CN102516381B - Natural sex storage protein-2 as well as preparation and use for same - Google Patents
Natural sex storage protein-2 as well as preparation and use for same Download PDFInfo
- Publication number
- CN102516381B CN102516381B CN201110426497XA CN201110426497A CN102516381B CN 102516381 B CN102516381 B CN 102516381B CN 201110426497X A CN201110426497X A CN 201110426497XA CN 201110426497 A CN201110426497 A CN 201110426497A CN 102516381 B CN102516381 B CN 102516381B
- Authority
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- China
- Prior art keywords
- storage protein
- centrifugal
- phe
- supernatant liquor
- sex storage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The invention relates to a preparation method for natural sex storage protein-2 and a use for the same, belonging to the field of biotechnology. The preparation method disclosed by the invention comprises the following steps of: using silkworm chrysalis as a raw material, homogenizing, filtering, centrifuging and collecting the supernatant, and then separating and purifying by the intrinsic characteristic of high temperature resistance of protein to obtain a high-purity natural sex storage protein-2 (SSP2), and using the same for cell culture. The method provided by the invention is easy to get raw material, low in production cost, simple and easy in operation, high in yield and suitable for large-scale production; additionally, the purified sex storage protein-2 can be used as a protective agent in cell culture for protecting cells from apoptosis due to cell stress.
Description
Technical field
The present invention relates to a kind of natural sex and store white 2 and its production and use, belong to biological technical field.
Background technology
Silkworm chrysalis is as unique insects food in " as the new resource for food list of bread and cheese management " of Ministry of Health's approval.It has high nutritive value, contains rich in protein, lipid acid and VITAMIN.Wherein (sex-specific storage-protein 2 SSP2), shows to have Cell protection to contained sex storage protein 2 through cell experiment, anti-apoptotic, somatotrophic effect.At present, bovine serum is necessary in the cell cultivation process, contain the necessary nutritive ingredient of abundant Growth of Cells, provide in conjunction with albumen, identify VITAMIN, lipid, metal ion, and be combined with toxic metal box heat source substance, play detoxification, the effect of potential of hydrogen damping fluid is played in the source that is cell attachment, sprawls the required factor of growth.But also there is more hidden danger in bovine serum, so its specification of quality is very strict, must prove from cows or country without mad cow disease, does not contain the inhibition to production vaccine virus, and without ground contaminations such as mycoplasma, viruses.And sex storage protein 2 preparation methods are simple, need not to consider the interference of above-mentioned pathogenic bacteria etc., and being expected to develop becomes the product that substitutes bovine serum, therefore has great research application prospect.
Summary of the invention
In view of this, the purpose of this invention is to provide a kind of natural sex and store white 2 and its production and use.The present invention with silkworm chrysalis as raw material, utilize the resistant to elevated temperatures characteristic of this albumen, obtain to have high biological activity by simple separation and purification means, highly purified sex storage protein 2, and replace serum to be used for cell cultures with it, and it is easy and simple to handle, and technical requirements is not high, output is large, is applicable to scale operation.
For achieving the above object, the invention provides a kind of natural sex storage protein 2, its aminoacid sequence is shown in SEQ ID NO:1.
The preparation method of above-mentioned natural sex storage protein 2 may further comprise the steps:
(1) silkworm chrysalis sample pretreatment: with 20-50mM phosphoric acid buffer or the distilled water of fresh silkworm chrysalis and 4-20 ℃ of precooling, join in the juice extractor homogenate 30 seconds by the mass volume ratio of 1:1, ice bath on ice 5 minutes, continue homogenate, repeat 3 times, the albumen in the silkworm chrysalis is fully dissolved; The homogenate sample is transferred in the Centrifuge Cup, 4 ℃, 8000-15000 rev/min, centrifugal 20-30 minute, centrifugal after supernatant liquor removes degrease by nine layers of filtered through gauze, repeated centrifugation and filtration step 3-5 time; Collect supernatant liquor, 4 ℃, 8000-15000 rev/min, centrifugal 20-30 minute, supernatant liquor was with 0.22 μ m or 0.45 μ m filter membrane vacuum filtration, 4 ℃ of preservations;
(2) initial gross separation purifying: the supernatant liquor that step (1) pre-treatment is obtained places 50 ℃ of waters bath with thermostatic control 30 minutes, 8000-15000 rev/min again, centrifugal 20-30 minute, get supernatant, nine layers of filtered through gauze, repeat 50 ℃ of water-baths, centrifugal and filtration step 3 times, collect supernatant liquor to place≤80 ℃ of waters bath with thermostatic control 30 minutes, 8000-15000 rev/min again, centrifugal 20-30 minute, get supernatant, nine layers of filtered through gauze, same step repeat (experimental results show that same temperature repeats 3 energy other foreign proteins are fully removed, but liquor capacity need to increase number of times greater than 2L) 3-5 time;
(3) further separation and purification: the supernatant liquor that step (2) initial gross separation purifying is obtained is with the ultrafiltration of 100kD ultra-filtration membrane bag, filtrate continues with the ultrafiltration of 10kD ultra-filtration membrane bag, respectively will be greater than 100kD, greater than 10kD, save backup less than 100kD with less than 4 ℃ of the filtrates of 10kD, get the filtrate sample Superdex greater than 100kD
TM200 separate, and rear concentrating collected at the peak that will contain target protein, and with the further separation and purification of Resource Q post, can obtain described natural sex storage protein 2.
Preferably, the acidity of phosphoric acid buffer is pH7.4-8.0 in the above-mentioned steps (1).
The present invention also provides the purposes of a kind of above-mentioned natural sex storage protein 2 in cell cultures, the natural sex storage protein 2 usefulness 30kD super filter tubes that above-mentioned further separation and purification is obtained concentrated quantitatively after, 4 ℃ are for subsequent use; Then prepare the cell culture medium that does not contain serum but contain the natural sex storage protein 2 of 25-1000 μ g/mL, concrete steps are as follows: sex storage protein 2 is carried out concentration determination; Cross 0.2 μ m or 0.22 μ m filter degerming after being diluted to certain concentration with serum free medium again; Cell needs to wash gently 2 times with serum free medium; The substratum that contains sex storage protein 2 for preparing is joined in the cell, cell is cultivated; Cultivate after five days, adopt mtt assay to detect cytoactive.
Preferably, the acidity of phosphoric acid buffer is pH7.4-8.0 in the above-mentioned steps (1).
The present invention also provides the purposes of above-mentioned natural sex storage protein 2 in cell cultures.
Preferably, after above-mentioned natural sex storage protein 2 usefulness 30kD super filter tubes concentrated quantitatively, 4 ℃ for subsequent use; Then prepare the cell culture medium that does not contain serum but contain the natural sex storage protein 2 of 25-100 μ g/mL, BmN cell, Hek293 cell are cultivated, and detected cytoactive with mtt assay.
The present invention can utilize silkworm chrysalis as raw material; obtain to have resistant to elevated temperatures sex storage protein by separation and purification; the method is easy and simple to handle; and output is high; be applicable to scale operation; the sex storage protein of purifying can be used as the protective material in the cell cultures, and Cell protection is in order to avoid cellular stress and cause apoptosis, and has no side effect.
Description of drawings
Fig. 1 is the electrophoretic analysis figure of natural sex storage protein 2 separation and purification; M: Low molecular weight proteins standard; 1: supernatant total protein after the dried silkworm chrysalis meal homogenate; 2:50 ℃ of water-bath 30min supernatant; 3:50 ℃ of water-bath 30min precipitation; 4:60 ℃ of water-bath 30min supernatant; 5:60 ℃ of water-bath 30min precipitation; 6:70 ℃ of water-bath 30min supernatant; 7:70 ℃ of water-bath 30min precipitation; 8:80 ℃ of water-bath 30min supernatant; 9:80 ℃ of water-bath 30min precipitation;
Fig. 2 is the Superdex of natural sex storage protein 2
TM200 analyze color atlas;
Fig. 3 is Superdex among Fig. 2
TMThe electrophoretic analysis figure of 200 separating natural sex storage proteins 2; M: Low molecular weight proteins standard; 1: former state (reductibility sample-loading buffer); 2: 1# peak among Fig. 2 (reductibility sample-loading buffer); 3: former state (irreducibility sample-loading buffer); 4: 1# peak among Fig. 2 (irreducibility sample-loading buffer);
Fig. 4 is the further analysis color atlas of Resource Q post; Wherein, 1# peak: not in conjunction with albumen; 2# peak: 280mM NaCl eluted protein;
Fig. 5 is the electrophoretic analysis figure at 2# peak among Fig. 4;
Fig. 6 is the evaluation mass spectrum of natural sex storage protein 2;
Fig. 7 is that the BmN cell non-serum is cultivated cell death inducing test MTT interpretation of result figure; Wherein, be serum-free without F, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, 1000 μ g/mL are respectively the final concentration of natural sex storage protein 2;
Fig. 8 is that the HEK293 cell non-serum is cultivated cell death inducing test MTT interpretation of result figure; Wherein, be serum-free without F, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, 1000 μ g/mL are respectively the final concentration of natural sex storage protein 2.
Embodiment
Be noted that following specifying all is exemplary, be intended to the invention provides further invention.Except as otherwise noted, all Science and Technology terms used herein have the identical meanings of usually understanding with the technical field of the invention personnel.
Elaborate particular content of the present invention below in conjunction with embodiment.
Embodiment 1:
The preparation method of a kind of natural sex storage protein 2 (SSP2) may further comprise the steps:
(1) silkworm chrysalis sample pretreatment: with the 50mM phosphoric acid buffer (pH7.4) of fresh silkworm chrysalis (available from Zhongqi Biological Pharmaceutical Co., Ltd., Zhejiang) with 4 ℃ of precoolings, joined in the juice extractor homogenate 30 seconds by the mass volume ratio of 1:1, ice bath on ice 5 minutes, continue homogenate, repeat 3 times, the albumen in the silkworm chrysalis is fully dissolved; The homogenate sample is transferred in the Centrifuge Cup, 4 ℃, 15000 rev/mins, centrifugal 20 minutes, centrifugal after supernatant liquor removes degrease by nine layers of filtered through gauze, repeated centrifugation and filtration step 3 times; Collect supernatant liquor, 4 ℃, 15000 rev/mins, centrifugal 20 minutes, supernatant liquor was with 0.45 μ m filter membrane vacuum filtration, 4 ℃ of preservations, and as shown in Figure 1, the protein electrophoresis figure that obtains is No. 1 swimming lane;
(2) initial gross separation purifying: the supernatant liquor that pre-treatment is obtained places 50 ℃ of waters bath with thermostatic control 30 minutes, and 12000 rev/mins again, centrifugal 20 minutes, get supernatant, nine layers of filtered through gauze repeat 50 ℃ of water-baths, centrifugal and filtration step 3 times; Collect supernatant liquor and place 80 ℃ of waters bath with thermostatic control 30 minutes, 15000 rev/mins again, centrifugal 20 minutes, get supernatant, nine layers of filtered through gauze, same step repeats 3 times, it is for subsequent use to collect 80 ℃ of 4 ℃ of pre-treatment supernatant liquors, as shown in Figure 1, the protein electrophoresis figure that obtains is No. 8 swimming lane (illustrate that sex storage protein 2 is present in 80 ℃ of supernatant liquors, the anti-80 ℃ of high temperature of sex storage protein also are described simultaneously);
(3) further separation and purification: the supernatant liquor that step (2) initial gross separation purifying is obtained is with the ultrafiltration of 100kD ultra-filtration membrane bag, filtrate continues with the ultrafiltration of 10kD ultra-filtration membrane bag, respectively will be greater than 100kD, greater than 10kD, save backup less than 100kD with less than 4 ℃ of the filtrates of 10kD; Get the filtrate sample Superdex greater than 100kD
TM200 separate, the peak that will contain target protein is collected rear concentrated, it is 1# marker peak shown in Figure 2, its electrophorogram is (band of arrow indication position is the electrophoretic band of sex storage protein 2) as shown in Figure 3, and with the further separation and purification (see figure 4) of Resource Q post, 2# peak among Fig. 4 collected and carry out electrophoresis, its electrophorogram is (arrow indication position is the electrophoretic band of sex storage protein 2) as shown in Figure 5, can obtain purer natural sex storage protein 2, the protein sample that this purifying is obtained, carry out mass spectrometric detection, result such as accompanying drawing 6, the albumen fraction of coverage that is numbered Bmb013502 in the peptide fingerprinting spectrum of this albumen and the white database of Southwestern silkworm egg reaches 53.9118%, peptide section coupling number is 41 sections, black character is concrete coupling peptide section in the square frame of Fig. 6 upper right corner, and obtains its aminoacid sequence shown in SEQ ID NO:1, proves that this albumen is natural sex storage protein 2.
The purposes of above-mentioned natural sex storage protein 2 in cell cultures: with above-mentioned steps (3) further natural sex storage protein 2 usefulness the 30kD super filter tubes that obtain of separation and purification concentrated quantitative after, 4 ℃ are for subsequent use; Because the homology of sex storage protein 2 and storage protein 2 reaches 90.65%, so design is to BmN cell (Institutes Of Technology Of Zhejiang's Life Science College biochemical research is presented), the cytosis test of Hek293 cell (available from Beijing gold amethyst biological medicine technology company limited), preparation does not contain serum, contain the cell culture medium of the natural sex storage protein 2 of finite concentration (i.e. 25 μ g/mL-1000 μ g/mL), concrete steps are as follows: this natural sex storage protein 2 is carried out concentration determination; Using serum free medium (BmN cell non-serum culture medium SF900 II is available from U.S. Gibico company, if Hek293 cell non-serum culture medium DMEM is available from Bioroc hundred gram medical biotechnology limited liability companys) to be diluted to respectively final concentration is to cross 0.2 μ m filter degerming behind 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, the 1000 μ g/mL again; BmN cell and Hek293 cell need wash 2 times gently with BmN cell non-serum culture medium and Hek293 cell non-serum culture medium; These substratum that contain this natural sex storage protein 2 that prepare are joined respectively in above-mentioned BmN cell and the Hek293 cell, these cells are cultivated; Cultivate after five days, adopt mtt assay to detect the activity of these cells.The results show; height (shown in Fig. 7,8) in the specific activity serum free medium of BmN cell and Hek293 cell under the condition that natural sex storage protein 2 final concentrations are 50 μ g/mL; can be applicable to cell cultures; therefore the natural sex storage protein 2 that obtains of this separation and purification; can Cell protection, have the effect of anti-apoptotic.
Embodiment 2:
The preparation method of a kind of natural sex storage protein 2 (SSP2) culturing cell may further comprise the steps:
(1) silkworm chrysalis sample pretreatment: with the distilled water of fresh silkworm chrysalis (available from Zhongqi Biological Pharmaceutical Co., Ltd., Zhejiang) with 20 ℃ of precoolings, joined in the juice extractor homogenate 30 seconds by the mass volume ratio of 1:1, ice bath on ice 5 minutes, continue homogenate, repeat 3 times, the albumen in the silkworm chrysalis is fully dissolved; The homogenate sample is transferred in the Centrifuge Cup, 4 ℃, 8000 rev/mins, centrifugal 30 minutes, centrifugal after supernatant liquor removes degrease by nine layers of filtered through gauze, repeated centrifugation and filtration step 3 times; Collect supernatant liquor, 4 ℃, 8000 rev/mins, centrifugal 30 minutes, supernatant liquor was with 0.45 μ m filter membrane vacuum filtration, 4 ℃ of preservations, and as shown in Figure 1, the protein electrophoresis figure that obtains is No. 1 swimming lane;
(2) initial gross separation purifying: the supernatant liquor that pre-treatment is obtained places 50 ℃ of waters bath with thermostatic control 30 minutes, and 8000 rev/mins again, centrifugal 30 minutes, get supernatant, nine layers of filtered through gauze repeat 50 ℃ of water-baths, centrifugal and filtration step 3 times; Collect supernatant liquor and place 80 ℃ of waters bath with thermostatic control 30 minutes, 8000 rev/mins again, centrifugal 30 minutes, get supernatant, nine layers of filtered through gauze, same step repeats 3 times, it is for subsequent use to collect 80 ℃ of 4 ℃ of pre-treatment supernatant liquors, and as shown in Figure 1, the protein electrophoresis figure that obtains is No. 8 swimming lane;
(3) further separation and purification: the supernatant liquor that step (2) initial gross separation purifying is obtained is with the ultrafiltration of 100kD ultra-filtration membrane bag, filtrate continues with the ultrafiltration of 10kD ultra-filtration membrane bag, respectively will be greater than 100kD, greater than 10kD, save backup less than 100kD with less than 4 ℃ of the filtrates of 10kD; Get the filtrate sample Superdex greater than 100kD
TM200 separate, the peak that will contain target protein is collected rear concentrated, it is 1# marker peak shown in Figure 2, its electrophorogram as shown in Figure 3, and with the further separation and purification (see figure 4) of Resource Q post, 2# peak among Fig. 4 collected and carry out electrophoresis, its electrophorogram as shown in Figure 5, can obtain purer natural sex storage protein 2, the protein sample that this purifying is obtained, carry out mass spectrometric detection, the result as shown in Figure 6, the albumen fraction of coverage that is numbered Bmb013502 in the peptide fingerprinting of this albumen spectrum and the white database of Southwestern silkworm egg reaches 53.9118%, peptide section coupling number is 41 sections, black character is concrete coupling peptide section in the square frame of Fig. 6 upper right corner, and obtains its aminoacid sequence shown in SEQ ID NO:1, proves that this albumen is natural sex storage protein 2.
The purposes of above-mentioned natural sex storage protein 2 in cell cultures: with above-mentioned steps (3) further natural sex storage protein 2 usefulness the 30kD super filter tubes that obtain of separation and purification concentrated quantitative after, 4 ℃ are for subsequent use; Because the homology of sex storage protein 2 and storage protein 2 reaches 90.65%, so design is to BmN cell (Institutes Of Technology Of Zhejiang's Life Science College biochemical research is presented), the cytosis test of Hek293 cell (available from Beijing gold amethyst biological medicine technology company limited), preparation does not contain serum, contain the cell culture medium of the natural sex storage protein 2 of finite concentration (i.e. 25 μ g/mL-1000 μ g/mL), concrete steps are as follows: this natural sex storage protein 2 is carried out concentration determination; Using serum free medium (BmN cell non-serum culture medium SF900 II is available from U.S. Gibico company, if Hek293 cell non-serum culture medium DMEM is available from Bioroc hundred gram medical biotechnology limited liability companys) to be diluted to respectively final concentration is to cross 0.22 μ m filter degerming behind 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, the 1000 μ g/mL again; BmN cell and Hek293 cell need wash 2 times gently with BmN cell non-serum culture medium and Hek293 cell non-serum culture medium; These substratum that contain this natural sex storage protein 2 that prepare are joined respectively in above-mentioned BmN cell and the Hek293 cell, these cells are cultivated; Cultivate after five days, adopt mtt assay to detect the activity of these cells.The results show; high in the specific activity serum free medium of BmN cell and Hek293 cell under the condition that natural sex storage protein 2 final concentrations are 50 μ g/mL; can be applicable to cell cultures; therefore the natural sex storage protein 2 that obtains of this separation and purification; can Cell protection, have the effect of anti-apoptotic.
In a word, the present invention can utilize silkworm chrysalis as raw material, obtains to have resistant to elevated temperatures natural sex storage protein 2 by separation and purification, and the natural sex storage protein 2 of purifying can be used as the protective material in the cell cultures, Cell protection is in order to avoid cellular stress and cause apoptosis, and has no side effect; The method is easy and simple to handle, and the result is stable, and repetition rate height and output are high, are applicable to scale operation.
The above is the preferred embodiments of the present invention only, it is pointed out that for the those of ordinary skill in the present technique, under the prerequisite that does not break away from core technology feature of the present invention, can also save and use Superdex in the step 3
TM200, the treatment process of Resource Q, the sample that separation and purification obtains does not affect the application in cell cultures.
SEQUENCE LISTING
＜110〉Tianjin Yaoyu Biotechnology Co., Ltd.
＜120〉natural sex storage protein 2 and its production and use
<130> 111707-I-CP-TJYU
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 703
<212> PRT
＜213〉the sex storage protein 2
<400> 1
Met Lys Ser Val Leu Ile Leu Ala Gly Leu Val Ala Val Ala Leu Ser
1 5 10 15
Ser Ala Val Pro Lys Pro Ser Thr Ile Lys Thr Lys Asn Val Asp Ala
20 25 30
Val Phe Val Glu Lys Gln Lys Lys Ile Leu Ser Phe Phe Gln Asp Val
35 40 45
Ser Gln Leu Asn Thr Asp Asp Glu Tyr Tyr Lys Ile Gly Lys Asp Tyr
50 55 60
Asp Ile Glu Met Asn Met Asp Asn Tyr Thr Asn Lys Lys Ala Val Glu
65 70 75 80
Glu Phe Leu Lys Met Tyr Arg Thr Gly Phe Met Pro Lys Asn Leu Glu
85 90 95
Phe Ser Val Phe Tyr Asp Lys Met Arg Asp Glu Ala Ile Ala Leu Phe
100 105 110
His Leu Phe Tyr Tyr Ala Lys Asp Phe Glu Thr Phe Tyr Lys Thr Ala
115 120 125
Cys Phe Ala Arg Val His Leu Asn Gln Gly Gln Phe Leu Tyr Ala Phe
130 135 140
Tyr Ile Ala Val Ile Gln Arg Ser Asp Cys His Gly Phe Val Val Pro
145 150 155 160
Ala Pro Tyr Glu Val Tyr Pro Lys Met Phe Met Asn Met Glu Val Leu
165 170 175
Gln Lys Ile Tyr Val Thr Lys Met Gln Asp Gly Leu Ile Asn Pro Glu
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Ala Ala Ala Lys Tyr Gly Ile His Lys Glu Asn Asp Tyr Phe Val Tyr
195 200 205
Lys Ala Asn Tyr Ser Asn Ala Val Leu Tyr Asn Asn Glu Glu Gln Arg
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Leu Thr Tyr Phe Thr Glu Asp Ile Gly Met Asn Ala Tyr Tyr Tyr Tyr
225 230 235 240
Phe His Ser His Leu Pro Phe Trp Trp Thr Ser Glu Lys Tyr Gly Ala
245 250 255
Leu Lys Glu Arg Arg Gly Glu Val Tyr Phe Tyr Phe Tyr Gln Gln Leu
260 265 270
Leu Ala Arg Tyr Tyr Phe Glu Arg Leu Thr Asn Gly Leu Gly Lys Ile
275 280 285
Pro Glu Phe Ser Trp Tyr Ser Pro Ile Lys Thr Gly Tyr Tyr Pro Leu
290 295 300
Met Leu Thr Lys Phe Thr Pro Phe Ala Gln Arg Pro Asp Tyr Tyr Asn
305 310 315 320
Leu His Thr Glu Glu Asn Tyr Glu Arg Val Arg Phe Leu Asp Thr Tyr
325 330 335
Glu Lys Thr Phe Val Gln Phe Leu Gln Lys Asp His Phe Glu Ala Phe
340 345 350
Gly Gln Lys Ile Asp Phe His Asp Pro Lys Ala Ile Asn Phe Val Gly
355 360 365
Asn Tyr Trp Gln Asp Asn Ala Asp Leu Tyr Gly Glu Glu Val Thr Lys
370 375 380
Asp Tyr Gln Arg Ser Tyr Glu Val Phe Ala Arg Arg Val Leu Gly Ala
385 390 395 400
Ala Pro Met Pro Phe Asp Lys Tyr Thr Phe Met Pro Ser Ala Met Asp
405 410 415
Phe Tyr Gln Thr Ser Leu Arg Asp Pro Ala Phe Tyr Gln Leu Tyr Asn
420 425 430
Arg Ile Val Glu Tyr Ile Val Glu Phe Lys Gln Tyr Leu Lys Pro Tyr
435 440 445
Thr Gln Asp Lys Leu Tyr Phe Asp Gly Val Lys Ile Thr Asp Val Lys
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Val Asp Lys Leu Thr Thr Phe Phe Glu Asn Phe Glu Phe Asp Ala Ser
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Asn Ser Val Tyr Phe Ser Lys Glu Glu Ile Lys Asn Asn His Val His
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Asp Val Lys Val Arg Gln Pro Arg Leu Asn His Ser Pro Phe Asn Val
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Asn Ile Glu Val Asp Ser Asn Val Ala Ser Asp Ala Val Val Lys Ile
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Phe Leu Ala Pro Lys Tyr Asp Asp Asn Gly Ile Pro Leu Thr Leu Glu
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Asp Asn Trp Met Lys Phe Phe Glu Leu Asp Trp Phe Thr Thr Lys Leu
545 550 555 560
Thr Ala Gly Gln Asn Lys Ile Ile Arg Asn Ser Asn Glu Phe Val Ile
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Phe Lys Glu Asp Ser Val Pro Met Thr Glu Ile Met Lys Met Leu Asp
580 585 590
Glu Gly Lys Val Pro Phe Asp Met Ser Glu Glu Phe Cys Tyr Met Pro
595 600 605
Lys Arg Leu Met Leu Pro Arg Gly Thr Glu Gly Gly Phe Pro Phe Gln
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Leu Phe Val Phe Val Tyr Pro Phe Asp Asn Lys Gly Lys Asp Leu Ala
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Pro Phe Glu Ser Phe Val Leu Asp Asn Lys Pro Leu Gly Phe Pro Leu
645 650 655
Asp Arg Pro Val Val Asp Ala Leu Phe Lys Val Pro Asn Met Tyr Phe
660 665 670
Lys Asp Ile Phe Ile Tyr His Glu Gly Glu Arg Phe Pro Tyr Lys Phe
675 680 685
Asn Ile Pro Ser Tyr Asp Thr Gln Ser Asn Val Val Pro Lys Asn
690 695 700
Claims (2)
1. the preparation method of a natural sex storage protein 2 is characterized in that, may further comprise the steps:
(1) silkworm chrysalis sample pretreatment: with 20-50mM phosphoric acid buffer or the distilled water of fresh silkworm chrysalis and 4 ℃ of-20 ℃ of precoolings, join in the juice extractor homogenate 30 seconds by the mass volume ratio of 1:1, ice bath on ice 5 minutes, continue homogenate, repeat 3 times, the albumen in the silkworm chrysalis is fully dissolved; The homogenate sample is transferred in the Centrifuge Cup, 4 ℃, 8000-15000 rev/min, centrifugal 20-30 minute, centrifugal after supernatant liquor removes degrease by nine layers of filtered through gauze, repeated centrifugation and filtration step 3 times; Collect supernatant liquor, 4 ℃, 8000-15000 rev/min, centrifugal 20-30 minute, supernatant liquor was with 0.22 μ m or 0.45 μ m filter membrane vacuum filtration, 4 ℃ of preservations;
(2) initial gross separation purifying: the supernatant liquor that step (1) pre-treatment is obtained places 50 ℃ of waters bath with thermostatic control 30 minutes, and 8000-15000 rev/min, centrifugal 20-30 minute, get supernatant, nine layers of filtered through gauze repeat 50 ℃ of water-baths, centrifugal and filtration step 3-5 time, collect supernatant liquor to place≤80 ℃ of waters bath with thermostatic control 30 minutes, 8000-15000 rev/min again, centrifugal 20-30 minute, get supernatant liquor, nine layers of filtered through gauze, same step repeats 3-5 time, until 80 ℃ of waters bath with thermostatic control are without Precipitation;
(3) further separation and purification: the supernatant liquor that step (2) initial gross separation purifying is obtained is with 100kD ultra-filtration membrane bag ultrafiltration and concentration, will save backup greater than 4 ℃ of the concentrated solutions of 100kD, gets the concentrated solution liquid sample Superdex greater than 100kD
TM200 separate, and rear concentrating collected at the peak that will contain target protein, and with the further separation and purification of Resource Q post, can obtain described natural sex storage protein 2, its aminoacid sequence such as SEQ ID NO:1.
2. method according to claim 1 is characterized in that, the acidity of phosphoric acid buffer is pH7.4-8.0 in the described step (1).
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