CN102816816A - Bacillus sphaericus parasporal crystal protein preparation method and application thereof - Google Patents
Bacillus sphaericus parasporal crystal protein preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Bacillus sphaericus parasporal crystal protein preparation method and application thereof. Wild type Bacillus sphaericus IAB 872 is cultured, and ultrasonic treatment, lysozyme treatment, DTT (dithiothreitol) treatment and compound cardiografin treatment are performed to improve the yield and activity of soluble crystal proteins, thereby obtaining high-effect soluble crystal proteins as many as possible. The Bacillus sphaericus parasporal crystal proteins have a certain inhibiting effect on human malignant tumor cells, and can be used for the preparation of antitumor drugs. The inhibition of the Bacillus sphaericus parasporal crystal proteins on the proliferation of human tumor cells includes the inhibition on tumor cells, the influence on apoptosis promotion of tumor cells, the influence on G2/M phase of cells and the arrest of cells in G2/M phase.
Description
Technical field
The invention belongs to microbial metabolites and applied technical field thereof, relate to a kind of Bacillus sphaericus parasporal crystal protein preparation method and application thereof.
Background technology
Spherical bacillus is the aerobic genus bacillus of strictness, and growth cycle can be divided into nourishing body phase and gemma phase, and its brood cell end or the proximal end of being born in cell, in the process that the brood cell forms, causes that thalline one end expands, and sporocyst is the drumstick shape.In the cell of spherical bacillus growth, be accompanied by brood cell's formation, the bacterial strain that has can produce one or more parasporal crystals, what have does not then produce parasporal crystal, and the form of parasporal crystal big or small with number also different and different along with bacterial strain.Parasporal crystal and brood cell the same end of being born in cell, and after the sporocyst maturation, crystal and brood cell still combine and be enclosed in the extine.Parasporal crystal have or not and form and spherical bacillus to kill mosquito active relevant; As in low virulent strain SSII-1,1404-924B and 1881, not finding parasporal crystal; Produce ellipse or oval parasporal crystal in the Kellen Q bacterial strain once in a while, do not have rhomboidan; High virulence 2297 bacterial strains mainly produce the rhombus parasporal crystal, also produce oval or oval parasporal crystal.Bacillus sphaericus is not to belonging to together, or belong to mosquito larvae not of the same race together different toxic actions is arranged, different serotypes, same serotype, or contain between the bacterial strain of identical toxin protein to kill the active difference of mosquito also bigger.
Bacillus sphaericus mainly is to be realized by the mosquito toxin that kills of its generation to the toxic action of different mosquito larvaes.Proved at present and in its growth and development process, can produce two types of different toxin: one type is the crystal toxin that is present in all supper toxic strains, forms with 51.4kDa albumen by 41.9; Another kind of be present in mosquito toxin extremely in low virulence and the part supper toxic strain (Mosquitocidal Toxin, Mtx), like Mtx1 (100kDa), Mtx2 (31.8kDa) and Mtx3 (35.8kDa) etc.
Virulent strain can form the parasporal crystal that is positioned at brood cell's epicyte in all high virulence and the part in its brood cell's forming process.This crystal is made up of 41.9 and 51.4kDa albumen (being designated as BinA and BinB respectively) of equivalent.BinA and BinB were synthesized in the bacterial spore formation phase, and formed the III phase through two proteic interactions and the folding formation crystal of assembling the brood cell.Exist in the time of BinA and BinB is that the formation parasporal crystal is necessary.Western Blot shows no cross reaction between BinA and BinB albumen, and the stronger homology of nothing between them is described.
Fragment subclone experiment shows that BinA albumen is poisonous to mosquito larvae separately, but that the virulence ratio contains two kinds of proteic crystalline virulence is much lower.Independent BinB is nontoxic to mosquito larvae, but it exists and obviously to strengthen the proteic toxicity of BinA, has only two kinds proteicly to exist simultaneously, and the toxin protein competence exertion goes out maximum cytotoxicity, be a kind of binary toxin (Binary toxin, Bin).This possibly be because mosquito clone lacks the peritrophic membrane barrier, take the photograph due to the mechanism in the binding site that has a low affinity and the different toxin cell.Although two proteic existence simultaneously were to guarantee that the complete activity of binary toxin is necessary, the amino acid whose difference of BinA, particularly amino acid determines it to kill the mosquito activity and kills the mosquito spectrum in the left and right sides, 100 position.
The Mtx1 toxin: the Mtx1 toxin is the soluble toxin of a kind of 100kDa, is made up of 870 amino acid.Its N-end has a sequence with G+ bacterium signal peptide characteristic, with ADP-ADP ribosyltransferase ADP catalytic subunit homology.The C-end has three terminal repeats.This 100kDa albumen can be by the albumen of mosquito larvae midgut proteinase degraded formation 27 and 70kDa.The 70kDa polypeptide has three about 90 amino acid whose Tumor-necrosis factor glycoproteinss, and its function is ominous.And 27kDa contains one with commentaries on classics corresponding zone, film district, and with several kinds of ADP-ribose transferring enzyme toxin weak homology is arranged.The disappearance experiment is proof also, 27kDa fragment ability self ADP-ribosylation, and the 70kDa fragment can make mosquito cell generation pathologic reaction, exists when having only two kinds of protein fragments, just mosquito larvae is shown toxicity.
Mtx2 toxin Mtx3 toxin: Mtx2 toxin and Mtx3 toxin all are isolatedly from the SSII-1 bacterial strain to be made up of 292 and 326 amino acid; Molecular weight be respectively 31.8 with the toxin protein of 35.8kDa; They do not have homology with 100kDa toxin and crystal toxin; And with the 33kDa of aerogenesis folder film carboxylic bacterium (Clostridium perfrigens)-the 31.68kDa cell toxicant of toxin and pseudomonas aeruginosa (Pseudomonas aeruginos) have homology (Liu et al; 1993, Thanabalu&Porter, 1996).38% homology is arranged between Mtx2 and Mtx3, all contain the signal peptide of a G+ bacterium and the commentaries on classics film district of supposition.To the comparative analysis of isolated M tx2 toxin protein from 6 different B .s virulent strains, the amino acid that proves 224 positions determined this toxin kill mosquito active with kill the mosquito spectrum.
Summary of the invention
The problem that the present invention solves is to provide a kind of Bacillus sphaericus parasporal crystal protein preparation method and application thereof; Through the parasporal crystal protein extraction of Bacillus sphaericus bacterial strain, the parasporal crystal albumen that is extracted can be applicable to treatment and/or suppresses tumour or tumour auxiliary diagnosis.
The present invention realizes through following technical scheme:
A kind of Bacillus sphaericus parasporal crystal protein preparation method may further comprise the steps:
1) with Bacillus sphaericus IAB 872 bacterial strains after activation on the LB solid medium, be inoculated in 30 ℃ of incubated overnight in the LB liquid nutrient medium, in the MBS substratum of transferring next day, concussion is cultured to the release fully from parent cell of brood cell and crystal;
2) collect fermented liquid, centrifugal, deposition fully disperses crystal, brood cell, cell debris with the ultrasonication suspension after washing with the NaCl solution centrifugal, fully shakes mixing then, and scumming is centrifugal, gets deposition;
3) deposition is added water and process suspension, adding N,O-Diacetylmuramidase to final concentration is 0.1～0.5mg/ml, handles 2～3h for 37 ℃, during soft mixing for several times, centrifugal collecting precipitation then;
4) deposition is added water and processes suspension, add DTT to its concentration be 50～100mM, fully behind the mixing, leave standstill 1h, centrifugal collecting precipitation in 27～30 ℃;
5) will be deposited in-20 ℃ with room temperature under freeze thawing once, add mass concentration 42～44% Compound Diatrzoatc Meglumlnes, fully centrifugal behind the mixing, collect supernatant, and with the supernatant 24～48h that in sterilized water, dialyses;
6) process lyophilized powder under the supernatant cryogenic vacuum freezing conditions after will dialysing, obtain Bacillus sphaericus parasporal crystal albumen.
Saidly in the MBS substratum, cultivate 70～74h, culture temperature is 28～30 ℃.
Being set at of ultrasonic generator during described ultrasonication: temperature is that 4 ℃, frequency are that 20kHz, power are 200W; Ultrasound procedures is: ultrasonic 5s, stop 5s, and 25min altogether circulates.
The freeze thawing of said step 5) is: keep down spending the night at-20 ℃, room temperature is dissolved down to it; Described centrifugal be the centrifugal 20min of 10000r/min.
The application of the parasporal crystal albumen of Bacillus sphaericus Bacillus sphaericus IAB 872 in the preparation antitumor drug.
Described parasporal crystal albumen comprises BinA and the BinB binary toxin that the protein polypeptide of 51kDa and 42kDa is formed.
Described antitumor drug is the medicine of short apoptosis of tumor cells.
Described antitumor drug is for suppressing the medicine of tumor cell proliferation.
The medicine of described inhibition tumor cell proliferation is arrested in tumour cell the medicine of G2/M phase.
Described antitumor drug is one or more in anti-cervical cancer, anti-liver cancer, anti-cancer of the stomach, the anti-lung cancer drugs.
Compared with prior art, the present invention has following beneficial technical effects:
Bacillus sphaericus parasporal crystal protein preparation method provided by the invention adopts the Bacillus sphaericus IAB 872 of wild-type to cultivate, and has especially improved the soluble crystal albumen that comprises binary toxin.In the prior art; Bacterial strain exists brood cell's fragmentation insufficient when cultivating extraction, and crystallin and gemma are wrapped in [J.Invertebr Pathol.2009 in the extine; 101:106 – 111.]; Can not fully extract albumen, to the defective that the proteic effect of soluble crystal also decreases, the present invention handles through ultrasonication, N,O-Diacetylmuramidase processing, DTT processing, Compound Diatrzoatc Meglumlne; Improve proteic output of soluble crystal and activity, thereby obtained soluble crystal albumen as much as possible, higher effect.The soluble crystal albumen that is obtained can reach 2.09mg/ml.
Include the 51kDa and the binary toxin of 42kDa that produce in the gemma growth later stage in the Bacillus sphaericus parasporal crystal albumen of the present invention preparation, and the gene order of binary toxin to kill the young preparation bacterial strain of mosquito Bacillus sphaericus 2362 (serotype H5a5b) highly consistent with being used as commercialization; And nourishing body does not produce any MTX toxin vegetative period.
The present invention provides Bacillus sphaericus parasporal crystal albumen that human malignant lesion's cell is had certain restraining effect first, can use preparing anti-tumor medicine.Bacillus sphaericus parasporal crystal albumen comprises the result of human tumor cells inhibition of proliferation:
Inhibiting rate to tumour cell: the parasporal crystal albumen that Bacillus sphaericus Bacillus sphaericus IAB 872 extracts all shows good inhibitory effect to five kinds of tumour cells such as HELA, liver cancer 7721,7402, cancer of the stomach 7901, lung cancer A549, and inhibiting rate is respectively: 78%, 61%, 56%, 53%, 79%.Select inhibiting rate higher H ELA and A549 tumour cell, make the drug level gradient and find that it has certain concentration dependent.
Influence to the short apoptosis of tumour cell: though small concentration administration group (0.167mg/mL) is early withered and the cell that withers evening amounts to 43.8%, compare to some extent with negative control group and increase, do not have evident difference; After but middle concentration administration group (0.25mg/mL) administration was handled, apoptosis rate was 58.8%.Big concentration administration group (0.375mg/mL) apoptosis rate is 62.2%, and comparing with negative control group all has significant difference; Show that (0.25mg/mL) dosage and above parasporal crystal albumen can promote the apoptosis of tumour cell.
Influence to tumour cell cycle: the flow cytometer detected result shows; Negative group G2 phase cell is 6.11%; Small concentration administration group (0.167mg/mL) G2 phase cell is 7.74%, and middle concentration administration group (0.25mg/mL) is 8.09%, and big concentration administration group (0.375mg/mL) is 16.18%; Parasporal crystal albumen can influence the G2/M phase of cell, makes cell be arrested in the G2/M phase.
Description of drawings
Fig. 1 is a Bacillus sphaericus Bacillus sphaericus IAB 872Bradford determination of protein concentration typical curve;
Fig. 2 carries out the apoptosis analytical results for two the dying of parasporal crystal albumen effect A549 cell 48h Annexin V-FITC/PI that the Flow cytometry Bacillus sphaericus is purified; Wherein, Q3: normal cell; Q4: cell early withers; Q2: cell withers evening; Q1: non-viable non-apoptotic cell a: negative control group; B:0.167mg/mL; C:0.25mg/mL; D:0.375mg/mL.
The parasporal crystal albumen effect A549 cell 48h cell cycle that Fig. 3 purifies for the Flow cytometry Bacillus sphaericus influence the result, wherein, a: negative control group; B:0.167mg/mL; C:0.25mg/mL; D:0.375mg/mL.
Embodiment
Bacillus sphaericus parasporal crystal protein preparation method provided by the invention and application thereof; Batch fermentation through the spherical bud bar of wild-type spore bacterium; Its cell is carried out the processing of the centrifugal grade of cracking; Obtain its meta-bolites parasporal crystal albumen, first this albumen is applied to anti-human tumor cells, the result shows can use preparing anti-tumor medicine.Below in conjunction with concrete embodiment the present invention is done further detailed description, said is to explanation of the present invention rather than qualification.
1, Bacillus sphaericus Bacillus sphaericus IAB 872 bacterial strains:
Bacillus sphaericus IAB 872 is a kind of existing Bacillus sphaericuses, and the toxin that it produced is usually used in mosquito extremely.Concrete; The present invention is that an isolated strain Bacillus sphaericus IAB 872 specifically describes from the pedotheque, and those skilled in the art also can adopt other channels to obtain Bacillus sphaericus IAB 872 and accomplish the present invention certainly.
2, Bacillus sphaericus Bacillus sphaericus IAB 872 produces the proteic extraction of parasporal crystal:
(1) with Bacillus sphaericus IAB 872 bacterial strains after activation on the LB solid medium; Picking list bacterium colony is 30 ℃ of incubated overnight in the LB liquid nutrient medium, and transfer with the ratio of 1:100 and cultivate about (200rpm) 72h to brood cell and crystal release fully from parent cell in 30 ℃ of concussions in the MBS substratum that shakes bottle greatly next day;
(2) collect fermented liquid, the centrifugal 10min of 10000g, deposition with the NaCl centrifuge washing of 0.5M is once used the ultrasonication suspension, being set at of ultrasonic generator: temperature is that 4 ℃, frequency are that 20kHz, power are 200W; Ultrasound procedures is: ultrasonic 5s, stop 5s, and 25min altogether circulates; Crystal, brood cell, cell debris are disperseed as far as possible, on eddy mixer, constantly shake mixing again, remove the foam of generation simultaneously, centrifugal, get deposition;
(3) process suspension by 70mg deposition/ml distilled water, press final concentration 0.1mg/ml and add N,O-Diacetylmuramidase (10mM EDTA configuration), handle 2hr for 37 ℃, during per half a hour take out soft mixing several, centrifugal collecting precipitation;
(4) process suspension by 70mg deposition/ml distilled water, add 50mM DTT (WR 34678), fully behind the mixing, 27 ℃ leave standstill processing 1hr, centrifugal collecting precipitation;
(5) will be deposited in-20 ℃ with room temperature under freeze thawing once; Add 42% Compound Diatrzoatc Meglumlne, centrifugally behind the mixing can fully separate gemma and nourishing body, gemma purity is improved; The centrifugal 20min of 10000rpm; Collect supernatant, and the 48h that in sterilized water, dialyses, the proteic concentration of solubility parasporal crystal detected then;
(6) process lyophilized powder under the supernatant cryogenic vacuum freezing conditions after will dialysing, obtain Bacillus sphaericus parasporal crystal albumen, preserve down with-4 ℃.
Parasporal crystal is meant binary toxin (the Binary Toxin that is formed at the protein polypeptide (BinA and BinB) of gemma growth later stage generation 51kDa and 42kDa; Bin) form, and the gene order of binary toxin is with kill the young preparation bacterial strain of mosquito Bacillus sphaericus 2362 (serotype H5a5b) as commercialization highly consistent.
The lysotype toxin protein that mosquito larvae is had toxic action that part supper toxic strain and all low virulence bacterial strains produce also that a kind of vegetative phase expresses, for the vegetative phase kill the mosquito toxin protein (Mosquitocidal Toxin, MTX).All supper toxic strains can both produce binary toxin, and wherein some bacterial strain also can produce the Mtx toxin simultaneously, and Bacillus sphaericus IAB 872 nourishing bodys do not produce any MTX toxin vegetative period.
3, Bradford determination of protein concentration:
G-250 is red-brown under acidic conditions, and be blue after the protein bound, and protein meets Beer's law in the finite concentration scope; The colorimetric at the 595nm place; 2-5min is maximum light absorption, stablizes 1hr (0.01-1.0mg albumen scope), the unavailable quartz curette of cuvette.
The concrete reagent that is adopted comprises:
Staining fluid: G-250100mg is dissolved in 95% ethanol, adds 85% phosphatase 11 00ml, and thin up is to 1000ml.(dye liquor is preserved the several months, and not adding water can prolonged preservation);
Standard protein solution: 0.5mg/ml bovine serum albumin BSA;
Concrete grammar:
Standard protein solution
Add 0ml successively, 0.05ml, 0.10ml, 0.15ml, 0.20ml, 0.25ml, 0.30ml, 0.35ml, 0.40ml, 0.45ml, the standard protein solution of 0.50ml adds the 3ml dye liquor, and shakes up, and places room temperature 20-25 ℃ 15min, measures absorbance value with the 595nm place.
With the A595 value is Y, and BSA content is X, and the production standard curve is obtained linear equations, and the detected result of mark curve is shown in Figure 1, combines absorbance value can know agnoprotein concentration again.
The detected result of parasporal crystal protein concentration is 2.09mg/ml.
4, tumor cell culture and inhibiting rate detect:
1) liver cancer 7721,7402, cancer of the stomach 7901, lung cancer A549, five kinds of cells of cervical cancer HELA in the RPMI-1640 that contains 10% foetal calf serum, place 37 ℃, 5%CO all available from U.S. ATCC cell bank
2Cultivate in the incubator.
2) growth conditions is good, in the MCF7,7721,7402,7901 of logarithmic phase growth, A549, HELA cell with 10
5Be inoculated in 96 orifice plates, spend the night and treat that cell is adherent naturally.With the spherical parasporal crystal protein extract that adds 0.5mg/ml concentration in the above-mentioned cell, then cell is put 37 ℃, 5%CO
2The middle 48h that cultivates.
3) mtt assay is measured the cell growth: after cultivating 48h, treat that the every hole of gaging hole adds MTT solution (5mg/ml) 20 μ l, 37 ℃ are continued to cultivate 4h.After stopping cultivating, the careful exhaustion treats that nutrient solution in the gaging hole, every hole add 150 μ l DMSO, and vibration 10min fully dissolves xln, measures absorbancy in ELIASA 490nm wavelength.Establish 5 parallel holes for every group, test equal triplicate for every group.
Inhibiting rate (%)=(survival rate of 1-cell) * 100% of cell growth
The parasporal crystal albumen that Bacillus sphaericus Bacillus sphaericus IAB 872 extracts all shows good inhibitory effect to five kinds of tumour cells such as HELA, liver cancer 7721,7402, cancer of the stomach 7901, lung cancer A549; Inhibiting rate is respectively: 78%, 61%, 56%, 53%, 79%, and specifically as shown in table 1.Select inhibiting rate higher H ELA and A549 tumour cell, make the drug level gradient and find that it has certain concentration dependent, specifically as shown in table 2.
Table 1 parasporal crystal albumen 0.5mg/mL is to the inhibiting rate of different tumour cells
| The tumour cell kind | 7901 | A549 | 7721 | 7402 | HELA |
| Inhibiting rate % | 70.93 | 78.12 | 59.53 | 56.32 | 78.10 |
Table 2 parasporal crystal albumen different concns is to the inhibiting rate of tumour cell A549 and HELA
| Crystallin concentration mg/mL | 0.0625 | 0.125 | 0.25 | 0.375 | 0.5 |
| Inhibiting rate % to A549 | 12.41 | 14.21 | 36.01 | 53.20 | 74.73 |
| Inhibiting rate % to HELA | 24.50 | 34.08 | 49.72 | 58.43 | 73.93 |
5, parasporal crystal albumen is to the influence of apoptosis of tumor cells:
1) the A549 cell in vegetative period of taking the logarithm, parallel inoculation, it is about 50% to treat behind the 24h that degrees of fusion reaches, and it is apoptosis-induced to use the parasporal crystal albumen of 0.125mg/mL, 0.187mg/mL, 0.25mg/mL concentration group to carry out, and negative control group adds isopyknic 1640 substratum.
2) behind the apoptosis-induced 48h administration group and negative control group cell are washed one time with PBS earlier, use 0.25% tryptic digestion dissociated cell again, obtain cell.
3) after cell dissociation gets off, add the cell culture fluid of collecting in the step (2), mixing is transferred in the centrifuge tube slightly, the centrifugal 5min of 1500r/min, and supernatant discarded, collecting cell, again with PBS re-suspended cell washing gently, and counting.
4) get 1 ~ 5 * 10
5Cell suspending liquid, 1500rpm abandons supernatant after centrifugal 5 minutes, adds 1 * Binding Buffer (with zero(ppm) water 1:9 dilution 10 * Binding Buffer) of 500 μ l.
5) add 5 μ L AnnexinV-FITC, add 10 μ LPropidium Iodide, mixings gently again.
6) lucifuge dyeing 5 ~ 15min at room temperature.
7) in 1 hour, carrying out flow cytometer detects.Excitation wavelength Ex=488nm; Emission wavelength Em=530nm.The green fluorescence FITC passage of Annexin V-FITC is FL1, and the PI red fluorescence is FL2 through the PI passage.Use as contrast, is carried out the fluorescence compensating regulation without the normal cell of apoptosis induction.
Use after the 0.167mg/mL, 0.25mg/mL, 0.375mg/mL parasporal crystal albumen effect A549 cell 48 hours, use two the dying of AnnexinV-FITC/PI to carry out the apoptosis analysis, investigate the shared ratio of apoptotic cell.Investigating extract is the situation of A549 apoptosis induction to gastric cells.
Detected result is as shown in Figure 2, and the result of cells were tested by flow cytometry shows that it is normal viable cell that each district of scatter diagram represents following implication: Q3 (Annexin-/PI-) respectively; Q4 (Annexin+/PI-) is a viable apoptotic cell; Q1 (Annexin+/PI+) is a non-viable apoptotic cell; Q2 (Annexin-/PI+) is mechanical injuries cell and downright bad cell.Negative control group is early withered and the cell that withers evening amounts to 40.1%.Small concentration administration group (0.167mg/mL) is early withered and the cell that withers evening amounts to 43.8%, compares to some extent with negative control group and increases, but do not have evident difference.After middle concentration administration group (0.25mg/mL) administration was handled, apoptosis rate was 58.8%.Big concentration administration group (0.375mg/mL) apoptosis rate is 62.2%, and comparing with negative control group all has significant difference.
6, parasporal crystal albumen is to the influence of tumour cell cycle:
1) the A549 cell in vegetative period of taking the logarithm; Parallel inoculation; It is about 50% to treat behind the 24h that degrees of fusion reaches, and it is apoptosis-induced to use the 4# genus bacillus crystallin of 0.125mg/mL, 0.187mg/mL, 0.25mg/mL concentration group to carry out, and negative control group adds isopyknic 1640 substratum.
2) behind the apoptosis-induced 48h administration group and negative control group cell are washed one time with PBS earlier, use 0.25% tryptic digestion dissociated cell again, obtain cell.
3) after cell dissociation gets off, add the cell culture fluid of collecting in the step (2), mixing is transferred in the centrifuge tube slightly, the centrifugal 5min of 1500r/min, and supernatant discarded, collecting cell, again with PBS re-suspended cell washing gently, and counting.
4) get 1 * 10
6Cell suspending liquid, 1500rpm abandons supernatant after centrifugal 5 minutes, adds the reagent A (PI that contains 50 μ g/mL, the RNase of 50 μ g/mL) of 500 μ l, 5 μ l reagent B (penetrating liquid), vortex 5 ~ 10s, room temperature lucifuge reaction 30min.
5) in 1 hour, carrying out flow cytometer detects.Sample filters with 200 eye mesh screens before detecting, in order to avoid stop up.
Use after the 0.167mg/mL, 0.25mg/mL, 0.375mg/mL parasporal crystal albumen effect A549 cell 48 hours, use PI singly to dye and carry out cell cycle analysis, investigate the influence of shared ratio in extract cell cycle each period.
The result of cells were tested by flow cytometry such as Fig. 3; Negative group G2 phase cell is 6.11%; Small concentration administration group (0.167mg/mL) G2 phase cell is 7.74%, and middle concentration administration group (0.25mg/mL) is 8.09%, and big concentration administration group (0.375mg/mL) is 16.18%; Parasporal crystal albumen can influence the G2/M phase of cell, makes cell be arrested in the G2/M phase.
In sum, the parasporal crystal albumen of Bacillus sphaericus IAB 872 has certain restraining effect to human malignant lesion's cell, comprises cervical cancer, liver cancer, stomach cancer cell; And human lung cancer cell A549 and cervical cancer cell HELA had high inhibitory, be respectively 78%, 79%.Inhibiting rate tumors of higher cell is further detected, select the inhibiting rate of parasporal crystal albumen different concns to tumour cell A549 and HELA, the result shows to have certain concentration dependent.
And, it being arrested in the G2/M phase in view of the influence and the inhibition tumor cell proliferation of parasporal crystal albumen to the short apoptosis of tumour cell, the parasporal crystal albumen of Bacillus sphaericus IAB 872 just can be applicable to preparing anti-tumor medicine so.
Claims (10)
1. a Bacillus sphaericus parasporal crystal protein preparation method is characterized in that, may further comprise the steps:
1) with Bacillus sphaericus IAB 872 bacterial strains after activation on the LB solid medium, be inoculated in 30 ℃ of incubated overnight in the LB liquid nutrient medium, in the MBS substratum of transferring next day, concussion is cultured to the release fully from parent cell of brood cell and crystal;
2) collect fermented liquid, centrifugal, deposition fully disperses crystal, brood cell, cell debris with the ultrasonication suspension after washing with the NaCl solution centrifugal, fully shakes mixing then, and scumming is centrifugal, gets deposition;
3) deposition is added water and process suspension, adding N,O-Diacetylmuramidase to final concentration is 0.1～0.5mg/ml, handles 2～3h for 37 ℃, during soft mixing for several times, centrifugal collecting precipitation then;
4) deposition is added water and processes suspension, add DTT to its concentration be 50～100mM, fully behind the mixing, leave standstill 1h, centrifugal collecting precipitation in 27～30 ℃;
5) will be deposited in-20 ℃ with room temperature under freeze thawing once, add mass concentration 42～44% Compound Diatrzoatc Meglumlnes, fully centrifugal behind the mixing, collect supernatant, and with the supernatant 24～48h that in sterilized water, dialyses;
6) process lyophilized powder under the supernatant cryogenic vacuum freezing conditions after will dialysing, obtain Bacillus sphaericus parasporal crystal albumen.
2. Bacillus sphaericus parasporal crystal protein preparation method as claimed in claim 1 is characterized in that, saidly in the MBS substratum, cultivates 70～74h, and culture temperature is 28～30 ℃.
3. Bacillus sphaericus parasporal crystal protein preparation method as claimed in claim 1 is characterized in that, being set at of ultrasonic generator during described ultrasonication: temperature is that 4 ℃, frequency are that 20kHz, power are 200W; Ultrasound procedures is: ultrasonic 5s, stop 5s, and 25min altogether circulates.
4. Bacillus sphaericus parasporal crystal protein preparation method as claimed in claim 1 is characterized in that the freeze thawing of said step 5) is: keep down spending the night at-20 ℃, room temperature is dissolved down to it; Described centrifugal be the centrifugal 20min of 10000r/min.
5. the application of the parasporal crystal albumen of Bacillus sphaericus Bacillus sphaericus IAB 872 in the preparation antitumor drug.
6. application as claimed in claim 5 is characterized in that, described parasporal crystal albumen comprises BinA and the BinB binary toxin that the protein polypeptide of 51kDa and 42kDa is formed.
7. application as claimed in claim 5 is characterized in that, described antitumor drug is the medicine of short apoptosis of tumor cells.
8. application as claimed in claim 5 is characterized in that, described antitumor drug is for suppressing the medicine of tumor cell proliferation.
9. application as claimed in claim 5 is characterized in that, the medicine of described inhibition tumor cell proliferation is arrested in tumour cell the medicine of G2/M phase.
10. application as claimed in claim 5 is characterized in that, described antitumor drug is one or more in anti-cervical cancer, anti-liver cancer, anti-cancer of the stomach, the anti-lung cancer drugs.
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| CN105664135A (en) * | 2016-02-03 | 2016-06-15 | 西安交通大学第一附属医院 | Extraction method of extracellular metabolism of bacillus sphaericus 2317-2 and anti-tumor application |
| CN107438617A (en) * | 2014-12-22 | 2017-12-05 | 农业生物群落股份有限公司 | Killing gene and application method |
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| CN1119877A (en) * | 1993-03-25 | 1996-04-03 | 希巴-盖吉股份公司 | Novel pesticidal proteins and strains |
| US6720167B1 (en) * | 2000-08-14 | 2004-04-13 | The Regents Of The University Of California | Insecticidal bacteria, and methods for making and using them |
| US20120207754A1 (en) * | 2011-02-15 | 2012-08-16 | Vaxiion Therapeutics, Inc. | Therapeutic compositions and methods for antibody and fc-containing targeting molecule-based targeted delivery of bioactive molecules by bacterial minicells |
-
2012
- 2012-08-29 CN CN201210312288.7A patent/CN102816816B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1119877A (en) * | 1993-03-25 | 1996-04-03 | 希巴-盖吉股份公司 | Novel pesticidal proteins and strains |
| US6720167B1 (en) * | 2000-08-14 | 2004-04-13 | The Regents Of The University Of California | Insecticidal bacteria, and methods for making and using them |
| US20120207754A1 (en) * | 2011-02-15 | 2012-08-16 | Vaxiion Therapeutics, Inc. | Therapeutic compositions and methods for antibody and fc-containing targeting molecule-based targeted delivery of bioactive molecules by bacterial minicells |
Non-Patent Citations (1)
| Title |
|---|
| 郭艾英 等: "苏云金芽孢杆菌晶体蛋白的制备方法", 《食品与发酵工业》, vol. 31, no. 8, 30 August 2005 (2005-08-30), pages 8 - 10 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107438617A (en) * | 2014-12-22 | 2017-12-05 | 农业生物群落股份有限公司 | Killing gene and application method |
| CN105664135A (en) * | 2016-02-03 | 2016-06-15 | 西安交通大学第一附属医院 | Extraction method of extracellular metabolism of bacillus sphaericus 2317-2 and anti-tumor application |
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| CN102816816B (en) | 2014-01-29 |
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