CN103509107B - Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic polypeptide - Google Patents
Method of preparing bombyx mori silk fibroin specific antibody by utilizing characteristic polypeptide Download PDFInfo
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- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 22
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- 238000002649 immunization Methods 0.000 claims abstract description 26
- 230000003053 immunization Effects 0.000 claims abstract description 26
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 25
- 239000008280 blood Substances 0.000 claims abstract description 18
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 8
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims abstract description 8
- 229940049954 penicillin Drugs 0.000 claims abstract description 8
- 230000008878 coupling Effects 0.000 claims abstract description 5
- 238000010168 coupling process Methods 0.000 claims abstract description 5
- 238000005859 coupling reaction Methods 0.000 claims abstract description 5
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 4
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- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 abstract 4
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 abstract 1
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- 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 polypeptide. The method comprises the following steps: synthesizing a "CGAGAGSGAGAGS" polypeptide sequence by utilizing an Fmoc method, coupling the polypeptide with keyhole limpet hemocyanin (KLH) through the cysteine on the N terminus of the polypeptide 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, adding streptomycin and penicillin to carry out an emulsion treatment so as to obtain a 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 adding streptomycin and penicillin to carry out an emulsion treatment so as to obtain the target product; collecting the blood of the immunized rabbit, when the antibody titer in the rabbit blood sample reaches 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.
Description
Technical field
The present invention relates to preparation method of polyclonal antibody, particularly relate to a kind of method preparing silk fibroin protein 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 polypeptide preparation silk fibroin protein 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 polypeptide preparation silk fibroin protein specific antibody to comprise the following steps:
Step one: utilize Fmoc method composition sequence to be the polypeptide of " CGAGAGSGAGAGS ", and make this polypeptide and carrier keyhole limpet hemocyanin (KLH) coupling obtain complete antigen by the halfcystine that the N of described polypeptide holds;
Step 2: with complete antigen described in normal saline dilution, complete antigen after dilution is mixed with complete Freund's adjuvant, and then add Streptomycin sulphate and penicillin solution and carry out emulsification and obtain initial immunity antigen emulsion, initial immunity antigen emulsion is used 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, and then add Streptomycin sulphate and penicillin solution and carry out emulsification and obtain; When the antibody in rabbit blood sample tire reach 1/10000 time, collect the blood of the rabbit after immunity, and clot fully shunk and antiserum(antisera) is separated out completely, then 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, every only injection 100 ul;
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, every only injection 100 ul.
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) the inventive method by silkworm (
bombyx mori) polyclonal antibody of feature polypeptide preparation of silk fibroin has very strong specificity, analyze known through ELISA method, 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, thus provide a kind of responsive special discrimination method efficiently for the silk goods residue in the early stage grave of China and ruins, for silk archaeology and Origin provide new scientific evidence, 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 " GAGAGSGAGAGS " polypeptide fragment frequency of occurrences is the highest, add up to 170 places, be the feature polypeptide of bombyx mori silk fibroin, therefore prepare antigen with this fragment.
Below illustrate preparation method of the present invention.
complete antigen synthesizes:
Utilize Fmoc method to synthesize haptens (i.e. " CGAGAGSGAGAGS " peptide sequence), add halfcystine at the N end of " CGAGAGSGAGAGS " peptide sequence and make this polypeptide and KLH coupling.By the effect of linking agent, the sulfydryl on the halfcystine that " CGAGAGSGAGAGS " N holds 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, add Streptomycin sulphate, penicillin solution carry out emulsification, then strengthened immunity time the antigen emulsion (booster immunization antigen emulsion) that uses.
2.
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.Once, each booster immunization is to the rear thigh of rabbit with subcutaneously carry out multi-point injection, every only injection 100 ul with booster immunization antigen emulsion to the 2nd week, 4 weeks, 6 weeks each booster immunizations after initial immunity.The 10th day venous blood sampling respectively after third time, four immunity detects antiserum titre.
When sero-fast in rabbit blood sample tire reach 1/10000 time, kill rabbit and collect blood, under a large amount of blood of collection put room temperature, 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 " CGAGAGSGAGAGS ") of synthesis and chromatographic stuffing Sulfo-link-gel are cross-linked, the halfcystine that " CGAGAGSGAGAGS " N is held is closed.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, wash polypeptide post containing the PBS of 0.02% merthiolate, obtains the antibody after purifying and in 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.Add TMB with 100 ul/ holes to develop the color in shady place, after colour developing 10min, add the H of 2M with 100ul/ hole
2sO
4stop.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 fibroin 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 2.006,1.840,2.149,2.339,2.445, the OD of blank
450value is 0.054.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 a method for feature polypeptide preparation silk fibroin protein specific antibody, it is characterized in that, comprise the following steps:
Step one: utilize Fmoc method composition sequence to be the polypeptide of " CGAGAGSGAGAGS ", and make this polypeptide and keyhole limpet hemocyanin coupling obtain complete antigen by the halfcystine that the N of described polypeptide holds;
Step 2: with complete antigen described in normal saline dilution, complete antigen after dilution is mixed with complete Freund's adjuvant, and then add Streptomycin sulphate and penicillin solution and carry out emulsification and obtain initial immunity antigen emulsion, initial immunity antigen emulsion is used 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, and then add Streptomycin sulphate and penicillin solution and carry out emulsification and obtain; When the antibody in rabbit blood sample tire reach 1/10000 time, collect the blood of the rabbit after immunity, and clot fully shunk and antiserum(antisera) is separated out completely, then 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, every only injection 100 ul;
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, every only injection 100 ul.
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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| 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 |
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| CN108387435B (en) * | 2018-01-30 | 2020-09-15 | 中国计量大学 | Trace silk fibroin enrichment method |
| CN111732657B (en) * | 2020-06-19 | 2021-12-14 | 西南大学 | Polypeptide antibody for directly detecting sericin protein and preparation method and application thereof |
| CN114805566B (en) * | 2022-05-16 | 2023-06-20 | 西南大学 | Mulberry silk fibroin heavy chain protein polyclonal antibody and preparation method and application thereof |
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