CN102558296A - Mytilus edulis enzymolysis polypeptide and preparation method and application thereof - Google Patents
Mytilus edulis enzymolysis polypeptide and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a mytilus edulis enzymolysis polypeptide. The mytilus edulis enzymolysis polypeptide is characterized by containing the following amino acid sequence: Asp Leu Tyr. The mytilus edulis enzymolysis polypeptide is prepared by adopting the following steps of: (1) preparing homogenate from mytilus edulis meat, adding alkaline protease, deactivating the protease, centrifuging, and taking clear solution of the upper layer; (2) performing ultra-filtration on the clear solution, collecting hydrolysate with the molecular weight of below 3K, concentrating, and performing freeze drying; (3) performing chromatographic separation by adopting a DEAE-SepharoseFF ion exchange column; (4) performing chromatographic separation by adopting a Sephadex G-25 gel column; and (5) performing high performance liquid chromatography purification. The invention also discloses application of the mytilus edulis enzymolysis polypeptide prepared by the steps in prostatic cancer resistance. Compared with the prior art, the invention has the advantages that: the mytilus edulis is subjected to enzymolysis and purification by adopting an optimal protease and an optimal technology, a strong cell proliferation inhibiting effect is achieved when the obtained target peptide is applied to prostatic cancer resistant cells, and a feasible research path is provided for resisting prostatic cancer.
Description
Technical field
The present invention relates to a kind of mussel enzymolysis polypeptide, the invention still further relates to the preparation method and the application on anti-prostate cancer thereof of this mussel enzymolysis polypeptide.
Background technology
Polypeptide can be used as neurotransmitter, interleukin-and ESC isoreactivity material in vivo, has the biology growing of keeping growth, immunomodulatory and metabolic isoreactivity function.Existing research shows, that the polypeptide that derives from organism has is antitumor, anti-oxidant, antibiotic, anti-ageing, hypertension isoreactivity function, especially anti-tumor activity have received various countries researchist's concern.At present, about the existing a large amount of reports of the anti-tumor activity of native peptides and synthetic peptide, and there is part of polypeptide to be applied to clinical as antitumor drug; The polypeptide in food proteins source is antibiotic, anti-oxidant for it, antihypertensive active research is a lot of, and less to the relevant report of its anti-tumor activity.
Sea life albumen is of a great variety, wide material sources, and an important source as bioactive peptide has received increasing concern.Mussel is under the jurisdiction of sea mollusk door (Mollusca) Bivalvia (Bivalvia) mussel order (Mytiloida) as a halobiontic important component part, claims Hai Hong in northern China, and the Jiangsu and Zhejiang Provinces is called mussel, and richness originates in marine site around the Shengsi, Zhejiang.Mussel is rich in crude protein, fat and glucide, contains various trace elements such as calcium, phosphorus, iron, VITAMINs in addition.Existing research shows; That mussel extract has is antitumor, anti-oxidant, anti-freezing, hypotensive, reducing blood-fat, raising immunizing power isoreactivity function; But referenced patent number is that (Granted publication number: CN100467030C), this patent discloses the application of extract on influenza of Mytilus crassitesta Lischke for the Chinese invention patent " extract of Mytilus crassitesta Lischke, method of manufacture and uses thereof " of ZL200510110096.8.Similarly can also referenced patent number be the Chinese invention patent of ZL200510024391.1 " a kind of Trachyostracous mussel extract that improves immunity function " (Granted publication number be CN100486593C); Application number is open " thick-shell mussel fat-soluble extract " (publication number: CN101606951A) of Chinese invention patent application of 200910101136.0.But do not see the relevant report of relevant mussel enzymolysis product so far to the prostate cancer cell effect.
Summary of the invention
Technical problem to be solved by this invention is to the above-mentioned state of the art a kind of mussel enzymolysis polypeptide to be provided.
Another technical problem to be solved by this invention provides a kind of preparation method of mussel enzymolysis polypeptide.
Another technical problem to be solved by this invention provides a kind of application of mussel enzymolysis polypeptide.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of mussel enzymolysis polypeptide is characterized in that comprising following aminoacid sequence:
Asp?Leu?Tyr。
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of mussel enzymolysis polypeptide is characterized in that adopting the following steps preparation:
1. get mussel meat, process homogenate, regulate pH value 9.5～10, solid-liquid ratio is 1: 2～1: 3, adds Sumizyme MP, and enzyme concentration is 1500～2000U/g, and enzymolysis time is 6～8 hours, and hydrolysis temperature is 45～50 ℃, and is centrifugal behind the enzyme that goes out, and gets supernatant liquid;
2. above-mentioned clear liquid is undergone ultrafiltration, collect the hydrolyzed solution below the molecular weight 3K, concentrate and lyophilize;
3. DEAE-SepharoseFF ion column exchange column chromatographic separation, with the lyophilize after product of step in 2. through DEAE-SepharoseFF ion column exchange column, 0.1～1mol/LNaCL gradient elution; Elution speed 1～1.5mL/min; Protein Detection appearance 280nm detects, and each elution peak is collected respectively, concentrates postlyophilization; Adopt mtt assay to carry out the anti-tumor activity experiment each peak component that obtains after the drying; Further confirm the elution peak at target peptide place, and this peak is collected in a large number, concentrate and lyophilize;
4. adopt Sephadex G-25 gel column chromatography to separate; With step 3. the lyophilize after product with high anti-tumor activity of gained cross Sephadex G-25 gel column; Moving phase is zero(ppm) water, and flow velocity is 1.5～2mL/min, and Protein Detection appearance 280nm detects; Each elution peak is collected respectively, concentrated postlyophilization and carry out the anti-tumor activity experiment with mtt assay;
5. high-efficient liquid phase chromatogram purification, with step obtain in 4. have high anti-tumor activity lyophilize after postpartum cross reversed phase high efficiency liquid phase post, chromatographic column is Zorbax SB C
18(250mm * 9.4mm, 5 μ m); Column temperature is a room temperature; Moving phase is acetonitrile and water, gradient elution: 0～20min, and acetonitrile is dense to change to 95%, elution speed 2.5～3.0mL/min, ultraviolet detection wavelength 280nm from 0%.
The application of described mussel enzymolysis polypeptide on anti-prostate cancer.Further, described mussel enzymolysis polypeptide suppresses the application on the propagation at anti-prostate cancer DU-145 cell; Described mussel enzymolysis polypeptide suppresses the application on the propagation at anti-prostate cancer PC-3 cell.
Compared with prior art; The invention has the advantages that: adopt best proteolytic enzyme and preferred technology that mussel is carried out the enzymolysis purifying; The target peptide of gained is applied to produce very strong cell inhibition proliferation function on the anti-prostate cancer cell, for anti-prostate cancer provides a FS approach; Integrated artistic is simple, and less input, is easy to apply.
Description of drawings
Fig. 1 is the histogram of the zymolyte of various proteolytic enzyme to the DU-145 cell proliferation inhibition rate.
Fig. 2 is the histogram of Sumizyme MP different molecular weight zymolyte to the DU-145 cell proliferation inhibition rate.
Fig. 3 is the DEAE Sepharose FF chromatography collection of illustrative plates of the following component of basic protein enzyme molecular weight 3K.
Fig. 4 is the Sephadex G-25 gel chromatography collection of illustrative plates at peak 2 among Fig. 3.
Fig. 5 is the RT-HPLC collection of illustrative plates of peak 2-2 among Fig. 4.
Fig. 6 is the RT-HPLC collection of illustrative plates of target peptide among Fig. 5.
Fig. 7 is a normal DU-145 cellular form HE dyeing back Photomicrograph.
Fig. 8 is a DU-145 cellular form HE dyeing back Photomicrograph behind the mussel polypeptide effect 24h.
Fig. 9 is a DU-145 cellular form HE dyeing back Photomicrograph behind the mussel polypeptide effect 48h.
Figure 10 is a normal PC-3 cellular form HE dyeing back Photomicrograph.
Figure 11 is a PC-3 cellular form HE dyeing back Photomicrograph behind the mussel polypeptide effect 24h.
Figure 12 is a PC-3 cellular form HE dyeing back Photomicrograph behind the mussel polypeptide effect 48h.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
1 material
1.1 animal: mussel (Mytilus edulis) is available from market, Zhoushan (identifying through professor Zhao Shenglong of Oceanography Institute Of Zhejiang), and after shelling ,-20 ℃ of refrigerations are subsequent use.
1.2 cell strain: Human Prostate Cancer Cells DU-145 and PC-3 are all available from Chinese Academy of Sciences's Shanghai cell bank.
1.3 main agents: stomach en-, trypsinase, papoid, Sumizyme MP, dextran gel SephadexG-25 and DEAE SepharoseFF anion-exchange column are all available from the permanent letter bio tech ltd in Asia-Pacific, Beijing; Foetal calf serum is available from Hangzhou SIJIQING biotechnology ltd; F12 powder culture medium and MTT are all available from U.S. SIGMA company; DMSO 99.8MIN. is available from U.S. AMRESCO company; Acetonitrile is available from the permanent grand experiment equipment ltd of sea, Ningbo daybreak; All the other reagent are analytical pure.
1.4 key instrument: BSA124S type electronic balance (Germany, Sartorius AG company); DS-1 type high-speed tissue mashing machine (Shanghai Sample Model Factory); CF16RXII high speed freezing centrifuge (HITACHI company of Hitachi); Automatic Fraction Collector (BSZ-40-LCD), Protein Detection appearance (HD-21-88) (the special Analytical Instrument Co., Ltd of Shanghai fine jade); SSW type ASIC micro computer electric heating thermostatic bath (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); Yi Lite P1201 type high performance liquid chromatograph (Dalian Yilite Analytical Instrument Co., Ltd); MSC300 ultrafiltration cup (ultra-filtration membrane: 3KDa and 5KDa) (Shanghai rub fast science equipment ltd).ZHJH-C1209C type Bechtop (the sincere analytical instrument of Shanghai intelligence Manufacturing Co., Ltd); Forma 3111 type CO
2Incubator (U.S. Thermo company); Inverted microscope (Japanese OLYMPUS company); ELIASA (U.S. Bio-Rad company).
2 methods
2.1 enzymolysis process flow process: select for use Sumizyme MP, trypsinase, stomach en-and 4 kinds of proteolytic enzyme of papoid respectively mussel meat to be carried out enzymolysis, the enzymatic hydrolysis condition of various proteolytic enzyme is seen table 1, and enzymolysis process is following successively:
Mussel is cleaned; Shell and get meat; Homogenate; 0.5mol/LNaOH transfer pH value with 0.1mol/LHCl solution; Add enzymic hydrolysis; Enzyme (90 ℃, heating 15min) goes out; Centrifugal (5000r/min, 20min); Get supernatant; Lyophilize; Mtt assay anti-tumor activity rough determination.
The enzymatic hydrolysis condition of several kinds of proteolytic enzyme of table 1
2.2 cell cultures: human prostata cancer DU-145 and PC-3 cell (former in Chinese Academy of Sciences's Shanghai cell bank) by the preservation of going down to posterity of this laboratory, with the F12 perfect medium that contains 10% calf serum in 37 ℃, 5%CO
2Be cultured to logarithmic phase in the incubator.
2.3 the mensuration of cell proliferation inhibition rate: adopt mtt assay to detect.Preparation PBS solution (the pH value is 7.2) and certain density enzymolysis polypeptide solution.The prostate cancer cell of taking the logarithm vegetative period is processed suspension, is seeded to 96 orifice plates, and every hole 200 μ L are in 5%CO
2, 37 ℃ of adherent 4h add enzymolysis polypeptide solution then respectively, and each concentration is established 3 parallel holes, establishes not dosing control group simultaneously, puts 5% CO
2, hatch in 37 ℃ of incubators, cultivate and finish to add 180 μ LMTT and 20 μ L PBS continuation cultivation 4h, inhale the liquid of abandoning 96 orifice plates, add the DMSO of 150 μ L, thorough mixing.Put enzyme-linked immunosorbent assay instrument and survey absorbancy, calculate cell inhibitory effect index (IR), calculate by following formula at 490nm.
IR=[(control group A value-drug group A value)/control group A value] * 100%
2.4 the initial gross separation of anti-tumor activity peptide: get the supernatant that the protease hydrolysis mussel meat obtains, use molecular weight cut-off to carry out ultrafiltration respectively, obtain molecular weight and be that 10K is above, 5K-10K, 3K-5K and the hydrolyzed solution below the 3K as the ultra-filtration membrane of 10K, 5K and 3K.Adopt mtt assay to measure the proliferation inhibition rate of each component respectively after the lyophilize, tentatively confirm to have molecular weight ranges than the powerful antitumor activity polypeptide to prostate cancer cell.Hydrolyzed solution to this molecular weight ranges is collected through ultrafiltration in a large number, concentrates and lyophilize, does further separation and purification.
2.5 the DEAE-SepharoseFF ion exchange column chromatography separates: the strongest part of activity that above-mentioned steps obtains is crossed the DEAE-SepharoseFF ion exchange column; Use PBS (PH is 7.4), 0.1mol/LNaCL, 0.3mol/LNaCL, 0.5mol/LNaCL and 1mol/LNaCL solution to carry out stepwise elution respectively; Elution speed is 1ml/min; Protein Detection appearance 280nm detects, and collects each elution peak respectively, and lyophilize.Adopt mtt assay to carry out the anti-tumor activity experiment each peak component that obtains after the drying, further confirm the elution peak at target peptide place, and this peak is collected in a large number, concentrate and lyophilize, as being further purified specimen in use.
2.6 Sephadex G-25 gel column chromatography separates: the strongest component of activity that is obtained by above-mentioned steps is crossed Sephadex G-25 gel column (80cm * 2.6cm); Moving phase is zero(ppm) water; Flow velocity is 2mL/min; Protein Detection appearance 280nm detects, and each elution peak is collected respectively, concentrates postlyophilization.Adopt mtt assay to carry out the anti-tumor activity experiment each peak component that obtains after the drying, confirm the elution peak at target peptide place, and this peak is collected in a large number, concentrate and lyophilize, as the performance liquid specimen in use.
2.7 performance liquid chromatography (HPLC) purifying and purity detecting: purifying chromatographic condition: chromatographic column is Zorbax SB C
18(250mm * 9.4mm, 5 μ m); Column temperature is a room temperature; Moving phase is acetonitrile and water, gradient elution: 0～20min, and acetonitrile concentration changes to 95% with 0%; Elution speed 3.0mL/m in; Ultraviolet detection wavelength 220nm.The purity detecting chromatographic condition: chromatographic column is Hypersil BDS C18 (250mm * 4.6mm, 5 μ m); Column temperature is a room temperature; Gradient elution: 0～20min, acetonitrile concentration changes to 95% by 0%; Elution speed 0.8mL/m in; Ultraviolet detection wavelength 220nm.
2.8 the aminoacid sequence of target peptide detects.Obtain this mussel enzymolysis polypeptide through detection, comprise following aminoacid sequence: Asp Leu Tyr.
2.9 cellular form is observed: adopt the HE dyeing process.DU-145 and PC-3 cell inoculation are being had on six orifice plates of deckglass, behind the cell climbing sheet 24h, are adding the target peptide of 20mg/ml, cultivate 24h and 48h respectively after, with deckglass taking-up, 95% alcohol fixation 15min.PBS washes 2 times, and Hematorylin dyes, and tap water is fully washed, and redye in Yihong, and tap water embathes, the dehydration of alcohol gradient, and YLENE is transparent, observes and take the photograph sheet under the neutral tree mounting, opticmicroscope.
3 results
3.1 confirming of proteolytic enzyme kind:
4 kinds of proteolytic enzyme all have certain inhibited proliferation to the resulting zymolyte of mussel meat enzymolysis to the DU-145 cell; Wherein the Sumizyme MP zymolyte is the strongest to the proliferation inhibition activity of DU-145 cell; Concentration is 20mg/ml, and behind the effect 48h, inhibiting rate is 28.4% (see figure 1).4 components that hydrolysis by novo liquid obtains after the ultra-filtration membrane ultrafiltration are 20mg/ml in concentration, and behind the effect 48h, the following component of 3K is maximum to the proliferation inhibition rate of DU-145 cell, is 30.3% (see figure 2).
3.2 the separation of anti-PCa bioactive peptide
The following component of 3K obtains 4 peak component (see figure 3)s altogether behind DEAE-Sepharose FF ion exchange column wash-out, wherein peak 2 anti-tumor activities are the strongest, and when concentration is 20mg/ml, behind DU-145 cytosis 48h, inhibiting rate is 41.2%.Peak 2 obtains 3 peak component (see figure 4)s behind Sephadex G-25 gel column wash-out; Wherein the anti-tumor activity of peak 2-2 is the strongest; In concentration is 20mg/ml, and behind DU-145 cytosis 48h, inhibiting rate is the purifying and the purity testing of the anti-PCa bioactive peptide of 46.5%3.3: peak 2-2 is through Zorbax SB C
18Behind the purifying, finally obtain 1 polypeptide, be the purified target peptide of this experiment, see Fig. 5.This peptide is crossed Hypersil BDS C18 chromatographic column detect its purity, simple spike when RT is about 15min, occurs, see Fig. 6, it is higher to explain that this tests resulting target peptide purity, is one-component.
3.4 target peptide is to DU-145 and the effect of PC-3 cell inhibitory effect: target peptide is provided with 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml and five concentration group of 25mg/ml; Respectively behind mensuration effect 24h, 48h and the 72h, to the influence of DU-145 and PC-3 cell proliferation.The result shows that target peptide is stronger than PC-3 cell to the proliferation inhibition activity of DU-145 cell, and the proliferation inhibition activity of two kinds of cells is all presented timeliness and dose-effect relationship (table 2).
Table 2 mussel enzymolysis polypeptide to the influence of human prostata cancer DU-145 and PC-3 cell proliferation (x ± s, n=3)
3.4 morphological observation: HE dyeing is extremely shown in Figure 14 like Fig. 7, normal cell kytoplasm dyeing homogeneous, and cell fission is obvious, and form is full, and nucleus is not of uniform size, and the kernel number is many.Behind the target peptide effect 24h, tenuigenin concentrates, and karyon begins pyknosis and diminishes, and the intercellular space increases.Behind the effect 48h, above-mentioned change aggravation, the intercellular space is strengthened obviously, and cell outline is fuzzy, and cell space sharply dwindles, parts of fine kytoplasm formation of vacuoles, the kernel reduced number, apoptotic body forms, the chromatin color burn.Morphological observation shows, proves that once more target peptide has time-effect relationship to the effect of DU-145 and PC-3 cell; Apoptotic body forms, and explains that the antitumor action of target peptide possibly realized through cell death inducing.
In the last few years, from natural product, sought the focus that antitumor drug novel, efficient, low toxicity is various countries' research always.Wherein, the anti-tumor activity peptide is because characteristics such as its multifunctionality, hypersensitivity and high stability also receive increasing concern.This experiment is at first chosen several kinds of proteolytic enzyme mussel protein is hydrolyzed, and relatively the anti-tumor activity of each zymolyte finds that its anti-tumor activity of different zymolytes is also inequality, and this should be relevant with the composition structure of zymolyte.Can infer that in view of the above when utilizing enzyme solution to obtain the anti-tumor activity peptide, the selection of enzyme is most important, under the prerequisite of conditions permit, should select for use several kinds of proteolytic enzyme to carry out screening active ingredients as much as possible.Secondly, obtain in the process of target peptide, detect the component of different molecular weight and different elution peaks in separation and purification; Its anti-tumor activity also has very big difference, and the strongest active part is the small molecular weight polypeptide below the 3K, and in the polypeptide in this scope; What play active function also mainly is the polypeptide fraction at certain peak; And along with the carrying out of separation and purification, the anti-tumor activity of polypeptide also strengthens gradually, and 28.4% before by separation and purification is increased to 47.6% to the inhibiting rate of tumour cell for it.Therefore, when preparation anti-tumor activity peptide, carrying out separation and purification, to obtain pure article also be very necessary.At last, according to existing result of study, same bioactive peptide to not of the same race be that the active function of cancer cells is also inequality; This is tested resulting active polypeptide and screens to prostate cancer DU-145 cell; Though this target peptide also has proliferation inhibition activity to the PC-3 cell, activity is starkly lower than the former, so; When carrying out the anti-tumor activity peptide screening, the selection of cancer cells kind system plays a key effect for the target peptide that finally obtains.
This research has confirmed that the utilization enzyme solution can extract the anti-tumor activity peptide from mussel protein; This bioactive peptide has significant proliferation inhibition activity to prostate cancer cell; This experiment is laid a good foundation for the research and development of food proteins source antitumor drug and healthcare products, but awaits further to study for its antitumor mechanism and toxicity thereof.
Claims (5)
1. mussel enzymolysis polypeptide is characterized in that comprising following aminoacid sequence:
Asp?Leu?Tyr。
2. the preparation method of mussel enzymolysis polypeptide according to claim 1 is characterized in that comprising the steps:
1. get mussel meat, process homogenate, regulate pH value 9.5～10, solid-liquid ratio is 1: 2～1: 3, adds Sumizyme MP, and enzyme concentration is 1500～2000U/g, and enzymolysis time is 6～8 hours, and hydrolysis temperature is 45～50 ℃, and is centrifugal behind the enzyme that goes out, and gets supernatant liquid;
2. with above-mentioned clear liquid process ultrafiltration, collect the hydrolyzed solution below the molecular weight 3K, concentrate and lyophilize;
3. DEAE-SepharoseFF ion column exchange column chromatographic separation, with the lyophilize after product of step in 2. through DEAE-SepharoseFF ion column exchange column, 0.1～1mol/LNaCL gradient elution; Elution speed 1～1.5mL/min; Protein Detection appearance 280nm detects, and each elution peak is collected respectively, concentrates postlyophilization; Adopt mtt assay to carry out the anti-tumor activity experiment each peak component that obtains after the drying; Further confirm the elution peak at target peptide place, and this peak is collected in a large number, concentrate and lyophilize;
4. adopt Sephadex G-25 gel column chromatography to separate; With step 3. the lyophilize after product with high anti-tumor activity of gained cross Sephadex G-25 gel column; Moving phase is zero(ppm) water, and flow velocity is 1.5～2mL/min, and Protein Detection appearance 280nm detects; Each elution peak is collected respectively, concentrated postlyophilization and carry out the anti-tumor activity experiment with mtt assay;
5. high-efficient liquid phase chromatogram purification, with step obtain in 4. have high anti-tumor activity lyophilize after postpartum cross reversed phase high efficiency liquid phase post, chromatographic column is Zorbax SB C
18(250mm * 9.4mm, 5 μ m); Column temperature is a room temperature; Moving phase is acetonitrile and water, gradient elution: 0～20min, and acetonitrile is dense to change to 95%, elution speed 2.5～3.0mL/min, ultraviolet detection wavelength 220nm from 0%.
3. claim 2 or 3 application of described mussel enzymolysis polypeptide on anti-prostate cancer.
4. claim 2 or 3 described mussel enzymolysis polypeptides suppress the application on the propagation at anti-prostate cancer DU-145 cell.
5. claim 2 or 3 described mussel enzymolysis polypeptides suppress the application on the propagation at anti-prostate cancer PC-3 cell.
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