, to close uncombined site.Pillar is balanced with PBS, Antibody Wash cleaning pillar adds PBS and 0.05%NaN after balancing pillar with PBS again3, by antigen affinity purification numbered cylinders and be stored in 4 DEG C it is standby.
4. the affinity purification of antibody
Using arteria carotis depletion method, collect rabbit whole blood and 2h is incubated at 37 DEG C, centrifuging and taking supernatant dispenses serum often pipe 1.5ml under aseptic condition.Antigen affinity purification post is placed in room temperature, 10 column volumes is balanced with PBS, to remove storing liquid.Take 2 pipe polyvalent antibodies and dilute 1 times, slow loading is carried out after being filtered with 0.22 μm of filter, be placed in room temperature shaker and be incubated 3h.10 column volumes are balanced with PBS after loading, to wash away uncombined antibody.With Antibody Wash (NaCl 8.6g/L, Glycine 7.5g/L, pH 3.0) wash-out, collect eluting peak, and neutralizer (1M Tris-HCl are added in time, pH 9.0) neutralize wash-out antibody to pH be 7.0 or so, it is ensured that the antibody of wash-out be in neutral environment in case influence antibody activity.Each pipe antibody elution is mixed, it is 0.5 μ l with 4 DEG C of ultrafiltration of 10KD Milipore ultra-filtration centrifuge tubes to final volume, sample 12%SDS-PAGE electrophoretic analysis purification effects are taken, with the AC after NanoDrop spectrophotometric determination affinity purifications, adds antibody to preserve liquid (0.02%NaN3, 50%Glycerol, 1mg/ml BSA) and in -20 DEG C of preservations.As a result：Through SDS-PAGE electroresis appraisals (Fig. 2) after antibody purification, the AC after NanoDrop spectrophotometric determination affinity purifications is 4.0mg/ml.
Embodiment 4：The identification of anti-apoC-III polyclonal antibodies
1. the ELISA identifications of the polyclonal antibody of anti-apoC-III
It is detection antigen with the apoC-III for synthesizing, coated elisa plate, rabbit anteserum is not immunized as negative control using 1: 2000 dilution, after antibody doubling dilution by rabbit anteserum and after purification, using ELISA method detection antibody potency, specific method is with embodiment 3.2.Result shows that anti-apoC-III antibody titers after purification are 1: 16000 (table 4).
Anti- apoC-III antibody titers ELISA the testing results of table 4
2. the Western-blot identifications of anti-apoC-III polyclonal antibodies
Human plasma and HepG2 cells are taken respectively, after being cracked, the protein concentration for extracting sample is measured with BCA determination of protein concentration kits, after SDS-PAGE electrophoresis being carried out with 20 μ g loadings, 200mA constant currents 1h goes to pvdf membrane, transfer terminates, and pvdf membrane, room temperature closing 60min are closed with TBST buffers 5% (M/V) skimmed milk power.The pvdf membrane that will have been closed is put into hybridization bag, and the anti-apoC-III polyclonal antibodies (primary antibody 1: 1000) for obtaining are purified in the embodiment 3 for adding the dilution of 5% (M/V) skimmed milk power, is sealed with film laminator, 4 DEG C of overnight incubations.Pvdf membrane takes out, and is put into TBST, is washed 4 times, every time 10 minutes on shaking table.5% skim milk of the goat-anti rabbit secondary antibody of HRP marks is made secondary antibody working solution by 1: 5000 dilution, and secondary antibody and pvdf membrane are incubated into 45min together with 37 DEG C, takes out pvdf membrane and is put into washing 4 times, every time 10 minutes in TBST buffer solutions.ECL develops the color to detect the expression of apoC-III.Result such as Fig. 3, the anti-apoC-III polyclonal antibodies in the present invention can detect the single band of molecular weight 11KD or so in human plasma and HcpG2 cell pyrolysis liquids, and being expected relative molecular weight with human apolipoprotein C-III is consistent.
Claims (6)
1. a kind of apoC-III antigen polypeptides, it is characterised in that amino acid sequence such as SEQ ID NO：Shown in 1.
2. a kind of polyclonal antibody of anti-apoC-III, it is characterised in that with apoC-III antigen polypeptides described in claim 1 be immunogene, immune animal is prepared from.
3. the polyclonal antibody of anti-apoC-III according to claim 2, it is characterised in that antibody titer is 1: 16000.
4. the preparation method of polyclonal antibody of anti-apoC-III described in claim 2, it is characterised in that step is as follows：After apoC-III antigen polypeptides described in claim 1 are coupled with ovalbumin OVA, as immunogene and adjuvant mixed immunity new zealand rabbit, five times are carried out to be immunized, detect serum antibody titer up to after 1: 16000 after ELISA, collection rabbit anteserum, after carrying out affinity purification, identified through ELISA and Western blot, obtain the polyclonal antibody of anti-apoC-III.
5. preparation method according to claim 4, it is characterised in that antigen affinity purification post is prepared with apoC-III antigen polypeptides described in claim 1, the affinity purification of polyclonal antibody is carried out.
6. application of the polyclonal antibody of anti-apoC-III described in claim 2 in hypertriglyceridemia diagnosis.
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Cited By (2)
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| CN108776215A (en) * | 2018-06-04 | 2018-11-09 | 中国医学科学院北京协和医院 | Systemic sclerosis diagnoses and the biomarker of PAH predictions |
| CN116789766A (en) * | 2023-07-05 | 2023-09-22 | 江苏帆博生物制品有限公司 | Preparation method of nano anti-affinity medium for apolipoprotein AI and purification method of apolipoprotein AI |
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| CN116789766A (en) * | 2023-07-05 | 2023-09-22 | 江苏帆博生物制品有限公司 | Preparation method of nano anti-affinity medium for apolipoprotein AI and purification method of apolipoprotein AI |
| CN116789766B (en) * | 2023-07-05 | 2023-11-21 | 江苏帆博生物制品有限公司 | Preparation method of nano anti-affinity medium for apolipoprotein AI and purification method of apolipoprotein AI |
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