CN102977200B - Bombyx natural immune protein Hemolin and purifying method thereof - Google Patents
Bombyx natural immune protein Hemolin and purifying method thereof Download PDFInfo
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- CN102977200B CN102977200B CN201210452712.8A CN201210452712A CN102977200B CN 102977200 B CN102977200 B CN 102977200B CN 201210452712 A CN201210452712 A CN 201210452712A CN 102977200 B CN102977200 B CN 102977200B
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Abstract
The invention relates to a bombyx natural immune protein Hemolin and a purifying method of the bombyx natural immune protein Hemolin and belongs to the field of protein purifying. Silkworms are taken as raw material, a component containing immune proteins is obtained with different centrifugation methods and then through ultrafiltration, supernatant liquid containing the target immune protein is subjected to separation and purification through a simple chromotography technique in an AKTA explorer 10 protein purification system, and the target immune protein Hemolin is obtained. The purifying method of the bombyx natural immune protein Hemolin mainly use Superdex 200 and Superdex 75 gel chromatography technique, provides a novel solution method for natural immune protein purification, and is of significant meaning.
Description
Technical field
The present invention relates to a kind of silkworm natural immunity albumen Hemolin and purification process thereof, belong to protein technical field.
Background technology
Hemolin, as the peculiar a kind of Buchner's bodies of insect, has important meaning in the immunologic mechanism of research insect.At present the research of its structure and function is all obtained to certain achievement, but less to its functional study in vitro, so this experiment obtains purer Hemolin by simple purifying, further the structure and function of this albumen of research lays the foundation, and also can do more deep exploration to its structure on this basis.
Because the complicated components of gene engineering product, to the purity requirement of product there is the trend of assembling sex change in higher and albumen in purge process, so its preparation and purification ratio common product are wanted the many of difficulty.Although people generally use preparation HPLC to carry out the final step purifying of genetic engineered product, but because HPLC has having relatively high expectations to software and hardware, early investment is large, purifying amount is limited, expensive, analyze biomacromolecule and mineral ion difficulty, the large and virose shortcoming such as in the majority of moving phase consumption.And Superdex 75 protein purification systems have the advantages such as purifying amount is large, flow velocity is fast, and charge capacity is larger, and chromatography condition is easy to control, and non-specific adsorption is little.
Summary of the invention
In view of this, the object of this invention is to provide a kind of silkworm natural immunity albumen Hemolin and purification process thereof, the silkworm chrysalis that the method infects taking silkworm baculovirus, as raw material, obtains the solution that contains natural immunity albumen by multiple centrifugal method; Mother liquor is carried out after determination of activity, and by mother liquor desugar high activity, desugar solution and mother liquor are crossed 300K film bag in 10:1 ratio by the method for grand suction and are removed sugar; The mother liquor of desaccharification is mixed by 1:2.5 with protein extract, in 4 DEG C of refrigerators, slowly stir and spend the night; Pass through afterwards weak anionic exchange column (DEAE) initial gross separation, then be further purified by sepharose post (SuperdexTM 200, SuperdexTM 75).
For achieving the above object, the invention provides a kind of silkworm natural immunity albumen Hemolin, its aminoacid sequence is as shown in SEQ ID NO.1.
The purification process of above-mentioned silkworm natural immunity albumen Hemolin, comprises the following steps:
(1) preparation of silkworm immune protein mother liquor;
(2) separation and purification of silkworm natural immunity albumen.
Further, wherein described in step (1), the preparation of silkworm immune protein mother liquor specifically comprises:
A. be stored in the silkworm chrysalis of-20 DEG C and the sterilizing ddH of 4 DEG C of precoolings
2o is blended in 2 min of homogenate on ice with the weight percent of 1:3, and homogenate is placed in and stops 5 min on ice, repeats 9 times;
B. in 15000 rpm, 4 DEG C of centrifugal 60 min, repeat 5 times, use for the last time 4 layers of filtered through gauze;
C. the supernatant liquor after filtering is got 200mL and water by the volume mixture of 1:1, with 0.22 μ m film packet filtering, repeats 10 times, and supernatant solution returns to the volume of 200mL;
D. step c gained supernatant liquor is in 50000rpm, after 10 DEG C of centrifugal 40min, and the resuspended band centrifugation that carries out of ultrafiltrated for the black group of appearance; Sample thief 300ml carries out sucrose density gradient centrifugation 3h;
E. sample under the sucrose solution that is 60% by the supernatant liquor of gained after centrifugal steps d 3h with mass body volume concentrations, collects by 35mL/ pipe, and in every pipe, gets 3mL and do specific activity mensuration.
Further, wherein the sucrose density gradient centrifugation of sample described in steps d is: the phosphate buffered saline buffer 200ml of paved pH 7.4, and sample 300ml, 30% sucrose 400ml, 55% sucrose is full of, 22000rpm density gradient centrifugation 3h.
Further, wherein the condition of band centrifugation described in steps d is: 50000rpm, 10 DEG C of centrifugal 40min; Sample is step c gained supernatant liquor; Sucrose concentration is mass body volume concentrations.
Further, wherein the preparation method of the phosphate buffered saline buffer of pH 7.4 described in steps d is: by 8g NaCl, 0.2g KCl, 3.58g Na
2hPO
412H
2o, 0.27g KH
2pO
4be dissolved in 800mL aseptic deionized water, be settled to 1000mL and get final product.
Further, wherein described in step (2), the separation and purification of silkworm natural immunity albumen specifically comprises:
A. activity is pressed to the volume ratio of 1:10 by the method for grand suction higher than 26 mother liquor and desugar component, cross 300K film bag and remove sugar;
B. the mother liquor of desaccharification is mixed by the volume ratio of 1:2.5 with protein extract, in 4 DEG C of refrigerators, slowly stir and spend the night;
C. after, by weak anionic exchange column DEAE initial gross separation, separating obtained solution is concentrated with the super filter tube of 100kD;
D. sepharose post separates, and obtains 8 peaks, and each different peak is in batches concentrated;
E. the concentrated peak containing target protein, is further purified and obtains target protein with sepharose post, and identifies with SDS-PAGE.
Further, wherein sepharose post described in step (d) is SuperdexTM 200 gel columns.
Further, wherein sepharose post described in step (e) is SuperdexTM 75 gel columns.
The present invention is using silkworm chrysalis as raw material, by different centrifugal methods, ultrafiltration, obtain the component that contains immune protein, the supernatant solution that contains target protein is so passed through to simple chromatographic technique separation and purification in AKTA explorer 10 protein purification systems (SuperdexTM 200, SuperdexTM 75) and obtain target protein Hemolin; In addition, the selected chromatography condition of the present invention not only effectively purifying target protein Hemolin, and effectively prevented the inactivation sex change of mark Hemolin, finally prepared the higher Hemolin with natural radioactivity of purity.
Brief description of the drawings
Fig. 1 is weak anionic exchange column (DEAE) the initial gross separation color atlas of silkworm natural immunity albumen Hemolin;
Fig. 2 A is one of SDS-PAGE collection of illustrative plates of weak anionic exchange column (DEAE) initial gross separation of silkworm natural immunity albumen Hemolin; Wherein 4,5,17,21,25,32 correspond respectively to the pipe that separation obtains;
Fig. 2 B be weak anionic exchange column (DEAE) initial gross separation of silkworm natural immunity albumen Hemolin SDS-PAGE collection of illustrative plates two; Wherein 37,38,63,73,75,76 etc. numerals correspond respectively to and separate the pipe obtaining;
Fig. 3 is the color atlas that SuperdexTM 200 separates;
Fig. 4 A is one of SDS-PAGE spectrogram after SuperdexTM 200 separates; Wherein 23,27,31,34,39,41,44,47 correspond respectively to the pipe that separation obtains;
Fig. 4 B is two of SDS-PAGE spectrogram after SuperdexTM 200 separates; Wherein the numerals such as 52,56,60,66,68,73,75 correspond respectively to and separate the pipe obtaining;
Fig. 5 is by the color atlas that separates with SuperdexTM 75 after the sample concentration of repeatedly collecting for No. 47;
Fig. 6 is to be the SDS-PAGE qualification figure of first peak of Fig. 4 after concentrated;
Fig. 7 is by the Mass Spectrometric Identification figure (one-level) sampling in the last item swimming lane of first glue in Fig. 3;
Fig. 8 is by the mass spectrum (secondary) sampling in the last item swimming lane of first glue in Fig. 3;
The peptide section sampling in the last item swimming lane of first glue in Fig. 3 is compared logarithm by Fig. 9.
Embodiment
Be noted that following illustrating is all example, be intended to further illustrate the invention provides, except as otherwise noted, it is identical that all Science and Technology terms used herein have the implication of conventionally understanding with the technical field of the invention personnel.
Below in conjunction with drawings and Examples, the present invention will be further described.
Embodiment 1: the preparation of silkworm immune protein mother liquor
Step 1: be stored in the silkworm chrysalis (being purchased from Zhongqi Biological Pharmaceutical Co., Ltd., Zhejiang) of-20 DEG C and the ddH of 4 DEG C of precoolings of sterilizing
2o is blended in 2 min of homogenate on ice with the weight percent of 1:3, and homogenate is placed in and stops 5 min on ice, repeats 9 times;
Step 2:15000 rpm, 4 DEG C of centrifugal 1h, supernatant discarded, the resuspended precipitation of water, then in 15000 rpm, 4 DEG C of centrifugal 1h, so repeat 5 times, use for the last time 4 layers of filtered through gauze;
Step 3: the supernatant liquor after filtration is got 200mL and water by the volume mixture of 1:1, with 0.22 μ m film packet filtering, repeats 10 times, and supernatant solution returns to the volume of 200mL;
Step 4: gained supernatant liquor is in 50000rpm, after 10 DEG C of centrifugal 40min, the resuspended band centrifugation (50000rpm, 10 DEG C of centrifugal 40min) that carries out of ultrafiltrated for the black group of appearance; Sample thief 300ml carries out sucrose density gradient centrifugation 3h, and (specific operation process is: paved PBS (phosphate buffered saline buffer of pH 7.4) 200ml; sample (step 3 gained supernatant liquor) 300ml; 30% (mass body volume concentrations) sucrose 400ml; 55% (mass body volume concentrations) sucrose is full of, 22000rpm density gradient centrifugation 3h);
Step 5: the supernatant liquor of gained after centrifugal step 4 3h is extruded the solution of different sucrose with the sucrose solution of 60% (mass body volume concentrations), be lower sample, collect by 35mL/ pipe, and get 3mL and do specific activity mensuration in every pipe.
Embodiment 2: the separation and purification of silkworm natural immunity albumen
(1) by activity higher than 2
6mother liquor (detect sample protein content by BCA method, activity=2n × 4/ sample concentration (mg/mL), the hole count of red cell agglutination occurs n=) get 200mL with desugar component by the volume ratio of 1:10 by the method for grand suction, 300K film bag removal sugar.Last volume returns to 200mL;
(2) mother liquor of desaccharification is mixed by the volume ratio of 1:2.5 with protein extract, in 4 DEG C of refrigerators, slowly stir and spend the night;
(3) afterwards by weak anionic exchange column (DEAE, be purchased from the company purchased from GE Healthcare) initial gross separation: the protein sample of gained in above-mentioned steps (2) is mixed by the volume ratio of 2:1 with DEAE, in 4 DEG C of refrigerators, slowly stir after 2h, DEAE filler is poured in pillar; Balance pillar also ensures that pillar is moist without bubble and filler; After being combined with system, separate as shown in Figure 1, the ultraviolet light absorption value of the protein sample of 10 to No. 50, up to 3000, illustrates that protein concentration is high; And the protein sample that absorption extraction is obtained carries out SDS-PAGE qualification, as shown in Fig. 2 A, Fig. 2 B, most albumen all concentrates between 25kDa to 60kDa, and protein concentration is the highest between 45kDa to 60kDa; The collection sample of 10 to 50 pipes is concentrated with the super filter tube of 100kD;
(4) sepharose post (SuperdexTM 200 is purchased from the company purchased from GE Healthcare) separates, and obtains 8 peaks (seeing Fig. 2 A, Fig. 2 B); As shown in Figure 3, obtain 8 peaks, collect by peak; SDS-PAGE qualification, as shown in Fig. 4 A, Fig. 4 B, protein sample can separate, and the albumen of No. 47 pipe only has two bands, illustrates that the protein ratio of gained is purer in the time managing for No. 47;
(5) repeating step (4) is all collected 47 pipes concentrated with the super filter tube of 30kD at every turn; Use afterwards sepharose post (SuperdexTM 75, purchased from GE Healthcare company) to be further purified, as shown in Figure 5, have 4 main peaks, collect by peak; First peak is identified to as shown in Figure 6, only have the band of an entry with SDS-PAGE, illustrated that separating obtained target protein is purer.
Embodiment 3: the Mass Spectrometric Identification of silkworm natural immunity albumen Hemolin
1. sample preparation: by after the second band target protein rubber tapping of No. 47 in Fig. 4 A, be cut into 1mm
3the fritter of left and right, packs in centrifuge tube.Deliver to Mass Spectrometric Identification company.Attention: ensure the clean of all article that sample contacts with it, prevent the pollution of foreign protein and other pollutents.
2. mass spectrum result is: identifying that above-mentioned sample is silkworm natural immunity albumen Hemolin, as shown in Figure 7, is one-level mass spectrum, as shown in Figure 8, is second order ms figure; The albumen fraction of coverage of numbering gi69146821 in peptide fingerprinting spectrum and the Southwestern large protein storehouse of this albumen reaches 49%, and obtains its aminoacid sequence as shown in Fig. 9 and SEQ ID NO.1, proves that this albumen is silkworm natural immunity albumen Hemolin.
The present invention is using silkworm chrysalis as raw material, by different centrifugal methods, ultrafiltration, obtain the component that contains immune protein, then after processing with protein extract, then through three step chromatographic separation purifying, obtain target protein Hemolin, the purifying of natural immunity albumen has been proposed to a kind of brand-new solution, significant.
The above, be only embodiments of the invention, should be understood that; for the those of ordinary skill in this technology; not departing under the prerequisite of core technology feature of the present invention, can also do some improvements and modifications, these retouchings and improvement also should belong to scope of patent protection of the present invention.
SEQUENCE LISTING
<110> Tianjin Yaoyu Biotechnology Co., Ltd.
<120> silkworm natural immunity albumen Hemolin and purification process thereof
<130> 122054-I-CP-TJYU
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 410
<212> PRT
<213> Hemolin albumen
<400> 2
Met Asn Ser Trp Thr Leu Leu Val Leu Gly Thr Cys Val Ile Tyr Thr
1 5 10 15
Thr Gly Gln Pro Val Asn Ser Gly Asp Lys Val Pro Val Leu Lys Glu
20 25 30
Ala Pro Ala Glu Val Leu Phe Arg Glu Gly Gln Ala Thr Arg Leu Glu
35 40 45
Cys Ala Thr Glu Gly Asp Asp Ser Gly Val Glu Tyr Ser Trp Arg Lys
50 55 60
Asp Gly Met His Phe Ser Val Gly Leu Asp Thr Leu Thr Thr Ile Asp
65 70 75 80
Ala Gly Ser Leu Val Phe Ser Gln Thr Lys Ala Ser Asp Glu Gly Glu
85 90 95
Tyr Gln Cys Phe Ala Lys Ser Asp Phe Gly Val Ala Ser Thr Arg Ala
100 105 110
Thr Lys Leu Arg Arg Thr Tyr Ile Glu Thr Pro Ala Phe Glu Glu Lys
115 120 125
Lys Val Thr Val Val Glu Gly Lys Pro Phe Glu Leu Arg Cys Pro Val
130 135 140
Pro Gly Gly Tyr Pro Lys Pro Thr Ile Ser Trp Met Arg His His Asp
145 150 155 160
Glu Asp Gly Ser Thr Glu Asn Phe Met Asp Arg Arg Ala Thr Tyr Ser
165 170 175
Pro Glu Gly Thr Leu Tyr Phe Ser Asn Ala Ser Leu Asp Asp Ala Asn
180 185 190
Asp Lys Thr Lys Leu Val Cys Met Ala Ser Ser Pro Ala Ala Asp Glu
195 200 205
Gly Val Pro Ile Val Thr Tyr Tyr Ile Thr Gln Val Thr Pro Ala Ser
210 215 220
Glu Pro Thr Tyr Gly Glu Leu Ile Pro Gln Tyr Leu Ser Asp His Val
225 230 235 240
Val Ala Lys Val Gly Asp Leu Thr Tyr Leu Tyr Cys Ile Tyr Gly Gly
245 250 255
Thr Pro Leu Ala His Pro Ser Trp Ser Lys Asp Gly Val Asn Val Asp
260 265 270
Asn Thr Tyr Lys Asp Arg Ile Thr Arg His Asn Arg Ser Ser Gly Arg
275 280 285
Arg Leu Val Ile Lys Glu Val Trp Ala Glu Asp Ala Gly Thr Tyr Thr
290 295 300
Cys Asp Val Asp Asn Gln Ala Gly Arg Arg Leu Gln His Thr Ile Thr
305 310 315 320
Phe Ser Val Val Ser Ala Pro Thr Phe Thr Thr Lys Pro Glu Lys Arg
325 330 335
Thr Leu Ala Thr Gln Gly Glu Asp Val Thr Ile Pro Cys Lys Ala Thr
340 345 350
Gly Ile Pro Ser Pro Leu Val Ser Trp Thr Tyr Asn Gly Glu Pro Val
355 360 365
Thr Glu Gly Val Thr Gly Asp Gly Leu Val Ile Lys Ala Val Asn Lys
370 375 380
Ser Asn Gln Gly Tyr Tyr Gly Cys Thr Ala Ser Asn Glu His Gly Ala
385 390 395 400
Glu Tyr Ala Glu Thr Ala Leu Gln Val Ala
405 410
Claims (2)
1. a purification process of silkworm natural immunity albumen Hemolin, is characterized in that, said method comprising the steps of:
(1) preparation of silkworm immune protein mother liquor:
A. be stored in the silkworm chrysalis of-20 DEG C and the sterilizing ddH of 4 DEG C of precoolings
2o mixes with the weight percent of 1:3, at 2 min of homogenate on ice, homogenate is placed in and stops 5 min on ice, repeats 9 times;
B. in 15000 rpm, 4 DEG C of centrifugal 1h, supernatant discarded, the resuspended precipitation of water, then in 15000 rpm, 4 DEG C of centrifugal 1h, so repeat 5 times, use for the last time 4 layers of filtered through gauze;
C. the supernatant liquor after filtering is got 200mL and water by the volume mixture of 1:1, with 0.22 μ m film packet filtering, repeats 10 times, and supernatant solution returns to the volume of 200mL;
D. step c gained supernatant liquor is in 50000rpm, and after 10 DEG C of centrifugal 40min, the black group of appearance is resuspended in 50000rpm with ultrafiltrated, and 10 DEG C are carried out band centrifugation 40min; Get gained supernatant liquor 300ml, the phosphate buffered saline buffer 200ml of paved pH 7.4,30% sucrose 400ml, 55% sucrose is full of, and carries out density gradient centrifugation 3h in 22000rpm, and wherein sucrose concentration is mass body volume concentrations;
E. be that 60% sucrose solution is extruded the solution of different sucrose by the supernatant liquor of gained after centrifugal steps d 3h with mass body volume concentrations, collect by 35mL/ pipe, and in every pipe, get 3mL and do specific activity mensuration;
(2) separation and purification of silkworm natural immunity albumen:
A. by activity higher than 2
6mother liquor and desugar component press the volume ratio of 1:10 by the method for siphon, cross 300K film bag and remove sugar;
B. the mother liquor of desaccharification is mixed by the volume ratio of 1:2.5 with protein extract, in 4 DEG C of refrigerators, slowly stir and spend the night;
C. after, by weak anionic exchange column DEAE initial gross separation, separating obtained solution is concentrated with the super filter tube of 100kD;
D. SuperdexTM 200 gel columns separate, and obtain 8 peaks, and each different peak is in batches concentrated;
E. the concentrated peak containing target protein, is further purified and obtains described silkworm natural immunity albumen Hemolin with SuperdexTM 75 gel columns, and identifies with SDS-PAGE; The aminoacid sequence of wherein said silkworm natural immunity albumen Hemolin is as shown in SEQ ID NO.1.
2. the method for claim 1, is characterized in that, the preparation method of the phosphate buffered saline buffer of described pH 7.4 is: by 8g NaCl, 0.2g KCl, 3.58g Na
2hPO
412H
2o, 0.27g KH
2pO
4be dissolved in 800mL aseptic deionized water, be settled to 1000mL and get final product.
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| CN201210452712.8A CN102977200B (en) | 2012-11-12 | 2012-11-12 | Bombyx natural immune protein Hemolin and purifying method thereof |
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| CN104711232B (en) * | 2013-12-16 | 2018-12-14 | 特菲(天津)生物医药科技有限公司 | A kind of surface display has the purification process of the silkworm with recombinant baculovirus of H5N1 Influenza virus HA protein |
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Non-Patent Citations (6)
| Title |
|---|
| hemolin precursor [Bombyx mori];Tanaka,H.等;《NCBI database》;20120825;NCBI Reference Sequence: NP_001037088.1 * |
| Tanaka,H.等.hemolin precursor [Bombyx mori].《NCBI database》.2012,NCBI Reference Sequence: NP_001037088.1. |
| 何芳青 等.家蚕类免疫球蛋白( hemolin)基因的克隆及表达特征和抗菌活性研究.《蚕业科学》.2010,第36卷(第1期),40-45. |
| 家蚕类免疫球蛋白( hemolin)基因的克隆及表达特征和抗菌活性研究;何芳青 等;《蚕业科学》;20101231;第36卷(第1期);40-45 * |
| 昆虫免疫防御分子Hemolin 的研究进展;李凤娟 等;《生命科学研究》;20120229;第16卷(第1期);90-94 * |
| 李凤娟 等.昆虫免疫防御分子Hemolin 的研究进展.《生命科学研究》.2012,第16卷(第1期),90-94. |
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