CN103102399B - Anti-tumor protein extracted from pholiota nameko and preparation method of antineoplastic protein - Google Patents
Anti-tumor protein extracted from pholiota nameko and preparation method of antineoplastic protein Download PDFInfo
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Abstract
The invention discloses an anti-tumor protein extracted from pholiota nameko and a preparation method of the anti-tumor protein. The anti-tumor protein is obtained by pulverizing pholiota nameko encarpium and then performing leaching, slating out, dialyzing, freeze-drying, ion exchange and gel chromatography. The anti-tumor protein is monomeric protein with the molecular weight of 17KDa, N-terminal amino acid sequence is as shown in SEQ ID NO:1: Ala Gly Aeg Thr Phe Ile Gly Tyr Asn Gly, and the anti-tumor protein has obvious inbibitional effect to growth of human breast cancer cell strain MCF7. The pholiota nameko anti-tumor protein is a natural extractive and is harmless to humans and the environment. The anti-tumor protein is simple in preparation method, relatively low in cost and suitable for industrial production.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of from Pholiota nameko (Pholiota nameko, popular name: anti-tumor protein extracting HUAZIGU) and preparation method thereof.
Background technology
In recent years, mushroom, as Chinese medicine or functional foodstuff, is paid close attention at food and medicine industry remarks.From mushroom, the separated many activeconstituentss that obtain, comprise small molecules, polysaccharide, albumen, polysaccharide-protein mixture etc., have good anti-oxidant, antitumor, antiviral, antimicrobial and immunoregulatory effect.Wherein, the research of relevant polysaccharide is the most extensive, yet, the biological activity protein of rich content in mushroom, comprise lectin, fungal immunomodulatory protein, ribosome inactivating protein, antimicrobial proteins, rnase and laccase etc. show huge potential in the application in food and medicine field.
Pholiota nameko (Pholiota nameko) belongs to Mycophyta, Basidiomycetes, Agaricales, Strophariaceae, Pholiota.It is the wood-decay fungi of the cap stick-slip that occurs in a kind of winter, spring, because there is the mucus HUAZIGU that is otherwise known as on its cap surface, is one of five large tame high-quality edible mushrooms in the world.
Pholiota nameko (Pholiota nameko) has sliding, fresh, tender, crisp feature, is rich in and is of value to the protein of HUMAN HEALTH, carbohydrate, mineral element (calcium, phosphorus, iron), VITAMIN (vitamins B
1, B
2, vitamins C, nicotinic acid) and the nutritive health-care material such as necessary other each seed amino acids of human body, be a kind of protective foods low in calories.According to one's analysis, the dry mushroom of every 100g is containing crude protein 33.76g, pure protein 15.13g, fatty 4.05g, total reducing sugar 38.89g, Mierocrystalline cellulose 14.23g, ash content 8.99g.Except edibleness, also there is certain pharmaceutical use.Its sporophore, containing abundant polysaccharide, can improve the immunizing power of body.
At present, except to Pholiota nameko (Pholiota nameko) growth characteristics, outside the research of cultivation technique, its bioactive research is mainly concentrated on polysaccharide, and still shallow to the biological activity protein research of rich content wherein.Zhou Rui etc. (Nanjing Forestry University's master thesis 2007) separation and purification from Pholiota nameko hypha fermentation liquid obtains the laccase that molecular weight is 55KDa.Yasuko Kawamura-Konishi etc. (Biosci.Biotechnol.Biochem., 71 (7): 1752-1760) from Pholiota nameko sporophore, separation and purification obtains tyrosine protein enzyme, molecular weight is about 42KDa.Gui-Ping Guan etc. (Journal of Bioscience and Bioengineering2011,111 (6): 641-645) from Pholiota nameko sporophore, separation and purification obtains serine protease, molecular weight is about 19KDa.Laccase can be used for lignin degrading, and tyrosine protein enzyme is mainly used in environmental improvement and comprises the waste water that contains phenols and the processing of contaminated soil.And the present invention first from Pholiota nameko separation and purification obtain a kind of new albumen with anti-tumor activity.
Summary of the invention
The object of the invention is to provide a kind of anti-tumor protein in Pholiota nameko (Pholiota nameko) and preparation method thereof, and this albumen has obvious restraining effect to the growth of human breast cancer cell strain MCF7.
The anti-tumor protein extracting from Pholiota nameko (Pholiota nameko) provided by the invention, molecular weight is 17KDa, n terminal amino acid sequence is sequence A la Gly Arg Thr Phe Ile Gly Tyr Asn Gly as shown in SEQ ID NO:1, and this anti-tumor protein is referred to as PNAP(Pholiota nameko Anti-tumor protein).
Anti-tumor protein in the Pholiota nameko the present invention relates to (Pholiota nameko) is prepared as follows:
(1) Pholiota nameko (Pholiota nameko) sporophore is pulverized, be dissolved in the NaCl solution of 0.2 ~ 0.5M, 2 ~ 6 ℃ of lixiviates 8 ~ 16 hours, the centrifugal 20 ~ 40min of 5000 ~ 8000rpm, collect supernatant liquor, i.e. Pholiota nameko Pholiota nameko sporophore crude extract;
(2) to adding ammonium sulfate to saturation ratio in Pholiota nameko (Pholiota nameko) the sporophore crude extract in step (1), be 70 ~ 100%, place 8 ~ 16 hours for 2 ~ 6 ℃, centrifugal 10 ~ the 40min of 10000 ~ 20000rpm, collecting precipitation, i.e. Pholiota nameko Pholiota nameko protein crude extract administration;
(3) the PBS damping fluid of the Pholiota nameko in step (2) (Pholiota nameko) 20 ~ 50mM, pH6.0 ~ 9.0 for protein crude extract administration is dissolved, then in the distilled water of 2 ~ 6 ℃, 1 ~ 5 times weight, dialyse 8 ~ 16 hours, 3 ~ 5 times repeatedly, after dialysis, carry out lyophilize, i.e. Pholiota nameko (Pholiota nameko) albumen meal;
(4) Pholiota nameko in step (3) (Pholiota nameko) albumen meal is dissolved in the MES A damping fluid of 25 ~ 50mM MES, pH5.0 ~ 6.0, loading ion exchange column, by the MES B buffer solution for gradient elution of 25 ~ 50mM MES, 0.5 ~ 1M NaCl, pH5.0 ~ 6.0, collect elution peak;
(5), by elution peak loading molecular sieve, with 25 ~ 50mM MES, 200 ~ 500mM NaCl, the MES C buffer solution elution of pH5.0 ~ 6.0, obtains anti-tumor protein.
Pholiota nameko in the present invention (Pholiota nameko) anti-tumor protein has obvious restraining effect to the growth of human breast cancer cell strain MCF7, and JC-1 staining analysis mitochondrial membrane potential proves that this albumen can inducing apoptosis of tumour cell.
The advantage that the present invention has and beneficial effect:
(1) in the present invention, Pholiota nameko (Pholiota nameko) anti-tumor protein is natural extract, has good security; (2) in the present invention, anti-tumor protein is single albumen, for obtaining amino acid and the encoding gene thereof of this albumen, provides the foundation; (3) in the present invention, anti-tumor protein has good antitumous effect; (4) in the present invention, anti-tumor protein preparation is simple, and cost is low, is suitable for scale operation.
Accompanying drawing explanation
Fig. 1 is the ion exchange chromatography figure of Pholiota nameko anti-tumor protein of the present invention.
Fig. 2 is the gel chromatography tomographic map of Pholiota nameko anti-tumor protein of the present invention.
Fig. 3 is the SDS-PAGE figure of Pholiota nameko anti-tumor protein of the present invention.
Fig. 4 is the restraining effect of Pholiota nameko anti-tumor protein of the present invention to MCF7 Growth of Cells.
Fig. 5 is that to the inducing action of apoptosis of tumor cells, (A. does not add the MCF7 cell of anti-tumor protein to Pholiota nameko anti-tumor protein of the present invention; B.5 the MCF7 cell that μ M anti-tumor protein is processed; C.10 the MCF7 cell that μ M anti-tumor protein is processed; D.15 the MCF7 cell that μ M anti-tumor protein is processed).
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
The preparation of anti-tumor protein in Pholiota nameko (Pholiota nameko)
(1) Pholiota nameko (Pholiota nameko) sporophore is pulverized, be dissolved in the NaCl solution of 0.5M, 4 ℃ of lixiviates 12 hours, the centrifugal 20min of 5000rpm, collects supernatant liquor, i.e. Pholiota nameko (Pholiota nameko) sporophore crude extract;
(2) to adding ammonium sulfate to saturation ratio in Pholiota nameko (Pholiota nameko) the sporophore crude extract in step (1), be 75%, place 12 hours for 4 ℃, the centrifugal 40min of 18000rpm, collecting precipitation, i.e. Pholiota nameko (Pholiota nameko) protein crude extract administration;
(3) the PBS damping fluid of the Pholiota nameko in step (2) (Pholiota nameko) 50mM, pH8.0 for protein crude extract administration is dissolved, then in the distilled water of 4 ℃, 5 times weight, dialyse 12 hours, 3 times repeatedly, after dialysis, carry out lyophilize, i.e. Pholiota nameko (Pholiota nameko) albumen meal;
(4) Pholiota nameko in step (3) (Pholiota nameko) albumen meal is dissolved in the MES A damping fluid of 50mM MES, pH6.0, loading ion exchange column, by the MES B buffer solution for gradient elution of 50mM MES, 0.8M NaCl, pH6.0, collect elution peak;
(5) by elution peak loading molecular sieve, with 50mM MES, 500mM NaCl, the MES C buffer solution elution of pH6.0, obtains anti-tumor protein, and the rate of recovery is about 0.05%.
Embodiment 2
The preparation of anti-tumor protein in Pholiota nameko (Pholiota nameko)
(1) Pholiota nameko (Pholiota nameko) sporophore is pulverized, be dissolved in the NaCl solution of 0.2M, 2 ℃ of lixiviates 8 hours, the centrifugal 40min of 5000rpm, collects supernatant liquor, i.e. Pholiota nameko (Pholiota nameko) sporophore crude extract;
(2) to adding ammonium sulfate to saturation ratio in Pholiota nameko (Pholiota nameko) the sporophore crude extract in step (1), be 70%, place 8 hours for 2 ℃, the centrifugal 40min of 10000rpm, collecting precipitation, i.e. Pholiota nameko (Pholiota nameko) protein crude extract administration;
(3) the PBS damping fluid of the Pholiota nameko in step (2) (Pholiota nameko) 20mM, pH9.0 for protein crude extract administration is dissolved, then in the distilled water of 2 ℃, 1 times weight, dialyse 16 hours, 5 times repeatedly, after dialysis, carry out lyophilize, i.e. Pholiota nameko (Pholiota nameko) albumen meal;
(4) Pholiota nameko in step (3) (Pholiota nameko) albumen meal is dissolved in the MES A damping fluid of 25mM MES, pH5.0, loading ion exchange column, by the MES B buffer solution for gradient elution of 25mM MES, 0.5M NaCl, pH5.0, collect elution peak;
(5) by elution peak loading molecular sieve, with 25mM MES, 200mM NaCl, the MES C buffer solution elution of pH5.0, obtains anti-tumor protein, and the rate of recovery is about 0.05%.
Embodiment 3
The preparation of anti-tumor protein in Pholiota nameko (Pholiota nameko)
(1) Pholiota nameko (Pholiota nameko) sporophore is pulverized, be dissolved in the NaCl solution of 0.5M, 6 ℃ of lixiviates 16 hours, the centrifugal 20min of 8000rpm, collects supernatant liquor, i.e. Pholiota nameko (Pholiota nameko) sporophore crude extract;
(2) to adding ammonium sulfate to saturation ratio in Pholiota nameko (Pholiota nameko) the sporophore crude extract in step (1), be 100%, place 16 hours for 6 ℃, the centrifugal 10min of 20000rpm, collecting precipitation, i.e. Pholiota nameko (Pholiota nameko) protein crude extract administration;
(3) the PBS damping fluid of the Pholiota nameko in step (2) (Pholiota nameko) 50mM, pH6.0 for protein crude extract administration is dissolved, then in the distilled water of 6 ℃, 5 times weight, dialyse 8 hours, 3 times repeatedly, after dialysis, carry out lyophilize, i.e. Pholiota nameko (Pholiota nameko) albumen meal;
(4) Pholiota nameko in step (3) (Pholiota nameko) albumen meal is dissolved in the MES A damping fluid of 50mM MES, pH6.0, loading ion exchange column, by the MES B buffer solution for gradient elution of 50mM MES, 1M NaCl, pH6.0, collect elution peak;
(5) by elution peak loading molecular sieve, with 50mM MES, 500mM NaCl, the MES C buffer solution elution of pH6.0, obtains anti-tumor protein, and the rate of recovery is about 0.05%.
The n terminal amino acid sequencing of embodiment 4, anti-tumor protein of the present invention
(1) SDS-PAGE electrophoresis: 15% gum concentration, adds and dye in advance marker, loading 20ug, applied sample amount 10ul;
(2) after electrophoresis finishes, will need the part of transferring film to cut, water cleans 2 times, and with transfering buffering liquid, (pH11) balance is 5 minutes for CAPS buffer:10mM CAPS, 10% methyl alcohol;
(3) prepare pvdf membrane, be cut into size the same as glue, rinsing 3 minutes in water after 100% wetted with methanol, by balance in transfering buffering liquid 15 minutes;
(4) gel is placed on the thick filter paper that is transferred damping fluid infiltration, pvdf membrane is overlying on gel, then covers the thick filter paper of an infiltration, they are sandwiched in electrotransfer groove together;
(5) gel is at negative pole, and pvdf membrane, at positive pole, adds the electrophoretic buffer of 1L, opens outer circulation refrigeration to 10 ℃, and opens the magnetic stirring apparatus under electroporation groove, low-speed running.
(6) opening power, is set to 180mA, 1 hour;
(7) after transferring film finishes, unload film, water cleans twice, puts into the ponceau dye liquor 30min that dyes, and film water is rinsed after lighter to background and dried, and cuts object band standby.
Edman edman degradation Edman records ten aminoacid sequence sequence A la Gly Arg Thr Phe Ile Gly Tyr Asn Gly as shown in SEQ ID NO:1 of Pholiota nameko of the present invention (Pholiota nameko) anti-tumor protein N end.
The antiproliferative effect of embodiment 5 anti-tumor protein of the present invention to MCF7 cell
(1) MCF7 cell is pressed to 1 * 10
5cfu/mL concentration is cultivated 24 hours 100 μ L/ holes for 37 ℃ in 96 orifice plates;
(2) to add 10 μ L be (embodiment 1,2 or 3 obtains) anti-tumor protein of 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M by the concentration of substratum successively gradient dilution in every hole, 3 of each concentration are parallel, and establish blank, cultivate 24 hours, 48 hours, 72 hours, 96 hours for 37 ℃.
(3) every hole adds 25 μ L through the 500g/L of 4 ℃ of precoolings Tricholroacetic Acid, and final concentration is 100g/L, places 1 hour for 4 ℃;
(4) distilled water wash is 5 times, and after dry air, every hole adds 4g/L SRB dyeing 30min;
(5) acetic acid washing is 5 times, and after dry air, every hole adds 10mmol/L Tris damping fluid 100 μ L to dissolve, and under 490nm, enzyme-linked immunosorbent assay instrument is measured light absorption value.
This albumen is to the antiproliferative effect of MCF7 cell as Fig. 4, and PNAP processes the IC after MCF724 hour, 48 hours, 72 hours, 96 hours, MCF7 cellular antiproliferative being done
50be respectively 15.52 μ M, 9.97 μ M, 4.99 μ M, 3.23 μ M.Illustrate that PNAP has good antiproliferative effect to MCF7 cell.
The inducing action of embodiment 6 anti-tumor protein of the present invention to apoptosis of tumor cells
(1) MCF7 cell is pressed to 1 * 10
5cfu/mL concentration is cultivated 24 hours 1mL/ hole for 37 ℃ in 12 orifice plates;
(2) every hole adds (embodiment 1,2 or 3 obtains) anti-tumor protein 10 μ L of 500 μ M, 1000 μ M, 1500 μ M, and 3 of each concentration are parallel, and establish blank, cultivate 24 hours for 37 ℃;
(3) get 1 * 10
5cfu cell, is resuspended in 0.5mL cell culture fluid, adds 0.5ml JC-1 staining fluid, hatches 20min for 37 ℃;
(4) 4 ℃ by cell collecting precipitation after the centrifugal 5min of 600g in step (3), JC-1 dyeing damping fluid washing 2 times, and flow cytometer detects (as Fig. 5).
The decline of mitochondrial membrane potential is an early stage significant event of apoptosis.Transformation by JC-1 from red fluorescence to green fluorescence can detect the decline of cell membrane potential at an easy rate.Right lower quadrant in figure represents green fluorescence.The MCF7 cell that 5 μ M, 10 μ M, 15 μ M Pholiota nameko (Pholiota nameko) anti-tumor proteins are processed, the ratio that sends green fluorescence cell is respectively 4.56%, 18.25%, 32.20%.As can be seen from the figure along with the increase of Pholiota nameko (Pholiota nameko) anti-tumor protein concentration, the ratio of apoptotic cell increases gradually, shows that Pholiota nameko of the present invention (Pholiota nameko) anti-tumor protein has inducing action to MCF7 apoptosis.
Claims (4)
1. the anti-tumor protein PNAP extracting in a Pholiota nameko (Pholiota nameko), described anti-tumor protein PNAP is from Pholiota nameko (Pholiota nameko), molecular weight is 17KDa, and n terminal amino acid sequence is sequence A la Gly Arg Thr Phe Ile Gly Tyr Asn Gly as shown in SEQ ID NO:1; Described anti-tumor protein PNAP obtains by the following method:
(1) Pholiota nameko (Pholiota nameko) sporophore is pulverized, be dissolved in the NaCl solution of 0.2～0.5M, 2～6 ℃ of lixiviates 8～16 hours, the centrifugal 20～40min of 5000～8000rpm, collects supernatant liquor, i.e. Pholiota nameko sporophore crude extract;
(2) to adding ammonium sulfate to saturation ratio to be 70～100%, 2～6 ℃ in the Pholiota nameko sporophore crude extract in step (1), place 8～16 hours the centrifugal 10～40min of 10000～20000rpm, collecting precipitation, i.e. Pholiota nameko protein crude extract administration;
(3) the PBS damping fluid of the Pholiota nameko in step (2) 20～50mM, pH6.0～9.0 for protein crude extract administration is dissolved, then in the distilled water of 2～6 ℃, 1～5 times weight, dialyse 8～16 hours, 3～5 times repeatedly, after dialysis, carry out lyophilize, i.e. Pholiota nameko albumen meal;
(4) the Pholiota nameko albumen meal in step (3) is dissolved in the MES A damping fluid of 25～50mM MES, pH5.0～6.0, loading ion exchange column, by the MES B buffer solution for gradient elution of 25～50mM MES, 0.5～1M NaCl, pH5.0～6.0, collect elution peak;
(5), by elution peak loading gel chromatography column, with 25～50mM MES, 200～500mM NaCl, the MES C buffer solution elution of pH5.0～6.0, obtains Pholiota nameko anti-tumor protein PNAP.
2. the preparation method of anti-tumor protein claimed in claim 1, is characterized in that comprising the steps:
(1) Pholiota nameko (Pholiota nameko) sporophore is pulverized, be dissolved in the NaCl solution of 0.2～0.5M 2～6 ℃ of lixiviates 8～16 hours, the centrifugal 20～40min of 5000～8000rpm, collect supernatant liquor, i.e. Pholiota nameko (Pholiota nameko) sporophore crude extract;
(2) to adding ammonium sulfate to saturation ratio in Pholiota nameko (Pholiota nameko) the sporophore crude extract in step (1), be 70～100%, place 8～16 hours for 2～6 ℃, centrifugal 10～the 40min of 10000～20000rpm, collecting precipitation, i.e. Pholiota nameko (Pholiota nameko) protein crude extract administration;
(3) the PBS damping fluid of the Pholiota nameko in step (2) (Pholiota nameko) 20～50mM, pH6.0～9.0 for protein crude extract administration is dissolved, then in the distilled water of 2～6 ℃, 1～5 times weight, dialyse 8～16 hours, 3～5 times repeatedly, after dialysis, carry out lyophilize, i.e. Pholiota nameko (Pholiota nameko) albumen meal;
(4) Pholiota nameko in step (3) (Pholiota nameko) albumen meal is dissolved in the MES A damping fluid of 25～50mM MES, pH5.0～6.0, loading ion exchange column, by the MES B buffer solution for gradient elution of 25～50mM MES, 0.5～1M NaCl, pH5.0～6.0, collect elution peak;
(5), by elution peak loading gel chromatography column, with 25～50mM MES, 200～500mM NaCl, the MES C buffer solution elution of pH5.0～6.0, obtains Pholiota nameko (Pholiota nameko) anti-tumor protein.
3. Pholiota nameko claimed in claim 1 (Pholiota nameko) anti-tumor protein is in the application in the growth-inhibiting of human breast cancer cell strain MCF7.
4. application according to claim 3, Pholiota nameko (Pholiota nameko) anti-tumor protein has obvious restraining effect to the growth of human breast cancer cell strain MCF7, and JC-1 staining analysis mitochondrial membrane potential proves that this albumen can inducing apoptosis of tumour cell.
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| CN106366169A (en) * | 2016-10-11 | 2017-02-01 | 南开大学 | Antineoplastic protein extract in Ramaria botrytis, and preparation method and application thereof |
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| CN108220258B (en) * | 2016-12-15 | 2023-06-27 | 天津市农业科学院 | Preparation method of stropharia rugoso-annulata active protein with laccase activity |
| CN107119096B (en) * | 2017-05-10 | 2022-11-18 | 天津市农业科学院 | Preparation method and application of pholiota nameko active peptide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106366169A (en) * | 2016-10-11 | 2017-02-01 | 南开大学 | Antineoplastic protein extract in Ramaria botrytis, and preparation method and application thereof |
| CN106366169B (en) * | 2016-10-11 | 2019-09-24 | 南开大学 | Anti-tumor protein extract and preparation method and application in a kind of Ramaria botrytis |
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