CN103509107A - 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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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, relate in particular to a kind of method of preparing silk fibroin protein specific antibody.
Background technology
Silkworm is the important economic insects of China, take and produces silk as object.Silk is mainly comprised of fibroin and two kinds of protein of silk gum, but sericin comes off in silk reeling process, and therefore, in silk goods, silk product etc., main composition 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, is necessary to set up the determination method of 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 that utilizes tradition to be prepared by natural protein macromole is difficult to detect the antigen protein having been destroyed like this, so just be difficult to seek the origin of silk, and more difficult the seeking of age evidence more early.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 protein detection is Western Blotting or the ELISA method based on antibody-antigen at present, 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 of 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 1: utilize Fmoc method composition sequence for the polypeptide of " CGAGAGSGAGAGS ", and the halfcystine of the N end by described polypeptide makes this polypeptide and carrier keyhole limpet hemocyanin (KLH) coupling obtain complete antigen;
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 to carry out emulsification to obtain initial immunity antigen emulsion, use initial immunity antigen emulsion to carry out initial immunity to rabbit, then rabbit is carried out to booster immunization, what booster immunization was used is booster immunization antigen emulsion; Described booster immunization antigen emulsion is to be mixed with incomplete Freund's adjuvant by the complete antigen after described dilution, and then adds Streptomycin sulphate and penicillin solution to carry out emulsification to obtain; Tiring while reaching 1/10000 of antibody in rabbit blood sample, collects the blood of the rabbit after immunity, and clot is fully shunk and antiserum(antisera) is separated out completely, then collects antiserum(antisera) and centrifugal treating obtains supernatant liquor.
Further, the present invention is in described step 2, and when preparation initial immunity antigen emulsion, the complete antigen after dilution is that 1:1 mixes by volume with complete Freund's adjuvant; When preparation booster immunization antigen emulsion, the complete antigen after dilution is that 1:1 mixes by volume with incomplete Freund's adjuvant.
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 the subcutaneous multi-point injection that carries out, every injection 100 ul;
The method of rabbit being carried out to described booster immunization is: after initial immunity, within the 2nd, 4,6 weeks, carry out respectively one time booster immunization, each booster immunization is to use booster immunization antigen emulsion to the rear thigh of rabbit and the subcutaneous multi-point injection that carries out, every 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: collected blood is put under room temperature and allowed it solidify, then solidificating blood is placed in 37 ℃ of incubators and places 30 min, then be placed in 4 ℃ 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, through ELISA method, analyze known, this antibody and silk fibroin protein have obvious immune response, sensitivity is very high, can be used in detection silk fibroin protein, be particularly suitable for the detection analysis of imperfect silk fibroin.
(2) antibody that utilizes the macromole of natural protein to prepare from traditional method is different, antibody prepared by the present invention not only can detect complete silk fibroin, also can be for detection of the silk fibroin molecular such as having ruptured in Ancient Silk Textile, thereby for the silk goods residue in the early stage grave of China and ruins provides a kind of sensitivity special discrimination method efficiently, 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/) upper 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 is synthetic:
Utilize Fmoc method to synthesize haptens (i.e. " CGAGAGSGAGAGS " peptide sequence), at the N of " CGAGAGSGAGAGS " peptide sequence end, add halfcystine and make this polypeptide and KLH coupling.By the effect of linking agent, sulfydryl and KLH primary amine on the halfcystine of " CGAGAGSGAGAGS " N end form covalent linkage, thereby polypeptide and KLH coupling are got up, and obtain complete antigen.
With appropriate normal saline dilution complete antigen, then by complete antigen and complete Freund's adjuvant after dilution by volume 1:1 mix, add Streptomycin sulphate, penicillin solution to carry out emulsification, the antigen emulsion using while obtaining initial immunity (initial immunity antigen emulsion).In addition, by complete antigen and incomplete Freund's adjuvant after dilution above by volume 1:1 mix, add Streptomycin sulphate, penicillin solution to carry out emulsification, the antigen emulsion using while being strengthened immunity (booster immunization antigen emulsion).
2.
antibody preparation:
Choose 2 White Rabbits (14-16 age in week) and carry out initial immunity and booster immunization.Before immunity, first extracting rabbit ear blood sample contrasts.Initial immunity be with initial immunity antigen emulsion to the rear thigh of rabbit and the subcutaneous multi-point injection that carries out, every injection 100 ul.The 2nd week, 4 weeks, 6 weeks each booster immunizations after initial immunity once, each booster immunization be with booster immunization antigen emulsion to the rear thigh of rabbit and the subcutaneous multi-point injection that carries out, inject 100 ul for every.The 10th day venous blood sampling for the third time, after four immunity detects antiserum titre respectively.
Sero-fast tiring while reaching 1/10000 in rabbit blood sample, kills rabbit and collects blood, and a large amount of blood of collection are put under room temperature and allowed it solidify; Solidificating blood is placed in 37 ℃ of incubators and places 30 min, then be placed in 4 ℃ of refrigerator overnight, clot is fully shunk and antiserum(antisera) is separated out completely.Collect antiserum(antisera), 4 ℃ of 3000 centrifugal 10 min of g, packing supernatant liquor, puts-70 ℃ and stores for future use.
antibody purification:
Utilize immunoaffinity chromatography technical antagonism serum to carry out purifying.First synthetic haptens (being polypeptide " CGAGAGSGAGAGS ") and chromatographic stuffing Sulfo-link-gel are cross-linked, make the halfcystine sealing of " CGAGAGSGAGAGS " N end.With the PBS coupled columns of 20ml, 50mM, pH7.4, carry out pre-treatment, 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.With the PBS coupled columns of 20ml, 50mM, pH7.4, clean, the flow velocity of cleaning is 60ml/h.With the glycine-HCL antagonist of pH3.0,0.1M, carry out wash-out.After wash-out completes, use is containing 50mM, pH7.4, containing the PBS washing polypeptide post of 0.02% merthiolate, obtains antibody after purifying and in 4 ℃ of storages.
antibodies specific effect measuring: antigen silk fibroin is coated with the concentration of 1ug/ml, 4 ℃ of coated spending the night.Antibody is diluted respectively to 20000 times, 10000 times, 5000 times, 1000 times, 200 times, the amount loading in 50ul/ hole, incubation 1h at 37 ℃.With TBST, with 200ul/ hole, wash 2 times.ELIAS secondary antibody is used with 1:5000 dilution, incubation 1h at 50ul/ hole, 37 ℃.With TBST, with 200ul/ hole, wash 3 times.With 100ul/ hole, add TMB to develop the color in shady place, after colour developing 10min, with 100ul/ hole, add the H of 2M
2sO
4stop.By microplate reader, measure OD
450value.According to OD
450value, judges the situations such as combination of antigen-antibody.Work as OD
450when value reaches positive criteria, just can determine and in sample, contain fibroin; If do not contain fibroin, OD in sample
450value will, lower than positive criteria, just can judge the existence of fibroin accordingly fast.
interpretation of result: result show except blank be colourless, sample well all presents yellow.By microplate reader, measure OD
450value result is: the antibody after purifying is carried out to the OD that obtains after 20000 times, 10000 times, 5000 times, 1000 times, 200 times dilutions
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 positive requirement, even by 20000 times of the antibody dilutions of preparation, the positive signal of silk fibroin still can be detected, develops the color clear, and result is clear and definite.From above result, can illustrate, the antibody of being prepared by the inventive method is high to the reaction sensitivity of fibroin, and required antigen amount is few, and easy and simple to handle, drop into little, should large-scale promotion utilization.
Claims (5)
1. a method of utilizing feature polypeptide preparation silk fibroin protein specific antibody, is characterized in that, comprises the following steps:
Step 1: utilize Fmoc method composition sequence for the polypeptide of " CGAGAGSGAGAGS ", and the halfcystine of the N end by described polypeptide makes this polypeptide and keyhole limpet hemocyanin coupling obtain complete antigen;
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 to carry out emulsification to obtain initial immunity antigen emulsion, use initial immunity antigen emulsion to carry out initial immunity to rabbit, then rabbit is carried out to booster immunization, what booster immunization was used is booster immunization antigen emulsion; Described booster immunization antigen emulsion is to be mixed with incomplete Freund's adjuvant by the complete antigen after described dilution, and then adds Streptomycin sulphate and penicillin solution to carry out emulsification to obtain; Tiring while reaching 1/10000 of antibody in rabbit blood sample, collects the blood of the rabbit after immunity, and clot is fully shunk and antiserum(antisera) is separated out completely, then collects antiserum(antisera) and centrifugal treating obtains supernatant liquor.
2. method according to claim 1 and 2, is characterized in that: in described step 2, when preparation initial immunity antigen emulsion, the complete antigen after dilution is that 1:1 mixes by volume with complete Freund's adjuvant; When preparation booster immunization antigen emulsion, the complete antigen after dilution is that 1:1 mixes by volume with incomplete Freund's adjuvant.
3. method according to claim 1 and 2, is characterized in that: in described step 2, the method for rabbit being carried out to initial immunity is: use initial immunity antigen emulsion to the rear thigh of rabbit and the subcutaneous multi-point injection that carries out, every injection 100 ul;
The method of rabbit being carried out to booster immunization is: after initial immunity, within the 2nd, 4,6 weeks, carry out respectively one time booster immunization, each booster immunization is to use booster immunization antigen emulsion to the rear thigh of rabbit and the subcutaneous multi-point injection that carries out, every injection 100 ul.
4. method according to claim 1 and 2, it is characterized in that: in described step 2, make the method that clot fully shrinks and antiserum(antisera) is separated out completely be: collected blood is put under room temperature and allowed it solidify, then solidificating blood is placed in 37 ℃ of incubators and places 30 min, then be placed in 4 ℃ of refrigerator overnight.
5. method according to claim 3, it is characterized in that: in described step 2, make the method that clot fully shrinks and antiserum(antisera) is separated out completely be: collected blood is put under room temperature and allowed it solidify, then solidificating blood is placed in 37 ℃ of incubators and places 30 min, then be placed in 4 ℃ 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 |
| CN104406992A (en) * | 2014-12-31 | 2015-03-11 | 浙江理工大学 | Method for detecting ancient silk fabrics by transmission electron microscope |
| CN104459104A (en) * | 2014-12-31 | 2015-03-25 | 浙江理工大学 | Method for detecting antique silk fabric through microscope |
| CN104459118A (en) * | 2014-12-31 | 2015-03-25 | 浙江理工大学 | Method for preparing double antibody sandwich method test paper for antique silk fabric |
| CN104447989A (en) * | 2014-12-31 | 2015-03-25 | 浙江理工大学 | Silkworm fibroin antibody preparing method |
| CN104459162A (en) * | 2014-12-31 | 2015-03-25 | 浙江理工大学 | Method for preparing indirect competition method test paper for antique silk fabric |
| CN104459163A (en) * | 2014-12-31 | 2015-03-25 | 浙江理工大学 | Method for detecting antique silk fabric |
| CN104483478A (en) * | 2014-12-31 | 2015-04-01 | 浙江理工大学 | Detection method for ancient silk fabrics |
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| CN107266573A (en) * | 2017-08-01 | 2017-10-20 | 山西农业大学 | The polyclonal antibody and preparation method of a kind of ox cross-film epididymal proteins 1 |
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