CN103509108B - Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic dodecapeptide - Google Patents
Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic dodecapeptide Download PDFInfo
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- 238000002649 immunization Methods 0.000 claims abstract description 26
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- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 21
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- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 18
- 239000008280 blood Substances 0.000 claims abstract description 14
- 210000004369 blood Anatomy 0.000 claims abstract description 14
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 4
- 230000036039 immunity Effects 0.000 claims description 25
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- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a method of preparing a bombyx mori silk fibroin specific antibody by utilizing a characteristic dodecapeptide. The method comprises the following steps: synthesizing a polypeptide with a "CGYGAGAGAGYGA" sequence, coupling the polypeptide with keyhole limpet hemocyanin (KLH) so as to obtain a complete antigen; diluting the complete antigen with normal saline, mixing the diluted complete antigen with a complete Freund's adjuvant, carrying out an emulsion treatment so as to obtain primary immunized antigen emulsion, subjecting a rabbit to a primary immunization by using the primary immunized antigen emulsion, then subjecting the rabbit to a strengthened immunization, wherein the strengthened immunization uses a strengthened immunized antigen emulsion, which is prepared by the following steps: mixing the diluted complete antigen with an incomplete Freund's adjuvant, and then carrying out an emulsion treatment so as to obtain the target product; collecting the blood of the immunized rabbit, when the antiserum titer of rabbit arrives at 1/10000; making the blood blocks fully contract to completely separate out the antiserum, then collecting the antiserum, and subjecting the antiserum to a centrifugation treatment so as to obtain a supernate. The antibody prepared by the invention has a strong specificity, and can be used for detection and analysis of silk fibroin in textile, and the like.
Description
Technical field
The present invention relates to polyclonal antibody preparation technology, particularly relate to a kind of technology preparing By Juvenile Hormone specific antibody.
Background technology
Silkworm is the important economic insects of China, to produce for the purpose of silk.Silk is primarily of fibroin and silk gum two kinds of protein compositions, but sericin comes off in silk reeling process, and therefore, composition main in silk goods, silk product etc. is silk fibroin.On the one hand, at present at piece market, personation silk product is more, has invaded consumer rights, has had a strong impact on China's silk good reputation for a long time, therefore, be necessary the determination method setting up scientific and precise.On the other hand, in silk ogigin study field, also in the urgent need to more scientific analytical procedure, because imbed silk goods in grave or ruins ancient times As time goes on, be subject to various factors and degraded, extraneous illumination, acid, alkali, the environmental factorss such as microorganism, silk protein generation sex change and macromole chain break are caused in capital, make it to be degraded into peptide section or amino acid, therefore the antibody utilizing tradition to be prepared by natural protein macromole is difficult to detect the antigen protein destroyed like this, so just be difficult to the origin seeking silk, and age evidence is more early more difficult seeks.Therefore how to adopt natural science applied advanced means, set up silk goods microscratch detection technique system, from impression, residue, soil, extract the information of ancient silk, very urgent to silk ogigin study.The modern technique of current protein detection is Western Blotting based on antibody-antigene or ELISA method, but Dispersal risk often adopts natural protein macromole, therefore, cannot meet above-mentioned research.
Summary of the invention
The object of the present invention is to provide a kind of method utilizing feature 12 polypeptide preparation By Juvenile Hormone specific antibody.
In order to achieve the above object, the technical solution used in the present invention is: the present invention utilizes the method for feature 12 polypeptide preparation By Juvenile Hormone specific antibody to comprise the following steps:
Step one: utilize Peptide synthesizer composition sequence to be the polypeptide of " CGYGAGAGAGYGA ", this polypeptide and keyhole limpet hemocyanin (KLH) coupling are got up to obtain complete antigen;
Step 2: with complete antigen described in normal saline dilution, the complete antigen after dilution is mixed with complete Freund's adjuvant, then carries out emulsification and obtain initial immunity antigen emulsion; Use initial immunity antigen emulsion to carry out initial immunity to rabbit, then carry out booster immunization to rabbit, booster immunization uses booster immunization antigen emulsion; Described booster immunization antigen emulsion be by described dilution after complete antigen mix with incomplete Freund's adjuvant, then carry out emulsification and obtain; After the antiserum titre of rabbit reaches 1/10000, collect the blood of the rabbit after immunity, and clot is fully shunk and antiserum(antisera) is separated out completely, collect antiserum(antisera) and centrifugal treating obtains supernatant liquor.
Further, the present invention is in described step 2, and when preparing initial immunity antigen emulsion, the complete antigen after dilution and complete Freund's adjuvant are that 1:1 mixes by volume; When preparing booster immunization antigen emulsion, the complete antigen after dilution and incomplete Freund's adjuvant are that 1:1 mixes by volume.
Further, the present invention is in described step 2, and the method for rabbit being carried out to described initial immunity is: use initial immunity antigen emulsion to the rear thigh of rabbit and subcutaneously carry out multi-point injection, often only injecting 100ul;
The method of rabbit being carried out to described booster immunization is: within the 2nd, 4,6 week after initial immunity, carry out a booster immunization respectively, each booster immunization uses booster immunization antigen emulsion to the rear thigh of rabbit and subcutaneously carries out multi-point injection, often only injects 100ul.
Further, the present invention, in described step 2, makes the method that clot fully shrinks and antiserum(antisera) is separated out completely be: allow it solidify under collected blood is put room temperature, be then placed in by solidificating blood in 37 DEG C of incubators and place 30 min, then be placed in 4 DEG C of refrigerator overnight.
Compared with prior art, the beneficial effect that the present invention has is:
(1) result that the inventive method is implemented shows, by the polyclonal antibody of silk fibroin protein feature 12 polypeptide preparation, there is very strong specificity, through elisa assay, this antibody and silk fibroin protein have obvious immune response, sensitivity is very high, can be used in detecting silk fibroin protein, be particularly suitable for the detection analysis of imperfect silk fibroin.
(2) antibody utilizing the macromole of natural protein to prepare from traditional method is different, antibody prepared by the present invention can not only detect complete silk fibroin, also can be used for detecting the silk fibroin molecular ruptured in such as Ancient Silk Textile, in archaeology, verification retrieval and other scientific researches, there is vital role.
Embodiment
At PubMeD(http: //www.ncbi.nlm.nih.gov/pubmed/) on download silkworm (
bombyx mori) heavy chain (H-chain) aminoacid sequence (as shown in SEQ No.1) of silk fibroin.By statistical study, determine that " GYGAGAGAGYGA " polypeptide fragment frequency of occurrences is very high, adding up to 47 places, is the feature polypeptide of bombyx mori silk fibroin, therefore synthesizes this fragment and carry out Dispersal risk as antigen.
Below illustrate preparation method of the present invention.
the synthesis of complete antigen
(halfcystine makes this polypeptide and KLH coupling to utilize Peptide synthesizer to synthesize haptens.By the effect of linking agent, the sulfydryl on the halfcystine of the N end of " CGYGAGAGAGYGA " polypeptide and KLH primary amine form covalent linkage, thus polypeptide and KLH coupling are got up, and obtain complete antigen.
With appropriate normal saline dilution complete antigen, then by complete antigen and the complete Freund's adjuvant after dilution by volume 1:1 mix, add Streptomycin sulphate, penicillin solution carries out emulsification, the antigen emulsion (initial immunity antigen emulsion) used when obtaining initial immunity.In addition, by complete antigen and the incomplete Freund's adjuvant after dilution above by volume 1:1 mix, then add Streptomycin sulphate, penicillin solution carry out emulsification, then strengthened immunity time the antigen emulsion (booster immunization antigen emulsion) that uses.
prepared by antibody:
Choose 2 White Rabbits (14-16 age in week) and carry out initial immunity and booster immunization.First extract rabbit ear blood sample before immunity to contrast.Initial immunity is to the rear thigh of rabbit with subcutaneously carry out multi-point injection, every only injection 100 ul with initial immunity antigen emulsion.Within the 2nd week, 4 weeks, 6 weeks after initial immunity, respectively carry out booster immunization once, each booster immunization uses booster immunization antigen emulsion to the rear thigh of rabbit and subcutaneously carries out multi-point injection, every only injection 100 ul.The 10th day venous blood sampling respectively after third time, four immunity detects antiserum titre.
After tiring and reach 1/10000 in sero-fast in rabbit blood sample, kill rabbit and collect under blood puts room temperature and allow it solidify; Solidificating blood is placed in 37 DEG C of incubators and places 30 min, then be placed in 4 DEG C of refrigerator overnight, clot is fully shunk and antiserum(antisera) is separated out completely; Collect antiserum(antisera), 4 DEG C of 3000 centrifugal 10 min of g, packing supernatant liquor, puts-70 DEG C and stores for future use.
antibody purification:
Immunoaffinity chromatography technical antagonism serum is utilized to carry out purifying.First the haptens (i.e. polypeptide " CGYGAGAGAGYGA ") of synthesis and chromatographic stuffing Sulfo-link-gel are cross-linked, close with halfcystine.Carry out pre-treatment with the PBS coupled columns of 20ml, 50mM, pH7.4, pretreated flow velocity is 60ml/h.With the PBS of 50mM, pH7.4, the antiserum(antisera) of 10ml is diluted to 20ml, loading after dilution.Repeat loading once.Clean with the PBS coupled columns of 20ml, 50mM, pH7.4, the flow velocity of cleaning is 60ml/h.Wash-out is carried out with the glycine-HCL antagonist of pH3.0,0.1M.After wash-out completes, use is containing 50mM, pH7.4, and the PBS containing 0.02% merthiolate washs polypeptide post, 4 DEG C of storages.
antibodies specific effect measuring: antigen silk fibroin is with the concentration bag quilt of 1ug/ml, and 4 DEG C of bags are spent the night.Antibody is diluted respectively 20000 times, 10000 times, 5000 times, 1000 times, 200 times, the amount loading in 50ul/ hole, incubation 1h at 37 DEG C.2 times are washed with 200ul/ hole with TBST.ELIAS secondary antibody uses with 1:5000 dilution, 50ul/ hole, incubation 1h at 37 DEG C.3 times are washed with 200ul/ hole with TBST.The colour developing of TMB nitrite ion is added with 100 ul/ holes.After color reaction 10min, with the H of 2M
2sO
4with the amount termination reaction in 100ul/ hole.OD is measured by microplate reader
450value.According to OD
450value, judges situations such as the combinations of antigen-antibody.Work as OD
450just can determine when value reaches positive criteria in sample containing fibroin; If not containing fibroin, then OD in sample
450value lower than positive criteria, just will can judge the existence of silk accordingly fast.
interpretation of result: result display except blank be colourless except, sample well all presents yellow.OD is measured by microplate reader
450value result is: the OD obtained after carrying out 20000 times, 10000 times, 5000 times, 1000 times, 200 times dilutions to the antibody after purifying
450value is respectively 0.847,1.050,1.606,2.303,2.378.According to platform OD
450value reaches 0.6 for positive requirement, even if by the antibody dilution 20000 times of preparation, the positive signal of silk fibroin still can be detected, develop the color clear, result is clear and definite.Can illustrate from above result, the reaction sensitivity of the antibody prepared by the inventive method to fibroin is high, and required antigen amount is few, and easy and simple to handle, drops into little, should large-scale promotion utilize.
Claims (5)
1. utilize the method for feature 12 polypeptide preparation silk fibroin protein specific antibody, comprise the following steps:
Step one: composition sequence is the polypeptide of " CGYGAGAGAGYGA ", gets up to obtain complete antigen by this polypeptide and keyhole limpet hemocyanin coupling;
Step 2: with complete antigen described in normal saline dilution, the complete antigen after dilution is mixed with complete Freund's adjuvant, then carries out emulsification and obtain initial immunity antigen emulsion; Use initial immunity antigen emulsion to carry out initial immunity to rabbit, then carry out booster immunization to rabbit, booster immunization uses booster immunization antigen emulsion; Described booster immunization antigen emulsion be by described dilution after complete antigen mix with incomplete Freund's adjuvant, then carry out emulsification and obtain; When the antiserum titre of rabbit reaches 1/10000, collect the blood of the rabbit after immunity, and clot is fully shunk and antiserum(antisera) is separated out completely, collect antiserum(antisera) and centrifugal treating obtains supernatant liquor.
2. method according to claim 1, is characterized in that: in described step 2, and when preparing initial immunity antigen emulsion, the complete antigen after dilution and complete Freund's adjuvant are that 1:1 mixes by volume; When preparing booster immunization antigen emulsion, the complete antigen after dilution and incomplete Freund's adjuvant are that 1:1 mixes by volume.
3. method according to claim 1 and 2, is characterized in that: in described step 2, and the method for rabbit being carried out to initial immunity is: use initial immunity antigen emulsion to the rear thigh of rabbit and subcutaneously carry out multi-point injection, often only injecting 100ul;
The method of rabbit being carried out to booster immunization is: within the 2nd, 4,6 week after initial immunity, carry out a booster immunization respectively, and each booster immunization uses booster immunization antigen emulsion to the rear thigh of rabbit and subcutaneously carries out multi-point injection, often only injects 100ul.
4. method according to claim 1 and 2, it is characterized in that: in described step 2, the method that clot fully shrinks and antiserum(antisera) is separated out completely is made to be: under collected blood is put room temperature, to allow it solidify, then solidificating blood is placed in 37 DEG C of incubators and places 30 min, then be placed in 4 DEG C of refrigerator overnight.
5. method according to claim 3, it is characterized in that: in described step 2, the method that clot fully shrinks and antiserum(antisera) is separated out completely is made to be: under collected blood is put room temperature, to allow it solidify, then solidificating blood is placed in 37 DEG C of incubators and places 30 min, then be placed in 4 DEG C of refrigerator overnight.
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| CN104059131A (en) * | 2014-07-04 | 2014-09-24 | 丝科普乐（北京）生物科技有限公司 | Anti-silk fibroin polyclonal antibody and preparation method thereof |
| CN104459104B (en) * | 2014-12-31 | 2016-02-17 | 浙江理工大学 | A kind of method of microscopic examination Ancient Silk Textile |
| CN104406992B (en) * | 2014-12-31 | 2016-03-02 | 浙江理工大学 | A kind of transmission electron microscope detects the method for Ancient Silk Textile |
| CN104459118B (en) * | 2014-12-31 | 2016-03-02 | 浙江理工大学 | A kind of preparation method of Ancient Silk Textile double-antibody method Test paper |
| CN104483479B (en) * | 2014-12-31 | 2016-02-17 | 浙江理工大学 | A kind of dot immuno gold filtration assay measures the method for Ancient Silk Textile |
| CN104483478B (en) * | 2014-12-31 | 2016-01-13 | 浙江理工大学 | A kind of detection method of Ancient Silk Textile |
| CN104459162B (en) * | 2014-12-31 | 2016-04-13 | 浙江理工大学 | A kind of preparation method of Ancient Silk Textile indirect competitive Test paper |
| CN105504056B (en) * | 2015-11-12 | 2019-03-05 | 浙江理工大学 | The method for preparing tussah silk fibroin antibody using feature hexapeptide |
| CN105504055B (en) * | 2015-11-12 | 2019-03-05 | 浙江理工大学 | The method for preparing tussah silk fibroin antibody using feature decapeptide |
| CN105693857A (en) * | 2016-02-24 | 2016-06-22 | 中国丝绸博物馆 | Method for preparing cattle hair detection antibody by feature diagnosis sequence |
| KR102012380B1 (en) * | 2016-08-26 | 2019-08-20 | (주)세원생명공학 | Peptide stimulating bone regeneration or formation and its use |
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| CN101066477A (en) * | 2007-05-17 | 2007-11-07 | 中国人民解放军第三军医大学 | Biological artificial blood vessel capable of in vivo capturing endothelial ancestral cell |
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| CF01 | Termination of patent right due to non-payment of annual fee |