CN102816816B - Bacillus sphaericus parasporal crystal protein preparation method and application thereof - Google Patents
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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
Bacillus sphaericus, is strict aerobic genus bacillus, 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, causes that thalline one end expands in the process of sporulation, and sporocyst is drumstick shape.In the cell of Bacillus sphaericus growth, be accompanied by brood cell's formation, some bacterial strains can produce one or more parasporal crystals, what have does not produce parasporal crystal, and the form of parasporal crystal big or small with number the also difference along with the difference of bacterial strain.Parasporal crystal and brood cell same one end of being born in cell, and after sporocyst maturation, crystal and brood cell still combine and are enclosed in extine.Parasporal crystal have or not and form and Bacillus sphaericus to kill mosquito active relevant, as do not found parasporal crystal in low virulent strain SSII-1,1404-924B and 1881, in Kellen Q bacterial strain, produce once in a while ellipse or oval parasporal crystal, there is no rhomboidan; High virulence 2297 bacterial strains mainly produce rhombus parasporal crystal, also produce oval or oval parasporal crystal.Bacillus sphaericus has different toxic actions to not belonging to or belong to together mosquito larvae not of the same race together, different serotypes, same serotype or also larger containing the difference of killing mosquito activity between the bacterial strain of identical toxin protein.
Bacillus sphaericus is mainly to be realized by the mosquito toxin that kills of its generation to the toxic action of different mosquito larvaes.Now proved and in its growth and development process, can produce the different toxin of two classes: a class has been the crystal toxin being present in all supper toxic strains, by 41.9 and 51.4kDa albumen form; Another kind of is to be present in to kill mosquito toxin (Mosquitocidal Toxin, Mtx) in low virulence and part supper toxic strain, as Mtx1 (100kDa), Mtx2 (31.8kDa) and Mtx3 (35.8kDa) etc.
All high virulence and sulfurization power bacterial strain can form the parasporal crystal that is positioned at brood cell's epicyte in its sporulation process.This crystal is comprised of 41.9 and the 51.4kDa albumen (being designated as respectively BinA and BinB) of equivalent.BinA and BinB were synthesized in the bacterial spore formation phase, and by the interaction of two albumen and folding assembling, formed crystal in the sporulation III phase.In the time of BinA and BinB, exist is that formation parasporal crystal is necessary.Western Blot shows no cross reaction between BinA and BinB albumen, illustrates between them without stronger homology.
Fragment subclone is tested and is shown, BinA albumen is poisonous to mosquito larvae separately, but virulence is more much lower than the virulence of the crystal containing two kinds of albumen.Independent BinB is nontoxic to mosquito larvae, but it exists the toxicity that obviously strengthens BinA albumen, when only having two kinds of albumen, exists, and toxin protein competence exertion goes out maximum cytotoxicity, is a kind of binary toxin (Binary toxin, Bin).This may be because mosquito clone lacks peritrophic membrane barrier, exists and take the photograph in the binding site of low affinity and different toxin cell due to mechanism.Although exist in the time of two albumen, be to guarantee that the complete activity of binary toxin is necessary, the amino acid whose difference of BinA, particularly determines that at 100 left and right, position amino acid it kills mosquito activity and kills mosquito spectrum.
Mtx1 toxin: Mtx1 toxin is the soluble toxin of a kind of 100kDa, is comprised of 870 amino acid.Its N-end has a sequence with G+ bacterium signal peptide feature, with ADP-ribosyltransferase catalytic subunit homology.C-end has three terminal repeats.This 100kDa albumen can be by the albumen of the formation 27 of mosquito larvae Midgut protein enzyme liberating and 70kDa.70kDa polypeptide has three approximately 90 amino acid whose tumor-necrosis factor glycoproteinss, and its function is ominous.And 27kDa contains corresponding region, Yi Getong transferring film district, and there is weak homology with several ADP-ribose transferring enzyme toxin.Disappearance experiment is proof also, 27kDa fragment energy self ADP-ribosylation, and 70kDa fragment can make mosquito cell generation pathologic reaction, when only having two kinds of protein fragments, exists, and just mosquito larvae is shown to toxicity.
Mtx2 toxin Mtx3 toxin: Mtx2 toxin and Mtx3 toxin are all isolatedly from SSII-1 bacterial strain to be comprised of 292 and 326 amino acid, molecular weight is respectively 31.8 and the toxin protein of 35.8kDa, they with 100kDa toxin and crystal toxin without homology, 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).Between Mtx2 and Mtx3, there is 38% homology, all contain the signal peptide of a G+ bacterium and the transferring film district of supposition.Comparative analysis to Mtx2 toxin protein separated from 6 different B .s virulent strains, proves that the amino acid of 224 positions has determined that this toxin kills mosquito activity and kills 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, by the parasporal crystal protein extraction of Bacillus sphaericus bacterial strain, the parasporal crystal albumen extracting can be applicable to treatment and/or suppresses tumour or tumour auxiliary diagnosis.
The present invention is achieved through the following technical solutions:
A parasporal crystal protein preparation method, comprises the following steps:
1) after Bacillus sphaericus IAB 872 bacterial strains are activated on LB solid medium, be inoculated in 30 ℃ of incubated overnight in LB liquid nutrient medium, transfer next day in MBS substratum, concussion is cultured to brood cell and crystal discharges completely from parent cell;
2) collect fermented liquid, centrifugal, precipitation, with after the washing of NaCl solution centrifugal, fully disperses crystal, brood cell, cell debris with ultrasonication suspension, and then fully concussion mixes, and scumming is centrifugal, gets precipitation;
3) precipitation is added to water and make suspension, adding N,O-Diacetylmuramidase to final concentration is 0.1～0.5mg/ml, processes 2～3h for 37 ℃, during softly mix for several times, centrifugal collecting precipitation then;
4) precipitation is added to water and makes suspension, add DTT to its concentration be 50～100mM, after fully mixing, in 27～30 ℃ of standing 1h, centrifugal collecting precipitation;
5) will be deposited in-20 ℃ with room temperature under freeze thawing once, add mass concentration 42～44% Compound Diatrzoatc Meglumlnes, fully mix rear centrifugally, collect supernatant 24～48h that supernatant is dialysed in sterilized water;
6) by making lyophilized powder under the supernatant cryogenic vacuum freezing conditions after dialysis, obtain Bacillus sphaericus parasporal crystal albumen.
Describedly in MBS substratum, cultivate 70～74h, culture temperature is 28～30 ℃.
Being set as 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 described step 5) is: at-20 ℃, keep spending the night, 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 preparing antitumor drug.
Described parasporal crystal albumen comprises BinA and the BinB binary toxin that the protein polypeptide of 51kDa and 42kDa forms.
Described antitumor drug is the medicine of short apoptosis of tumor cells.
Described antitumor drug is the medicine of inhibition tumor cell propagation.
The medicine of described inhibition tumor cell propagation is arrested in tumour cell the medicine of G2/M phase.
Described antitumor drug is one or more in the medicine of anti-cervical cancer, anti-liver cancer, anti-cancer of the stomach, anti-lung cancer.
Compared with prior art, the present invention has following useful technique effect:
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 is when cultivating extraction, exist brood cell's fragmentation insufficient, crystallin and gemma are wrapped in [J.Invertebr Pathol.2009 in extine, 101:106 – 111.], can not fully extract albumen, the defect that the effect of soluble crystal albumen is also decreased, the present invention processes by ultrasonication, N,O-Diacetylmuramidase processing, DTT processing, Compound Diatrzoatc Meglumlne, improve output and the activity of soluble crystal albumen, thereby obtained soluble crystal albumen as much as possible, higher effect.The soluble crystal albumen obtaining can reach 2.09mg/ml.
In Bacillus sphaericus parasporal crystal albumen prepared by the present invention, include the 51kDa that produces in the gemma growth later stage and the binary toxin of 42kDa, and the gene order of binary toxin with as commercialization, kill the young preparation bacterial strain of mosquito Bacillus sphaericus 2362(serotype H5a5b) highly consistent; And nourishing body does not produce any MTX toxin vegetative period.
The present invention provides Bacillus sphaericus parasporal crystal albumen to have certain restraining effect to human malignant lesion's cell first, can apply the preparation of antitumor drug.Bacillus sphaericus parasporal crystal albumen comprises the result of the inhibition of human tumor cells propagation:
Inhibiting rate to tumour cell: the parasporal crystal albumen that Bacillus sphaericus Bacillus sphaericus IAB 872 extracts all shows good restraining 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 higher HELA and the A549 tumour cell of inhibiting rate, make drug level gradient and find that it has certain concentration dependent.
Impact on the short apoptosis of tumour cell: amount to 43.8% although small concentration administration group (0.167mg/mL) is early withered with the cell withering evening, compare to some extent and increase with negative control group, there is no obvious difference; After but middle concentration administration group (0.25mg/mL) administration is processed, apoptosis rate is 58.8%.Large concentration administration group (0.375mg/mL) apoptosis rate is 62.2%, compares and all has significant difference with negative control group; Show that (0.25mg/mL) dosage and above parasporal crystal albumen can promote the apoptosis of tumour cell.
Impact on tumour cell cycle: 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%, middle concentration administration group (0.25mg/mL) is 8.09%, large concentration administration group (0.375mg/mL) is 16.18%, parasporal crystal albumen can affect the G2/M phase of cell, makes cell block in the G2/M phase.
Accompanying drawing explanation
Fig. 1 is Bacillus sphaericus Bacillus sphaericus IAB 872Bradford determination of protein concentration typical curve;
Fig. 2 is that two the dying of parasporal crystal albumen effect A549 cell 48h Annexin V-FITC/PI that Flow cytometry Bacillus sphaericus is purified carried out apoptosis analytical results; 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.
Fig. 3 is the result that affects of the Flow cytometry Bacillus sphaericus parasporal crystal albumen effect A549 cell 48h cell cycle of purifying, wherein, and 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, by the batch fermentation of the spherical bud bar of wild-type spore bacterium, its cell is carried out to the processing of the centrifugal grade of cracking, obtain its meta-bolites parasporal crystal albumen, first this albumen is applied to anti-human tumour cell, result shows to apply the preparation of antitumor drug.Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
1, Bacillus sphaericus Bacillus sphaericus IAB 872 bacterial strains:
Bacillus sphaericus IAB 872 is a kind of existing Bacillus sphaericuses, and its toxin producing is usually used in killing mosquito.Concrete, the present invention is that an isolated strain Bacillus sphaericus IAB 872 specifically describes from pedotheque, and those skilled in the art also can adopt other channels to obtain Bacillus sphaericus IAB 872 and complete the present invention certainly.
2, the extraction that Bacillus sphaericus Bacillus sphaericus IAB 872 produces parasporal crystal albumen:
(1) after Bacillus sphaericus IAB 872 bacterial strains are activated on LB solid medium, picking list bacterium colony is 30 ℃ of incubated overnight in LB liquid nutrient medium, and transfer in the MBS substratum of large shaking flask and in 30 ℃ of concussions, to cultivate (200rpm) 72h left and right and from parent cell, discharge completely to brood cell and crystal next day with the ratio of 1:100;
(2) collect fermented liquid, the centrifugal 10min of 10000g, precipitation with the NaCl centrifuge washing of 0.5M once, is used ultrasonication suspension, being set as 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, more constantly shake and mix on eddy mixer, remove the foam of generation simultaneously, centrifugal, get precipitation;
(3) by 70mg precipitation/ml distilled water, make suspension, by final concentration 0.1mg/ml, add N,O-Diacetylmuramidase (10mM EDTA configuration), process 2hr for 37 ℃, during per half an hour take out and softly mix for several times, centrifugal collecting precipitation;
(4) by 70mg precipitation/ml distilled water, make suspension, add 50mM DTT(dithiothreitol (DTT)), after fully mixing, 27 ℃ of standing processing 1hr, centrifugal collecting precipitation;
(5) will be deposited in-20 ℃ with room temperature under freeze thawing once, add 42% Compound Diatrzoatc Meglumlne, mix rear centrifugal can abundant separated gemma and nourishing body, gemma purity is improved, the centrifugal 20min of 10000rpm, collect supernatant, and the 48h that dialyses in sterilized water, then detect the concentration of solubility parasporal crystal albumen;
(6) by making lyophilized powder under the supernatant cryogenic vacuum freezing conditions after dialysis, obtain Bacillus sphaericus parasporal crystal albumen, with-4 ℃ at preserve.
Parasporal crystal refers in the gemma growth later stage and produces binary toxin (the Binary Toxin that the protein polypeptide (BinA and BinB) of 51kDa and 42kDa forms, Bin) form, and the gene order of binary toxin with as commercialization, kill the young preparation bacterial strain of mosquito Bacillus sphaericus 2362(serotype H5a5b) highly consistent.
What part supper toxic strain and all Hypovirulent strains also produced that a kind of vegetative phase expresses has the lysotype toxin protein of toxic action to mosquito larvae, is vegetative phase Mosquitocidal toxin (Mosquitocidal Toxin, MTX).All supper toxic strains can produce binary toxin, and wherein some bacterial strain also can produce 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 after protein bound, is blue, and protein meets Beer's law within the scope of finite concentration, in 595nm place colorimetric, 2-5min is maximum light absorption, stablizes 1hr(0.01-1.0mg albumen scope), the unavailable quartz curette of cuvette.
The concrete reagent adopting 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 successively 0ml, 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 3ml dye liquor, and shakes up, and is placed in room temperature 20-25 ℃ 15min, measures absorbance value with 595nm place.
Take A595 value as Y, and BSA content is X, and production standard curve, obtains linear equations, shown in detected result Fig. 1 of mark curve, then in conjunction with the known agnoprotein concentration of absorbance value.
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, all purchased from U.S. ATCC cell bank, in containing in the RPMI-1640 of 10% foetal calf serum, are placed in 37 ℃, 5%CO
2in incubator, cultivate.
2) growth conditions is good, MCF7,7721,7402,7901, the A549 growing in logarithmic phase, HELA cell are with 10
5be inoculated in 96 orifice plates, spend the night and treat that cell is naturally adherent.By adding the spherical parasporal crystal protein extract of 0.5mg/ml concentration in above-mentioned cell, then cell is put to 37 ℃, 5%CO
2middle cultivation 48h.
3) mtt assay is measured Growth of Cells: cultivate after 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, careful exhaustion treats that nutrient solution in gaging hole, every hole add 150 μ l DMSO, and vibration 10min, fully dissolves xln, measures absorbancy in microplate reader 490nm wavelength place.Establish 5 parallel holes for every group, every group of experiment all in triplicate.
The inhibiting rate of Growth of Cells (%)=(survival rate of 1-cell) * 100%
The parasporal crystal albumen that Bacillus sphaericus Bacillus sphaericus IAB 872 extracts all shows good restraining 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 higher HELA and the A549 tumour cell of inhibiting rate, make drug level gradient and find that it has certain concentration dependent, specifically as shown in table 2.
The inhibiting rate of table 1 parasporal crystal albumen 0.5mg/mL to different tumour cells
| Tumour cell kind | 7901 | A549 | 7721 | 7402 | HELA |
| Inhibiting rate % | 70.93 | 78.12 | 59.53 | 56.32 | 78.10 |
The inhibiting rate of table 2 parasporal crystal albumen different concns to 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, the impact of parasporal crystal albumen on apoptosis of tumor cells:
1) the A549 cell in vegetative period of taking the logarithm, parallel inoculation, treats after 24h that degrees of fusion reaches approximately 50%, uses the parasporal crystal albumen of 0.125mg/mL, 0.187mg/mL, 0.25mg/mL concentration group to carry out apoptosis-induced, negative control group adds isopyknic 1640 substratum.
2) after apoptosis-induced 48h, administration group and negative control group cell are first washed to one time with PBS, then use 0.25% tryptic digestion dissociated cell, obtain cell.
3) after cell dissociation gets off, add the cell culture fluid of collecting in step (2), slightly mix, transfer in centrifuge tube, the centrifugal 5min of 1500r/min, supernatant discarded, collecting cell, then 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 distilled water 1:9 dilution 10 * Binding Buffer) of 500 μ l.
5) add 5 μ L AnnexinV-FITC, then add 10 μ LPropidium Iodide, mix gently.
6) at room temperature, lucifuge dyeing 5 ~ 15min.
7) in 1 hour, carry out flow cytometer detection.Excitation wavelength Ex=488nm; Emission wavelength Em=530nm.The green fluorescence FITC passage of Annexin V-FITC is FL1, and PI red fluorescence is FL2 by PI passage.Use without the normal cell of apoptosis induction in contrast, is carried out fluorescence compensating regulation.
After using 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 apoptosis analysis, investigate the shared ratio of apoptotic cell.Investigating extract is the situation of A549 apoptosis induction to gastric cells.
As shown in Figure 2, the result of cells were tested by flow cytometry shows that each district of scatter diagram represents respectively following implication to detected result: be Q3(Annexin-/PI-) normal viable cell; Q4(Annexin+/PI-) be viable apoptotic cell; Q1(Annexin+/PI+) be non-viable apoptotic cell; Q2(Annexin-/PI+) be mechanical injuries cell and downright bad cell.Negative control group is early withered and is amounted to 40.1% with the cell withering evening.Small concentration administration group (0.167mg/mL) is early withered and is amounted to 43.8% with the cell withering evening, compares to some extent and increases, but there is no obvious difference with negative control group.After middle concentration administration group (0.25mg/mL) administration is processed, apoptosis rate is 58.8%.Large concentration administration group (0.375mg/mL) apoptosis rate is 62.2%, compares and all has significant difference with negative control group.
6, the impact of parasporal crystal albumen on tumour cell cycle:
1) the A549 cell in vegetative period of taking the logarithm, parallel inoculation, after 24h, treat that degrees of fusion reaches approximately 50%, the 4# genus bacillus crystallin of use 0.125mg/mL, 0.187mg/mL, 0.25mg/mL concentration group carries out apoptosis-induced, and negative control group adds isopyknic 1640 substratum.
2) after apoptosis-induced 48h, administration group and negative control group cell are first washed to one time with PBS, then use 0.25% tryptic digestion dissociated cell, obtain cell.
3) after cell dissociation gets off, add the cell culture fluid of collecting in step (2), slightly mix, transfer in centrifuge tube, the centrifugal 5min of 1500r/min, supernatant discarded, collecting cell, then 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 (containing the PI of 50 μ g/mL, the RNase of 50 μ g/mL) of 500 μ l, the penetrating liquid of 5 μ l reagent B(), vortex 5 ~ 10s, room temperature lucifuge reaction 30min.
5) in 1 hour, carry out flow cytometer detection.Before detecting, sample filters with 200 eye mesh screens, in order to avoid stop up.
After using 0.167mg/mL, 0.25mg/mL, 0.375mg/mL parasporal crystal albumen effect A549 cell 48 hours, use mono-the dying of PI to carry out cell cycle analysis, investigate the impact of shared ratio in extract cell cycle each period.
The result of cells were tested by flow cytometry is as Fig. 3, negative group G2 phase cell is 6.11%, small concentration administration group (0.167mg/mL) G2 phase cell is 7.74%, middle concentration administration group (0.25mg/mL) is 8.09%, large concentration administration group (0.375mg/mL) is 16.18%, parasporal crystal albumen can affect the G2/M phase of cell, makes cell block 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 are had to higher inhibiting rate, be respectively 78%, 79%.The tumour cell higher to inhibiting rate further detects, and selects the inhibiting rate of parasporal crystal albumen different concns to tumour cell A549 and HELA, and result shows to have certain concentration dependent.
And in view of parasporal crystal albumen is on the impact of the short apoptosis of tumour cell and inhibition tumor cell propagation, being arrested in the G2/M phase, the parasporal crystal albumen of Bacillus sphaericus IAB 872 just can be applicable to the preparation of antitumor drug so.
Claims (1)
1. a Bacillus sphaericus parasporal crystal protein preparation method, is characterized in that, comprises the following steps:
1) will
bacillus sphaericusafter IAB 872 bacterial strains activate on LB solid medium, be inoculated in 30 ℃ of incubated overnight in LB liquid nutrient medium, transfer next day in MBS substratum, concussion is cultured to brood cell and crystal discharges completely from parent cell;
2) collect fermented liquid, centrifugal, precipitation, with after the washing of NaCl solution centrifugal, fully disperses crystal, brood cell, cell debris with ultrasonication suspension, and then fully concussion mixes, and scumming is centrifugal, gets precipitation;
3) precipitation is added to water and make suspension, adding N,O-Diacetylmuramidase to final concentration is 0.1～0.5mg/ml, processes 2～3h for 37 ℃, during softly mix for several times, centrifugal collecting precipitation then;
4) precipitation is added to water and makes suspension, add DTT to its concentration be 50～100mM, after fully mixing, in 27～30 ℃ of standing 1h, centrifugal collecting precipitation;
5) will be deposited in-20 ℃ with room temperature under freeze thawing once, add mass concentration 42～44% Compound Diatrzoatc Meglumlnes, fully mix rear centrifugally, collect supernatant 24～48h that supernatant is dialysed in sterilized water;
6) by making lyophilized powder under the supernatant cryogenic vacuum freezing conditions after dialysis, obtain Bacillus sphaericus parasporal crystal albumen.
2
.bacillus sphaericus parasporal crystal protein preparation method as claimed in claim 1, is characterized in that, describedly in 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 as 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 described step 5) is: at-20 ℃, keep spending the night, 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 preparing antitumor drug, described parasporal crystal albumen comprises BinA and the BinB binary toxin that the protein polypeptide of 51kDa and 42kDa forms; Described antitumor drug is one or more in the medicine of anti-cervical cancer, anti-liver cancer, anti-cancer of the stomach, anti-lung cancer.
6
.application as claimed in claim 5, is characterized in that, described antitumor drug is the medicine of short apoptosis of tumor cells.
7
.application as claimed in claim 5, is characterized in that, described antitumor drug is the medicine of inhibition tumor cell propagation.
8
.application as claimed in claim 7, is characterized in that, the medicine of described inhibition tumor cell propagation is tumour cell to be arrested in to the medicine of G2/M phase.
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| DK2675474T3 (en) * | 2011-02-15 | 2019-04-23 | Vaxiion Therapeutics Llc | THERAPEUTIC COMPOSITIONS AND PROCEDURES FOR TARGETED ADMINISTRATION OF BIOACTIVE MOLECULES BY BACK-TERIAL MINICLES BASED ON ANTIBODY AND FC-CONTAINING TAR-GETING MOLECULES |
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2012
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| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 苏云金芽孢杆菌晶体蛋白的制备方法;郭艾英 等;《食品与发酵工业》;20050830;第31卷(第8期);第8-10页 * |
| 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》.2005,第31卷(第8期),第8-10页. |
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