CN103739690A

CN103739690A - Method for separation preparation of anti-tumor polypeptide compound from ArcainflataReeve, and uses of anti-tumor polypeptide compound

Info

Publication number: CN103739690A
Application number: CN201310619247.7A
Authority: CN
Inventors: 于荣敏; 徐建; 宋丽艳
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-11-29
Filing date: 2013-11-29
Publication date: 2014-04-23

Abstract

The present invention relates to separation preparation of an active polypeptide compound from ArcainflataReeve, and medicinal uses of the active polypeptide compound. The preparation method comprises: shelling ArcainflataReeve, washing content, adding a buffer solution to homogenize, extracting, carrying out centrifugation to remove residue, taking the supernatant, carrying out fractional precipitation with ammonium sulfate, carrying out dialysis desalting to obtain an anti-tumor active component, carrying out dialysis desalting and freeze drying on the active component, carrying out ion-exchange column chromatography, respectively collecting active peaks, and carrying out hydrophobic column chromatography to achieve separation purification so as to obtain the anti-tumor activity polypeptide compound with the purity of 99%. According to the present invention, the modern extraction separation technology is creatively adopted to separate the active polypeptide compound having clear material basis and molecular weight of 20538.0 Da and containing secondary structures such as 45.2% of alpha-helix, 2.9% of beta-sheet, 26.0% of beta-turn and 25.9% of random coil from the ArcainflataReeve, wherein the compound has the significant anti-tumor activity. In addition, in vitro anti-tumor experiment results show that the inhibition rate on human lung adenocarcinoma cells (A549) is 85%, and the compound can be used for development and application of novel anti-tumor drugs.

Description

From stalwart blood clam, separate method and the purposes of a kind of tumor protein p53 compound of preparation

Technical field

The invention belongs to medical technical field, relate to a kind of polypeptide compound, its method for separating and preparing and this compound anti-tumor medical application that obtains a kind of tool anti-tumor activity from marine organisms chief blood clams (Arca inflata Reeve) through steps such as a series of extractions, separation, chromatography purifications.

Background technology

Chief blood clam ( arca inflatareeve) belong to animal door, lamellibranchiata, blood clam order, blood clam section, is one of halobios that china natural resources is abundant.Chief blood clam fertilizer substantially, delicious meat, in ancient book, recording stalwart blood clam has " make us eating, beneficial color, disappear clot and reduce phlegm long-pending " effect, nutritive value and economic worth are very high, are one of important economic shellfishes in China and coastland, Asia.

By consulting data of literatures, find, foreign scholar characterizes and has expressed a kind of polypeptide with anti-microbial activity and immunoregulatory activity from stalwart blood clam, and the research of extraction active substance has no report.Domestic scholars mainly concentrates on the research of stalwart blood clam enzymolysis product activity and relevant aquatic products cultivation aspect to the research of stalwart blood clam, but the research to stalwart blood clam protein and peptide material is little, as Chinese Patent Application No. 200610036488.9 discloses " polypeptide protein class actives and its production and use in stalwart blood clam ", this patent is mainly for being studied in molecular weight 3-150KDa polypeptide active plural components, this patent documentation has described in detail and from stalwart blood clam, has extracted active crude protein, the process of polypeptides matter, it extracts product is thick composite parts, purity is not high and active substance is basic indefinite, also have no any extracts and is separated to tumor protein p53 compound and the physico-chemical property thereof that purity is high and basic substance is clear and definite from stalwart blood clam, the research report of structure elucidation.Therefore working substance basis clear and definite in stalwart blood clam is further furtherd investigate and is significant, for rationally utilizing oceanic resources that theoretical foundation is provided.

Summary of the invention

The object of the invention is to, by modern extraction separation and purification technology, from marine organisms, extract protein isolate polypeptide active substance, a kind of active polypeptide compound that derives from stalwart blood clam is particularly provided, comprise preparation method, medicinal use and structure elucidation etc.

The present invention realizes by the following technical solutions: by fresh stalwart blood clam peel off, take out content, clean, add damping fluid, homogenate, extraction, centrifugal, get supernatant liquor, ammonium sulfate precipitation, get precipitation dialysis desalting, employing ion-exchange chromatography separates, collect respectively Peak Activity, after dialysis desalting, through hydrophobic chromatography post, separate, obtain purity and be 99%, molecular weight is the anti-tumour active polypeptide compound of 20538.0 Da, and its structure resolved.

This polypeptide compound is the material that natural extract separates, and has significant anti-tumor activity, and anticancer experiment in vitro shows that its inhibiting rate to human lung adenocarcinoma cell (A549) has reached 85%.The method of extracting activity polypeptid substance from stalwart blood clam of the present invention, to obtain a polypeptide compound through ion exchange chromatography and hydrophobic chromatography separation and purification, this polypeptide compound purity is high, and anti-tumor active substance basis is clear and definite, therefore can be used for anti-tumor medical application.

Accompanying drawing explanation

Fig. 1 is the relative molecular weight collection of illustrative plates of measuring active polypeptide compound in example 1 with electrospray ionization mass spectrometry.

Fig. 2 is the circular dichroism spectrogram of polypeptide compound in example 1.

Fig. 3,4,5,6 is the partial amino-acid series collection of illustrative plates that the attached ionization time-of-flight mass spectrometry of example 1 mesostroma assisted laser desorption is measured polypeptide compound.

embodiment:

Below in conjunction with technical scheme and chart, describe specific embodiments of the invention in detail.

embodiment 1

Stalwart blood clam is peeled off, get its content, with low-temperature distillation water cleaning three times, after control is dry, weigh, according to the part by weight of 1:3, add Extraction buffer (pH=8.0, 0.03 mol/L, sodium phosphate buffer), adopt high-speed tissue mashing machine, block to fine and smooth nothing with 8000 rpm interval homogenate 3 min, homogenate is placed in to low frequency ultrasound instrument supersound extraction 40 min, use low-temperature and high-speed whizzer, 10000 rpm, 4 ℃, centrifugal 30 min remove slag, after measuring the volume of supernatant liquor, be placed on after the ammonium sulfate that adds 70% saturation ratio in ice bath environment, continue to stir and saltout after 1 h, high speed centrifugation is removed precipitation and is got supernatant liquor, measure the ammonium sulfate that adds 100% saturation ratio after volume, continue again to stir to saltout after 1 h, 10000 rpm, 4 ℃, centrifugal 30 min, collecting precipitation.Precipitation is dissolved to be placed in 3000 D dialysis tubings with a small amount of extracting solution and dialyse 24 h with desalination, at interval of 6 h, change periphery distilled water.Use DEAE-Sepharose Fast Flow anion-exchange chromatography to separate the active ingredient after dialysis desalting, the Tris-HCl damping fluid of 0.03 mol/L that is 8.0 with pH is as initial elutriant, by the concentration that increases sodium-chlor, carry out stepwise elution, absorption peak is detected at 280 nm places, collect the elutriant of 0.1 mol/L sodium-chlor wash-out, after dialysis desalting, re-use Phenyl Sepharose CL-4B hydrophobic chromatography post and separate.First use 1.0 mol/L, pH is that 8.0 ammonium sulfate phosphoric acid buffer carries out wash-out, then successively with carrying out stepwise elution containing 0.5 mol/L and the ammonium sulfate of 0 mol/L, the sodium phosphate buffer that pH is 8.0, absorption peak is detected at 280 nm places, collect the not elutriant of the sodium phosphate buffer wash-out of liquid containing ammonium sulfate, after dialysis desalting, concentrated frozen is dry, has obtained the active polypeptide compound in stalwart blood clam.The purity that detects this polypeptide with RP-HPLC (RPLC) has reached 99%.Adopting ESI-MS(electrospray ionization mass spectrometry) molecular weight of measuring this polypeptide is 20538.0 Da, by circular dichroism spectrum (CD spectrum) and Jasco albumen secondary structure assessment software, analyzes its secondary structure and forms.With MALDI-TOF-MS(matrix assisted laser desorption ionization ionization time-of-flight mass spectrometry) detected the Partial Fragment aminoacid sequence of polypeptide.

Table 1. polypeptide compound albumen secondary structure composition

Figure 2013106192477100002DEST_PATH_IMAGE001

embodiment 2

Adopt mtt assay to carry out antitumor activity screening to whole extraction sepn process.

By the tumour cell in logarithmic phase (attached cell need be used trysinization), make containing cell 3～4 × 10 ³individual/ml single cell suspension (cell density), is inoculated in respectively in aseptic 96 well culture plates, and 100 μ l/ holes, in 5% CO ₂, in 37 ℃ of incubators, cultivate.After cell cultures 12 h, establish blank group (adding equal-volume RPMI1640), positive control (cis-platinum) and administration group, 3 repeating holes of each concentration group, add medicine, and every hole total reaction volume is 200 μ l, and cell is put and in incubator, cultivated 48 h.Experiment stops front 4 h and adds people MTT 20 μ l(5 mg/ml) continue to cultivate 4 h, abandon supernatant liquor, add DMSO 200 μ l/ holes, be placed in 10 min that vibrate on shaker, by microplate reader in wavelength 570 nm places mensuration optical densitys.With the positive contrast of cis-platinum, use pharmacological method to calculate IC ₅₀value (drug level when inhibiting rate reaches 50%).Experimental result is as shown in table 1, and with the positive contrast medicine of cis-platinum, polypeptide compound is to human A549 cell lines, the restraining effect of human liver cancer cell HepG2 and Human colorectal cancer cells HT-29.

The anti tumor activity in vitro test result of table 2. polypeptide compound (

± SD, n=3)

?

Figure 2013106192477100002DEST_PATH_IMAGE003

In sum, the stalwart blood clam polypeptide compound that the present invention separates preparation has very strong antitumor action, because of preparation process natural, pollution-free, the feature that this polypeptide compound has efficiently, purity is high, and effective substance structure is clear and definite, be expected to become a kind of safe, effective, quality controllable new type antineoplastic medicine.

Claims

A stalwart blood clam ( arca inflatareeve) anti-tumour active polypeptide compound in, its molecular weight is 20538.0 Da, iso-electric point is 5.4, containing alpha-helix 45.2%, beta sheet 2.9%, β-corner 26.0%, random coil 25.9%, its amino acid fragment sequence comprises I/LSMEDVEESR, KNGMHSI/LDVNHSGR, AMKI/LI/LNPKKGI/LVPI/LR and AMGAHKPPKGNEI/LGHR.
2. the method for separating and preparing of the anti-tumour active polypeptide compound in the stalwart blood clam of claim 1, it is characterized in that: content is peeled off, taken out to fresh stalwart blood clam and clean, add damping fluid homogenate to extract, centrifugal, get supernatant liquor, ammonium sulfate precipitation, get precipitation dialysis desalting, adopt ion-exchange chromatography to separate, collect respectively Peak Activity, after dialysis desalting, through hydrophobic chromatography post, separate, obtain the active polypeptide compound of purity 99%.
3. the method for separating and preparing of anti-tumour active polypeptide compound in the stalwart blood clam of stating according to claim 2, it is characterized in that the damping fluid adding is 0.03 mol/L, pH is 8.0 sodium phosphate buffer, adding the volume of damping fluid and the weight ratio of stalwart blood clam is 1:3, carries out homogenate with high-speed tissue mashing machine.
4. the method for separating and preparing of anti-tumour active polypeptide compound in stalwart blood clam according to claim 2, it is characterized in that adopting ammonium sulfate precipitation, first make to extract supernatant liquor and reach 70% saturation ratio ammonium sulfate, centrifugal, get supernatant, add again ammonium sulfate precipitation to reach 100% saturation ratio, centrifugal, stay precipitation.
5. the method for separating and preparing of anti-tumour active polypeptide compound in stalwart blood clam according to claim 2, it is characterized in that a small amount of 0.03mol/L of precipitation, pH is after 8.0 Tris damping fluid dissolves, with after dialysis tubing dialysis desalting, separate with DEAE-Sephorose Fast Flow anion-exchange chromatography post, its wash-out initial buffer liquid is pH8.0 Tris-HCl damping fluid, and 0～2.0 mol/L NaCl carries out stepwise elution, and absorption peak is detected at 280 nm places.
6. the method for separating and preparing of anti-tumour active polypeptide compound in stalwart blood clam according to claim 2, it is characterized in that collecting the elutriant with 0.1 mol/L sodium-chlor wash-out, after dialysis desalting, separate with Phenyl Sepharose CL-4B hydrophobic chromatography post, its elutriant is 0.03 mol/L, pH8.0 sodium phosphate buffer, the ammonium sulfate of concentration 1.0～0 mol/L carries out stepwise elution, and absorption peak is detected at 280 nm places.
7. the method for separating and preparing of anti-tumour active polypeptide compound in stalwart blood clam according to claim 2, it is characterized in that the not elutriant of the sodium phosphate buffer wash-out of liquid containing ammonium sulfate of collection, after dialysis desalting, concentrated frozen is dry, has obtained the active polypeptide compound in stalwart blood clam.
8. the method for separating and preparing of anti-tumour active polypeptide compound in stalwart blood clam according to claim 2, the molecular weight that it is characterized in that the stalwart blood clam polypeptide compound obtaining is 20538.0 Da, iso-electric point is 5.4, containing alpha-helix 45.2%, beta sheet 2.9%, β-corner 26.0%, random coil 25.9%.
9. in the stalwart blood clam described in claim 1 or 2, tumor protein p53 compound can be used for preparing antitumor drug.