CN108341866A - A method of the extraction separation ferritin from silkworm blood - Google Patents
A method of the extraction separation ferritin from silkworm blood Download PDFInfo
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- CN108341866A CN108341866A CN201810290080.7A CN201810290080A CN108341866A CN 108341866 A CN108341866 A CN 108341866A CN 201810290080 A CN201810290080 A CN 201810290080A CN 108341866 A CN108341866 A CN 108341866A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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Abstract
The method that the invention discloses a kind of to extract separation ferritin from silkworm blood, chooses the Fecl that silkworm feeding a certain concentration of specific silkworm age is first3Solution, continuous feeding break silkworm caudal horn after 24 days, silkworm blood is collected, a small amount of phenylthiourea is added thereto, adds protein lysate, after mixing well, frozen-thawed, then ultrasonic disruption are carried out, it is then centrifuged for detaching, takes supernatant, heating water bath, it centrifuges again, takes supernatant, concentrated with super filter tube, collection is shut off liquid, and the verified liquid that shuts off is the silkworm ferritin of concentration, and the ferritin of high concentration is further obtained in the method for PAGE glue recycling.The method of the present invention is at low cost, and material source is abundant and extraction process is simple, and a kind of new way is provided for the acquisition of ferritin.
Description
Technical field
The present invention relates to the separation technology fields of insect ferritin, and in particular to one kind extraction separation iron from silkworm blood
The method of albumen.
Background introduction
Ferritin is a kind of iron-binding protein of generally existing, and important angle is play in the storage of iron and transportational process
Color.Ferritin is made of 24 subunits, these subunits are grouped together into the hollow ball of 4,3,2 point symmetries.Insect iron
The globular protein complex that albumen is made of heavy chain subunit (H chains) and light chain subunits (L chains), wherein H chains contain conservative iron oxygen
Change enzymatic activity, Fe2+ can be oxidized to Fe3+ in conjunction with iron atom.Though without ferroxidase activity but its acidic amino acid on L chains
It can promote the formation of the hydrolysis and iron core of iron.It can apply to nanometer using the cavity structure structure Nano medication of ferritin to cure
It learns.At present mainly by using FeS, the biosynthesis ferritin such as CdS, the difficulty of synthesis is larger, also, in the ferritin synthesized
Some natural structures and function often change.
Separation ferritin is extracted in present invention research from the blood of silkworm, and extracts the object isolated by experimental identification
Contain ferritin complex and its subunit in matter, this method is at low cost, and material source is abundant, and extraction process is simple, extracts
Ferritin natural structure and function do not change, the ferritin levels that extract are high, a concentration of 5 μ g/ μ L.
Invention content
The present invention provides a kind of method of the extraction separation ferritin from silkworm blood, this extracting method is at low cost, efficiency
Height, energy high efficiency extraction detach the ferritin in silkworm blood, and the ferritin levels extracted are high, this method extraction purification
Ferritin contributes to the utilization and biological study of ferritin.
The technical solution adopted by the present invention is as follows：
A method of the extraction separation ferritin from silkworm blood, which is characterized in that concrete operation step is as follows：
A, the Fecl of a concentration of 9%-11% of silkworm feeding of specific silkworm age is chosen3Solution, continuous feeding are cut after 2-4 days
It destroys family himself silkworm caudal horn, collects silkworm blood, a small amount of phenylthiourea is added in collecting pipe, shakes up；
B, protein lysate is added in the blood collected to step A, 0.8-1.2ml lysates are added in the blood of every 100 μ L,
It mixes well, obtains mixing sample liquid；
C, the mixing sample liquid for obtaining step B, first frozen-thawed, then ultrasonication 9-11 minutes, then at 3-4 DEG C
At a temperature of, with the rotating speed of 11000-13000rpm, 14-16mins is centrifuged, supernatant is taken；
D, the obtained supernatants of step C are placed in 74-76 DEG C of water-bath and are heated 1.5-2.5 hours, then in 3-5 DEG C of temperature
Under, with 11000-13000rpm rotating speeds, 14-16min is centrifuged, supernatant is taken；
E, select molecular weight that centrifuge tube is concentrated by ultrafiltration for the Millipore albumen of 95-105kDa, it will be upper obtained by step D
Clear liquid is concentrated, and the liquid that shuts off is collected, and the verified liquid that shuts off is the silkworm ferritin crude extract of concentration；
F, the ferritin crude extract that step E is obtained cuts yellow band glue after Native-PAGE, is returned with PAGE glue albumen
It receives kit to recycle up to silkworm ferritin, quantitative using Bradford methods, it is 5 μ g/ μ L to measure ferritin levels.
The method of extraction separation ferritin in the slave silkworm blood, it is characterised in that：Specific silkworm age described in step A
Silkworm refer to the 1st day 5 ages silkworm.
The method of extraction separation ferritin in the slave silkworm blood, which is characterized in that give specific silkworm described in step A
The silkworm feeding Fecl in age3The specific method of solution is：Choose the Fecl of the 1st day 5 ages continuous 10% concentration of feeding of silkworm3Solution,
By Fecl3Solution is evenly coated on mulberry leaf, average every 5 μ L of silkworm, continuous feeding 3 days, daily feeding 3 times.
The method of extraction separation ferritin in the slave silkworm blood, it is characterised in that：Lysate described in step B
Ingredient is 0.5M Tris-HCL (PH6.8), 0.1%Triton X-100,1mM PMSF, 0.2%Ferene S.
The method of extraction separation ferritin in the slave silkworm blood, it is characterised in that：The item of concentration described in step E
Part is 4 DEG C, 12000rpm, centrifuges 2min.
The method of extraction separation ferritin in the slave silkworm blood, it is characterised in that：Verification method described in step E
For：By the liquid that shuts off of collection, through Native-PAGE electrophoresis detections, visible yellow color band before being unstained, it is iron egg to verify the band
White band.
The method of extraction separation ferritin in the slave silkworm blood, it is characterised in that：Verification method described in step E
For：One-dimensional Native-PAGE and SDS-PAGE combinations LC-MS/MS analyses are respectively adopted to the silkworm ferritin of separation and Extraction
Albumen verification is carried out, analytical proof silkworm ferritin obtains good separation and Extraction.
Advantageous effect
The method of the present invention has efficiently extracted out the ferritin in silkworm blood, and extraction cost is low, and material source is abundant, and
Extraction process is simple, through Native-PAGE, SDS-PAGE analyses and mass spectrum verification, it was demonstrated that silkworm ferritin and its subunit
Good separating effect, and do not pollute.Therefore, the ferritin of the method for the present invention extraction purification contributes to utilization and the biology of ferritin
Research is learned, the method for the present invention provides a kind of new way for the source of ferritin.
Below by way of specific implementation mode, the above of the present invention is described in further detail again.But it should not incite somebody to action
This above-mentioned subject area for being interpreted as the present invention is only limitted to following instance verification.All skills realized based on the above of the present invention
Art all belongs to the scope of protection of the present invention.
Description of the drawings
Fig. 1 is extraction and the identification Native-PAGE electrophoretograms of silkworm ferritin complex.
Figure 1A is Native-PAGE analysis silkworm blood ferritins (before dyeing)；Figure 1B is Native-PAGE analysis silkworms
After ferritin dyeing, the yellow band in Figure 1A is ferritin band, the M in Fig. 1：Albumen Marker, 1：The iron egg of separation
In vain.
Fig. 2 is that SDS-PAGE analyzes silkworm ferritin subunit electrophoretogram.
M in figure：Albumen Marker；a:Ferritin light chain subunit；b:Ferritin heavy chain subunit.
Specific implementation mode
A method of extraction separation ferritin, concrete operation step are as follows from silkworm blood：
(1) Fecl of the 1st day silkworm feeding a concentration of 10% of 5 ages is chosen3Solution, continuous feeding break silkworm tail after 3 days
Angle collects silkworm blood, a small amount of phenylthiourea is added in collecting pipe, shakes up, anti-Hemostatic Oral Liquid melanism, silkworm strain is made greatly
(P50) it is provided by Life Science College bombycology system of Agricultural University Of Anhui；
(2) protein lysate of precooling is added in the silkworm blood of step (1) collection, 1ml is added in every 100 μ L blood
Protein extract, after mixing well, fast freeze-thaw is primary, the ingredient of lysate：0.5M Tris-HCL (PH6.8), 0.1%
Triton X-100,1mM PMSF, 0.2%Ferene S；
(3) albumen is crushed with the power ultrasonic of 30W, operating condition is ultrasound 3s, pause 4s, ice-bath ultrasonic 10mins, 4
DEG C, 12000rpm, centrifugation 15mins；
(4) supernatant being moved in another clean centrifuge tube, 75 DEG C of water-baths 2 hours, 4 DEG C, 12000rpm centrifuges 15mins,
Collect supernatant；
(5) super filter tube that selective retention molecular weight is 100kDa concentrates the supernatant of collection, and concentration condition is 4 DEG C,
12000rpm centrifuges 2min；
(6) trapped fluid is taken out, the liquid that will shut off cuts yellow band glue after Native-PAGE electrophoresis, with PAGE glue albumen
QIAquick Gel Extraction Kit recycling is to get silkworm ferritin, with the concentration of protein in Bradford method determination samples, remaining sample storage
It is spare at -80 DEG C.
Silkworm ferritin extraction result is tested below by Native-PAGE and SDS-PAGE combinations mass spectral analysis
Card.
1 experiment material
1.1 reagent
Biochemical reagents SDS, acrylamide, Ferene S, Tris, ammonium persulfate, TEMED, methene acrylamide,
The chemical reagent such as Bradford reagent, coomassie brilliant blue R_250, TrionX-100, DTT, PMSF, absolute ethyl alcohol are purchased from
Sangon Biotech (Shanghai) Co., Ltd.；
1.2.1 protein quantification
Silkworm ferritin levels are quantified using Bradford methods, the results show that ferritin levels are 5 μ g/ μ L.
1.2.2Native-PAGE analysis
(1) 12.5 μ 5 × loading of L buffer (no SDS) mixings are added in the albumen for taking 50 μ L concentrated, add per hole
Enter 10 μ L albumen samples and protein Marker；
It (2), will be electric when first migrating out concentration glue arrival separation with 70V progress electrophoresis to bromophenol blue under conditions of precooling
Pressure is transferred to 100V until bromophenol blue transport glue bottom, stops electrophoresis；
(3) it because containing Ferene S in protein extract, can be combined to form coloured complexing with the iron ion in ferritin
Object, therefore have faint yellow band before being unstained, after coomassie brilliant blue R_250 dyeing and destainer decoloration, by comparing the band
Positioned at 670kDa or so.
1.2.3 SDS-PAGE is analyzed
There is yellow band before being unstained through Native-PAGE analyses, cuts the band and suitable 5 × loading is added
Buffer (containing SDS and 2-ME) is incubated at room temperature 15mins, boils 10mins, carries out the SDS-PAGE separation of binomial, dyeing mirror
Determine ferritin subunit.
1.2.4 mass spectral analysis
Native-PAGE cuts protein band and carries out LC-MS/MS mass spectral analyses, and SDS-PAGE digs out a progress MALDI-
TOF/TOF MS mass spectral analyses.
1.2.5 the separation of silkworm ferritin complex and identification
The molecular weight that silkworm ferritin complex is analyzed according to Native-PAGE is about 670kDa, the yellow item in Figure 1A
Band as ferritin band cuts the progress LC-MS/MS mass spectral analyses of yellow band Song Sheng works biotech firm and identifies two iron eggs
Bai Yaji, it was demonstrated that the yellow band is ferritin, concrete outcome such as table 1.
Table 1 is based on LC-MS/MS and identifies silkworm ferritin
1.2.6 the separation and identification of silkworm ferritin subunit
Silkworm ferritin subunit is detached according to SDS-PAGE, two subunits of silkworm ferritin is as a result isolated, sees Fig. 2,
Through two the subunits BmFerLCH and BmFerHCH that MALDI-TOF/TOF MS Mass Spectrometric Identifications are silkworm ferritin, further prove
The yellow band of Fig. 1 separation is ferritin.Concrete outcome such as table 2.
Table 2 is based on MALDI-TOF/TOF MS and identifies silkworm ferritin subunit
Conclusion：
The method of extraction separation silkworm ferritin in the slave silkworm blood provided by inventor so that iron in silkworm blood
Albumen obtains good separation and Extraction.Through Native-PAGE, SDS-PAGE analyses and mass spectrum verification, it was demonstrated that silkworm iron egg
Its subunit good separating effect of bletilla, and do not pollute.Therefore, the ferritin of the method for the present invention extraction purification contributes to ferritin
With and biological study, the method for the present invention provide a kind of new way for the source of ferritin.
Claims (7)
1. a kind of method of the extraction separation ferritin from silkworm blood, which is characterized in that concrete operation step is as follows：
A, the Fecl of a concentration of 9%-11% of silkworm feeding of specific silkworm age is chosen3Solution, continuous feeding break silkworm after 2-4 days
Caudal horn collects silkworm blood, a small amount of phenylthiourea is added in collecting pipe, shakes up；
B, protein lysate is added in the blood collected to step A, 0.8-1.2ml lysates are added in the blood of every 100 μ L, fully
Mixing obtains mixing sample liquid；
C, the mixing sample liquid for obtaining step B, first frozen-thawed, then ultrasonication 9-11 minutes, then in 3-4 DEG C of temperature
Under, with the rotating speed of 11000-13000rpm, 14-16mins is centrifuged, supernatant is taken；
D, the obtained supernatants of step C are placed in 74-76 DEG C of water-bath and are heated 1.5-2.5 hours, then at a temperature of 3-5 DEG C,
With 11000-13000rpm rotating speeds, 14-16min is centrifuged, supernatant is taken；
E, select molecular weight that centrifuge tube is concentrated by ultrafiltration for the Millipore albumen of 95-105kDa, by the supernatant obtained by step D
It is concentrated, collects the liquid that shuts off, the verified liquid that shuts off is the silkworm ferritin crude extract of concentration；
F, the ferritin crude extract that step E is obtained cuts yellow band glue after Native-PAGE, is tried with PAGE glue protein salvage
Agent box recycles up to silkworm ferritin, quantitative using Bradford methods, and it is 5 μ g/ μ L to measure ferritin levels.
2. the method for the extraction separation ferritin according to claim 1 from silkworm blood, it is characterised in that：Step A institutes
The silkworm for the specific silkworm age stated refers to the 1st day 5 ages silkworm.
3. the method for the extraction separation ferritin according to claim 1 from silkworm blood, which is characterized in that step A institutes
The silkworm feeding Fecl to specific silkworm age stated3The specific method of solution is：It is dense to choose the 1st day 5 ages continuous feeding 10% of silkworm
The Fecl of degree3Solution, by Fecl3Solution is evenly coated on mulberry leaf, average every 5 μ L of silkworm, continuous feeding 3 days, daily feeding 3 times.
4. the method for the extraction separation ferritin according to claim 1 from silkworm blood, it is characterised in that：Step B institutes
The ingredient for the lysate stated is 0.5M Tris-HCL (PH6.8), 0.1%Triton X-100,1mM PMSF, 0.2%Ferene
S。
5. the method for the extraction separation ferritin according to claim 1 from silkworm blood, it is characterised in that：Step E institutes
The condition for the concentration stated is 4 DEG C, 12000rpm, centrifuges 2min.
6. the method for the extraction separation ferritin according to claim 1 from silkworm blood, it is characterised in that：Step E institutes
The verification method stated is：By the liquid that shuts off of collection, through Native-PAGE electrophoresis detections, visible yellow color band before being unstained, verification
The band is the band of ferritin.
7. the method for the extraction separation ferritin according to claim 1 from silkworm blood, it is characterised in that：Step E institutes
The verification method stated is：One-dimensional Native-PAGE and SDS-PAGE is respectively adopted to the silkworm ferritin of separation and Extraction to combine
LC-MS/MS analyses carry out albumen verification, and analytical proof silkworm ferritin obtains good separation and Extraction.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102505033A (en) * | 2012-01-05 | 2012-06-20 | 上海海洋大学 | Method for preparing lactoferricin and method for applying lactoferricin in bacterial inhibition of foods |
| CN102875669A (en) * | 2012-10-29 | 2013-01-16 | 天津商业大学 | Method for separating and extracting ovotransferrin |
| CN104130321A (en) * | 2014-07-10 | 2014-11-05 | 中国水产科学研究院南海水产研究所 | Crude extraction method for abalone ferritin |
| CN105769923A (en) * | 2016-05-27 | 2016-07-20 | 厦门市医药研究所 | Bombyx mori extract and extraction method and application thereof |
-
2018
- 2018-04-03 CN CN201810290080.7A patent/CN108341866A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102505033A (en) * | 2012-01-05 | 2012-06-20 | 上海海洋大学 | Method for preparing lactoferricin and method for applying lactoferricin in bacterial inhibition of foods |
| CN102875669A (en) * | 2012-10-29 | 2013-01-16 | 天津商业大学 | Method for separating and extracting ovotransferrin |
| CN104130321A (en) * | 2014-07-10 | 2014-11-05 | 中国水产科学研究院南海水产研究所 | Crude extraction method for abalone ferritin |
| CN105769923A (en) * | 2016-05-27 | 2016-07-20 | 厦门市医药研究所 | Bombyx mori extract and extraction method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| BEOM SU KIM ET AL.: "Characterization and immunological analysis of ferritin from the hemolymph of Galleria mellonella", 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY PART A》 * |
| H.NICHOL ET AL.: "The characterization of ferritin in an insect", 《INSECT BIOCHEMISTRY》 * |
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