CN102816816B - Bacillus sphaericus parasporal crystal protein preparation method and application thereof - Google Patents

Abstract

The invention discloses a method for preparing Bacillus sphaericus parasporal crystal protein and its application. The wild-type Bacillus sphaericus IAB 872 is used for cultivation, and through ultrasonic treatment, lysozyme treatment, DTT treatment and compound diatrizoate meglumine treatment, the The production and activity of soluble crystal protein, so as to obtain as much soluble crystal protein with higher efficacy as possible. The Bacillus parasporal crystal protein has a certain inhibitory effect on human malignant tumor cells, and can be used in the preparation of antitumor drugs. The results of the inhibition of human tumor cell proliferation by Bacillus sphaericus parasporal crystal protein include: the inhibition rate of tumor cells, the effect on tumor cell apoptosis, can affect the G2/M phase of cells, and make cells block in G2/M phase. M period.

Description

A kind of Bacillus sphaericus parasporal crystal protein preparation method and application thereof

technical field

The invention belongs to the technical field of microbial metabolites and applications thereof, and relates to a method for preparing Bacillus sphaericus parasporal crystal protein and its application.

Background technique

Bacillus sphaericus is a strictly aerobic bacillus. The growth cycle can be divided into vegetative phase and spore phase. The spores are born at or near the end of the cell. During the process of spore formation, one end of the thallus expands, and the sporangia Drumstick-shaped. In the cells of Bacillus sphaericus, with the formation of spores, some strains can produce one or more paraspore crystals, while others do not produce paraspore crystals, and the shape, size and number of paraspore crystals also vary with the strain. different from each other. Paraspore crystals and spores are attached to the same end of the cell. After the sporangia matures, the crystals and spores are still combined and wrapped in the outer membrane of the spore. The presence or absence of parasporal crystals and their shape are related to the mosquito-killing activity of Bacillus sphaericus. For example, no parasporal crystals were found in attenuated strains SSII-1, 1404-924B, and 1881, and occasionally oval or oval crystals were produced in Kellen Q strains Shaped paraspore crystals, no rhomboid crystals; the highly virulent 2297 strain mainly produced rhombic paraspore crystals, and also produced oval or oval paraspore crystals. Bacillus sphaericus has different poisonous effects on mosquito larvae of different genera, or different species of the same genus, and the mosquito-killing activity of different serotypes, the same serotype, or strains containing the same toxin protein is also quite different.

The poisonous effect of Bacillus sphaericus on different mosquito larvae is mainly realized by the mosquito-killing toxin produced by it. It has been proved that two different toxins can be produced during its growth and development: one is a crystal toxin present in all highly virulent strains, consisting of 41.9 and 51.4 kDa proteins; the other is present in low virulence and some Mosquitocidal Toxin (Mtx) in highly virulent strains, such as Mtx1 (100kDa), Mtx2 (31.8kDa) and Mtx3 (35.8kDa), etc.

All highly virulent strains and some moderately virulent strains could form parasporal crystals located in the outer membrane of the spore during sporulation. The crystals are composed of equal amounts of 41.9 and 51.4 kDa proteins (denoted as BinA and BinB, respectively). BinA and BinB are synthesized in the sporulation phase of bacteria and assembled to form crystals through the interaction and folding of the two proteins in the sporulation phase III. The simultaneous presence of BinA and BinB is required for the formation of parasporal crystals. Western Blot showed that there was no cross-reaction between BinA and BinB proteins, indicating that there was no strong homology between them.

Fragment subcloning experiments showed that the BinA protein alone was toxic to mosquito larvae, but much less virulent than crystals containing both proteins. BinB alone is non-toxic to mosquito larvae, but its presence significantly enhances the toxicity of BinA protein. Only when the two proteins exist at the same time can the toxin protein exert its maximum killing activity. It is a binary toxin (Binary toxin, Bin) . This may be due to the lack of a peritrophic membrane barrier in mosquito cell lines, the presence of low-affinity binding sites, and a different mechanism of toxin endocytosis. Although the simultaneous presence of the two proteins is necessary to ensure the complete activity of the binary toxin, the amino acid difference of BinA, especially the amino acid at position 100, determines its mosquito-killing activity and mosquito-killing spectrum.

Mtx1 toxin: Mtx1 toxin is a soluble toxin of 100 kDa consisting of 870 amino acids. Its N-terminus has a sequence characteristic of a G+ bacterial signal peptide, homologous to the catalytic subunit of ADP-ribosyltransferase. There are three terminal repeats at the C-terminus. This 100 kDa protein can be degraded by mosquito larval midgut proteases to form 27 and 70 kDa proteins. The 70kDa polypeptide has three repeats of approximately 90 amino acids, and its function is ominous. The 27kDa contains a region corresponding to the transmembrane region and has weak homology with several ADP-ribosyltransferase toxins. Deletion experiments also proved that the 27kDa fragment can self-ADP-ribosylate, and the 70kDa fragment can cause pathological reactions in mosquito cells. Only when the two protein fragments exist at the same time can they show toxicity to mosquito larvae.

Mtx2 toxin Mtx3 toxin: Both Mtx2 toxin and Mtx3 toxin are isolated from SSII-1 strains, composed of 292 and 326 amino acids, with molecular weights of 31.8 and 35.8kDa toxin proteins, and they have no homology with 100kDa toxin and crystal toxin It is homologous to the 33kDa-toxin of Clostridium perfrigens and the 31.68kDa cytotoxin of Pseudomonas aeruginos (Liu et al, 1993, Thanabalu & Porter, 1996). There is 38% homology between Mtx2 and Mtx3, both contain a G+ bacterial signal peptide and putative transmembrane region. The comparative analysis of Mtx2 toxin proteins isolated from 6 different B.s virulent strains proved that the amino acid at position 224 determines the mosquito-killing activity and mosquito-killing spectrum of the toxin.

Contents of the invention

The problem to be solved by the present invention is to provide a method for preparing Bacillus sphaericus parasporal crystal protein and its application. By extracting the parasporal crystal protein from Bacillus sphaericus strains, the extracted parasporal crystal protein can be used to treat and/or inhibit tumors Or tumor auxiliary diagnosis.

The present invention is achieved through the following technical solutions:

A method for preparing Bacillus sphaericus parasporal crystal protein, comprising the following steps:

1) After activating the Bacillus sphaericus IAB 872 strain on LB solid medium, inoculate it in LB liquid medium for overnight culture at 30°C, transfer it to MBS medium the next day, and culture with shaking until the spores and crystals are completely removed from the mother cells freed;

2) Collect the fermentation broth, centrifuge, and wash the precipitate with NaCl solution, then use ultrasonic treatment to disperse the crystals, spores, and cell fragments, then shake and mix well, remove the foam, centrifuge, and take the precipitate;

3) Add water to the precipitate to make a suspension, add lysozyme to a final concentration of 0.1-0.5mg/ml, treat at 37°C for 2-3 hours, mix gently several times during this period, and then centrifuge to collect the precipitate;

4) Add water to the precipitate to make a suspension, add DTT to a concentration of 50-100mM, mix well, let stand at 27-30°C for 1 hour, and collect the precipitate by centrifugation;

5) Freeze and thaw the precipitate once at -20°C and room temperature, add compound meglumine diatrizoate with a mass concentration of 42-44%, mix well and centrifuge, collect the supernatant, and dialyze the supernatant in sterile water for 24-48 hours ;

6) The dialysis supernatant was made into freeze-dried powder under the condition of low-temperature vacuum freezing to obtain Bacillus sphaericus parasporal crystal protein.

The culture is carried out in MBS medium for 70-74 hours, and the culture temperature is 28-30°C.

The settings of the ultrasonic generator during the ultrasonic treatment are as follows: the temperature is 4° C., the frequency is 20 kHz, and the power is 200 W; the ultrasonic program is: ultrasonic for 5 s, stop for 5 s, and cycle for 25 min in total.

The freezing and thawing in step 5) is as follows: keep overnight at -20°C, and dissolve at room temperature; the centrifugation is 10000r/min for 20min.

Application of the parasporal crystal protein of Bacillus sphaericus IAB 872 in the preparation of antitumor drugs.

The parasporal crystal protein includes BinA and BinB binary toxins composed of 51kDa and 42kDa protein polypeptides.

The antitumor drug is a drug that promotes tumor cell apoptosis.

The antitumor drug is a drug that inhibits tumor cell proliferation.

The drug for inhibiting tumor cell proliferation is a drug for arresting tumor cells in G2/M phase.

The antineoplastic drugs are one or more of the drugs against cervical cancer, liver cancer, stomach cancer and lung cancer.

Compared with the prior art, the present invention has the following beneficial technical effects:

The method for preparing Bacillus sphaericus parasporal crystal protein provided by the present invention adopts wild-type Bacillus sphaericus IAB 872 for cultivation, especially improves the soluble crystal protein containing binary toxin. In the prior art, when the strain is cultured and extracted, there is insufficient spore fragmentation, crystal protein and spores are wrapped in the outer membrane of the spore [J.Invertebr Pathol.2009,101:106–111.], and the protein cannot be fully extracted. The effect of soluble crystal protein is also reduced to some extent. The present invention improves the output and activity of soluble crystal protein by ultrasonic treatment, lysozyme treatment, DTT treatment and compound diatrizoate meglumine, thereby obtaining as much as possible, higher Efficacy of soluble crystal protein. The obtained soluble crystal protein can reach 2.09mg/ml.

The Bacillus sphaericus parasporal crystal protein prepared by the present invention contains 51kDa and 42kDa binary toxins produced in the late stage of spore growth, and the gene sequence of the binary toxins is the same as that used as commercial mosquito larvicide strain Bacillus sphaericus 2362 (serum type H5a5b) were highly consistent; and no MTX toxin was produced during the growth phase of the vegetative body.

The invention provides for the first time that the parasporal crystal protein of Bacillus sphaericus has a certain inhibitory effect on human malignant tumor cells and can be used for the preparation of antitumor drugs. Results of inhibition of human tumor cell proliferation by Bacillus sphaericus parasporal crystals include:

Inhibitory rate of tumor cells: parasporal crystal protein extracted from Bacillus sphaericus IAB 872 showed good inhibitory effect on five kinds of tumor cells including HELA, liver cancer 7721, 7402, gastric cancer 7901, and lung cancer A549, and the inhibition rates were respectively : 78%, 61%, 56%, 53%, 79%. Select HELA and A549 tumor cells with high inhibition rate, and make drug concentration gradient to find that it has a certain concentration dependence.

The effect on tumor cell apoptosis: Although the small concentration administration group (0.167mg/mL) had a total of 43.8% of early and late withering cells, compared with the negative control group, there was no significant difference; In the concentration administration group (0.25mg/mL), the apoptosis rate was 58.8%. The apoptotic rate of the high-concentration administration group (0.375mg/mL) was 62.2%, which was significantly different from that of the negative control group; it indicated that (0.25mg/mL) administration dose and above paraspitial crystal protein could promote tumor Apoptosis of cells.

Effects on tumor cell cycle: The results of flow cytometry showed that the G2 phase cells in the negative group were 6.11%, the G2 phase cells in the small concentration administration group (0.167mg/mL) were 7.74%, and the medium concentration administration group (0.25mg/mL) was 7.74%. /mL) was 8.09%, and the high-concentration administration group (0.375mg/mL) was 16.18%. Paracystic crystallin can affect the G2/M phase of cells and make cells arrest in G2/M phase.

Description of drawings

Fig. 1 is Bacillus sphaericus IAB 872 Bradford protein concentration determination standard curve;

Figure 2 shows the results of flow cytometry detection of parasporal crystal protein purified from Bacillus sphaericus, A549 cells 48h Annexin V-FITC/PI double staining for apoptosis analysis results; among them, Q3: normal cells; Q4: premature apoptotic cells; Q2: Late apoptotic cells; Q1: necrotic cells a: negative control group; b: 0.167mg/mL; c: 0.25mg/mL; d: 0.375mg/mL.

Figure 3 is the result of flow cytometry detection of parasporal crystal protein purified from Bacillus sphaericus acting on A549 cells for 48 hours, wherein, a: negative control group; b: 0.167 mg/mL; c: 0.25 mg/mL; d: 0.375 mg/mL.

Detailed ways

The method for preparing Bacillus sphaericus parasporal crystal protein and its application provided by the present invention, through batch fermentation of wild-type Bacillus sphaericus, the cells are lysed and centrifuged to obtain its metabolite parasporal crystal protein. The protein is applied to anti-human tumor cells, and the result shows that it can be applied to the preparation of anti-tumor drugs. The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations.

1. Bacillus sphaericus IAB 872 strain:

Bacillus sphaericus IAB 872 is an existing strain of Bacillus sphaericus that produces a toxin that is commonly used to kill mosquitoes. Specifically, the present invention is specifically described from a strain of Bacillus sphaericus IAB 872 isolated from a soil sample. Of course, those skilled in the art can also use other channels to obtain Bacillus sphaericus IAB 872 to complete the present invention.

2. Extraction of parasporal crystal protein produced by Bacillus sphaericus IAB 872:

(1) After activating the Bacillus sphaericus IAB 872 strain on LB solid medium, pick a single colony and culture it in LB liquid medium at 30°C overnight, and transfer it to MBS culture in a large shake flask at a ratio of 1:100 the next day Incubate at 30°C with shaking (200rpm) for about 72 hours until the spores and crystals are completely released from the mother cells;

(2) Collect the fermentation broth, centrifuge at 10,000g for 10min, wash the precipitate once with 0.5M NaCl, and treat the suspension with ultrasonic waves. The settings of the ultrasonic generator are: temperature 4°C, frequency 20kHz, power 200W; ultrasonic program For: Ultrasound for 5s, stop for 5s, and cycle for 25min in total; disperse the crystals, spores, and cell fragments as much as possible, then shake and mix on the vortex mixer continuously, remove the generated foam at the same time, centrifuge, and take the precipitate;

(3) Make a suspension with 70mg precipitate/ml double distilled water, add lysozyme (10mM EDTA configuration) at a final concentration of 0.1mg/ml, treat at 37°C for 2 hours, take it out every half hour during the period, gently mix several times, and centrifuge collect sediment;

(4) Make a suspension with 70mg of precipitate/ml double distilled water, add 50mM DTT (dithiothreitol), mix well, let stand at 27°C for 1 hour, and collect the precipitate by centrifugation;

(5) Freeze and thaw the precipitate once at -20°C and room temperature, add 42% compound meglumine diatrizoate, mix well and centrifuge to fully separate the spores and vegetative bodies to improve the purity of the spores, centrifuge at 10,000rpm for 20min, and collect the supernatant. And dialyzed in sterile water for 48h, then detect the concentration of soluble parasporal crystal protein;

(6) The supernatant after dialysis was made into freeze-dried powder under the condition of low-temperature vacuum freezing to obtain the parasporal crystal protein of Bacillus sphaericus, and stored at -4°C.

Parasporal crystals refer to the formation of binary toxins (Binary Toxin, Bin) composed of 51kDa and 42kDa protein polypeptides (BinA and BinB) in the late stage of spore growth, and the gene sequence of the binary toxin is the same as that used to kill mosquito larvae commercially The preparation strain Bacillus sphaericus 2362 (serotype H5a5b) was highly identical.

Some high-virulence strains and all low-virulence strains also produce a soluble toxin protein expressed in the vegetative period that has a poisonous effect on mosquito larvae, which is the vegetative period mosquito-killing toxin protein (Mosquitocidal Toxin, MTX). All highly virulent strains can produce binary toxins, and some of them can also produce Mtx toxins at the same time, while Bacillus sphaericus IAB 872 does not produce any MTX toxins during the vegetative phase.

3. Determination of Bradford protein concentration:

G-250 is brownish-red under acidic conditions, and turns blue when combined with protein, and the protein conforms to Beer's law within a certain concentration range. It is colorimetric at 595nm, with maximum light absorption in 2-5min, stable for 1hr (0.01-1.0 mg protein range), quartz cups are not available for cuvettes.

The specific reagents used include:

Staining solution: Dissolve 100mg of G-250 in 95% ethanol, add 100ml of 85% phosphoric acid, and dilute to 1000ml with water. (The dye solution can be stored for several months, and it can be stored for a long time without adding water);

Standard protein solution: 0.5mg/ml bovine serum albumin BSA;

specific method:

standard protein solution

Add 0ml, 0.05ml, 0.10ml, 0.15ml, 0.20ml, 0.25ml, 0.30ml, 0.35ml, 0.40ml, 0.45ml, 0.50ml of standard protein solution in turn, add 3ml of dye solution, shake well, and place at room temperature 20-25°C for 15 minutes, and measure the light absorption value at 595nm.

Taking the A595 value as Y and the BSA content as X, make a standard curve and obtain the linear equation. The detection results of the marked curve are shown in Figure 1. Combined with the light absorption value, the unknown protein concentration can be known.

The detection result of parasporal crystal protein concentration was 2.09mg/ml.

4. Tumor cell culture and inhibition rate detection:

1) Liver cancer 7721, 7402, gastric cancer 7901, lung cancer A549, and cervical cancer HELA cells were purchased from the ATCC cell bank in the United States and cultured in 1640 culture medium containing 10% fetal bovine serum at 37°C and 5% CO ₂ Cultivated in a box.

2) MCF7, 7721, 7402, 7901, A549, HELA cells in good growth state and growing in the logarithmic phase were inoculated in 96-well plates at 10 ⁵ , and the cells naturally adhered to the wall overnight. Add 0.5 mg/ml parasporal crystal protein extract to the above cells, and then culture the cells at 37°C and 5% CO ₂ for 48 hours.

3) Determination of cell growth by MTT method: After culturing for 48 hours, add 20 μl of MTT solution (5 mg/ml) to each well to be tested, and continue culturing at 37°C for 4 hours. After terminating the culture, carefully absorb the culture medium in the wells to be tested, add 150 μl DMSO to each well, shake for 10 min to fully dissolve the crystals, and measure the absorbance at a wavelength of 490 nm on a microplate reader. Five parallel wells were set up in each group, and each experiment was repeated three times.

Inhibition rate of cell growth (%) = (1- cell survival rate) × 100%

The parasporal crystal protein extracted from Bacillus sphaericus IAB 872 showed good inhibitory effect on HELA, liver cancer 7721, 7402, gastric cancer 7901, lung cancer A549, and the inhibition rates were 78%, 61%, 56%, 53%, 79%, as shown in Table 1. The HELA and A549 tumor cells with higher inhibition rate were selected, and the drug concentration gradient was used to find that they had a certain concentration dependence, as shown in Table 2.

Table 1 Inhibitory rate of paracystic crystal protein 0.5mg/mL to different tumor cells

Tumor cell type 7901 A549 7721 7402 HELA Inhibition rate% 70.93 78.12 59.53 56.32 78.10

Table 2 Inhibition rate of different concentrations of paracystic crystal protein on tumor cells A549 and HELA

Crystal protein concentration mg/mL 0.0625 0.125 0.25 0.375 0.5 Inhibition rate to A549% 12.41 14.21 36.01 53.20 74.73 Inhibition rate of HELA % 24.50 34.08 49.72 58.43 73.93

5. The effect of paracystic crystal protein on tumor cell apoptosis:

1) Take A549 cells in the logarithmic growth phase and inoculate them in parallel. After 24 hours, the degree of confluence reaches about 50%, and induce apoptosis with 0.125mg/mL, 0.187mg/mL, and 0.25mg/mL concentration groups of paraspitial crystal protein. An equal volume of 1640 medium was added to the negative control group.

2) After 48 hours of induction of apoptosis, the cells of the administration group and the negative control group were washed with PBS, and then digested and dissociated with 0.25% trypsin to obtain cells.

3) After the cells are digested, add the cell culture medium collected in step (2), mix slightly, transfer to a centrifuge tube, centrifuge at 1500r/min for 5min, discard the supernatant, collect the cells, and gently resuspend the cells in PBS for washing , and count.

4) Take 1~5×10 ⁵ cell suspension, centrifuge at 1500rpm for 5 minutes, discard the supernatant, add 500μl of 1×Binding Buffer (dilute 10×Binding Buffer 1:9 with distilled water).

5) Add 5 μL AnnexinV-FITC, then add 10 μL Propidium Iodide, and mix gently.

6) Stain in the dark for 5-15 minutes at room temperature.

7) Perform flow cytometry detection within 1 hour. Excitation wavelength Ex=488nm; emission wavelength Em=530nm. The green fluorescence FITC channel of Annexin V-FITC is FL1, and the PI red fluorescence through the PI channel is FL2. Normal cells without induction of apoptosis were used as controls for fluorescence compensation adjustments.

After using 0.167mg/mL, 0.25mg/mL, and 0.375mg/mL paracystic crystal protein to treat A549 cells for 48 hours, use AnnexinV-FITC/PI double staining for apoptosis analysis to investigate the proportion of apoptotic cells. To investigate the effect of the extract on apoptosis induction of gastric cell line A549.

The detection results are shown in Figure 2. The flow cytometry results show that each area of the scatter plot represents the following meanings: Q3 (Annexin-/PI-) is normal living cells; Q4 (Annexin+/PI-) is early apoptosis Cells; Q1 (Annexin+/PI+) is late apoptotic cells; Q2 (Annexin-/PI+) is mechanically injured cells and necrotic cells. In the negative control group, 40.1% of cells were prematurely withered and late withered. In the small concentration administration group (0.167mg/mL), the total number of early and late withered cells was 43.8%, which was increased compared with the negative control group, but there was no significant difference. In the middle concentration administration group (0.25mg/mL), the apoptosis rate was 58.8%. The apoptotic rate of the high-concentration administration group (0.375mg/mL) was 62.2%, which was significantly different from that of the negative control group.

6. The effect of parasporal crystal protein on tumor cell cycle:

1) Take A549 cells in the logarithmic growth phase and inoculate them in parallel. After 24 hours, the fusion degree reaches about 50%. Use 4# Bacillus crystal protein of 0.125mg/mL, 0.187mg/mL, and 0.25mg/mL concentration groups to induce apoptosis. In the negative control group, an equal volume of 1640 medium was added.

2) After 48 hours of induction of apoptosis, the cells of the administration group and the negative control group were washed with PBS, and then digested and dissociated with 0.25% trypsin to obtain cells.

3) After the cells are digested, add the cell culture medium collected in step (2), mix slightly, transfer to a centrifuge tube, centrifuge at 1500r/min for 5min, discard the supernatant, collect the cells, and gently resuspend the cells in PBS for washing , and count.

4) Take 1× ¹⁰⁶ cell suspension, centrifuge at 1500rpm for 5 minutes, discard the supernatant, add 500μl reagent A (containing 50μg/mL PI, 50μg/mL RNase), 5μl reagent B (permeabilization solution), Vortex for 5-10s, and react for 30min at room temperature in the dark.

5) Perform flow cytometry detection within 1 hour. Samples were filtered with a 200-mesh sieve before testing to avoid clogging.

After using 0.167mg/mL, 0.25mg/mL, 0.375mg/mL paracystic crystal protein to act on A549 cells for 48 hours, use PI single staining for cell cycle analysis to investigate the effect of the extract on the proportion of each phase of the cell cycle.

The results determined by flow cytometry are shown in Figure 3. The G2 phase cells in the negative group were 6.11%, the G2 phase cells in the small concentration administration group (0.167mg/mL) were 7.74%, and the medium concentration administration group (0.25mg/mL) was 8.09%, and 16.18% in the high-concentration administration group (0.375mg/mL). Paracystic crystallin can affect the G2/M phase of cells and make cells arrest in G2/M phase.

In summary, the paracystic crystal protein of Bacillus sphaericus IAB 872 has a certain inhibitory effect on human malignant tumor cells, including cervical cancer, liver cancer, and gastric cancer cells; it also has a high inhibitory rate on human lung cancer cell A549 and cervical cancer cell HELA , respectively 78% and 79%. The tumor cells with higher inhibition rate were further detected, and the inhibition rate of tumor cell A549 and HELA by different concentrations of paraspitial crystal protein was selected, and the results showed that there was a certain concentration dependence.

Moreover, in view of the effect of paraspitial crystal protein on tumor cell apoptosis and inhibition of tumor cell proliferation, and arresting it in the G2/M phase, the paraspial crystal protein of Bacillus sphaericus IAB 872 can be applied to the preparation of anti-tumor drugs.

Claims (1)

1. a method for preparing bacillus sphaericus parasporal crystal protein, is characterized in that, comprises the following steps: 1) After activating the Bacillus sphaericus IAB 872 strain on LB solid medium, inoculate it in LB liquid medium for overnight culture at 30°C, transfer it to MBS medium the next day, and culture with shaking until the spores and crystals are completely removed from the mother cells freed; 2) Collect the fermentation broth, centrifuge, and wash the precipitate with NaCl solution, then use ultrasonic treatment to disperse the crystals, spores, and cell fragments, then shake and mix well, remove the foam, centrifuge, and take the precipitate; 3) Add water to the precipitate to make a suspension, add lysozyme to a final concentration of 0.1-0.5mg/ml, treat at 37°C for 2-3 hours, mix gently several times during this period, and then centrifuge to collect the precipitate; 4) Add water to the precipitate to make a suspension, add DTT to a concentration of 50-100mM, mix well, let stand at 27-30°C for 1 hour, and collect the precipitate by centrifugation; 5) Freeze and thaw the precipitate once at -20°C and room temperature, add compound meglumine diatrizoate with a mass concentration of 42-44%, mix well and centrifuge, collect the supernatant, and dialyze the supernatant in sterile water for 24-48 hours ; 6) The dialysis supernatant was made into freeze-dried powder under the condition of low-temperature vacuum freezing to obtain Bacillus sphaericus parasporal crystal protein. 2 . The method for preparing Bacillus sphaericus parasporal crystal protein according to claim 1 , characterized in that the culture is carried out in MBS medium for 70-74 hours at a culture temperature of 28-30° C. 3 . 3. The method for preparing Bacillus sphaericus parasporal crystal protein as claimed in claim 1, wherein the setting of the ultrasonic generator during the ultrasonic treatment is as follows: the temperature is 4°C, the frequency is 20kHz, and the power is 200W; Ultrasound program: ultrasound 5s, stop 5s, a total of 25min cycle. 4. The method for preparing Bacillus sphaericus parasporal crystal protein according to claim 1, characterized in that, the freezing and thawing in step 5) is: keep overnight at -20°C, and dissolve at room temperature; Centrifuge at 10000r/min for 20min. 5. The application of the parasporal crystal protein of Bacillus sphaericus IAB 872 in the preparation of antitumor drugs, the parasporal crystal protein includes BinA and BinB binary toxins composed of protein polypeptides of 51kDa and 42kDa; the The antineoplastic drug is one or more of the drugs against cervical cancer, liver cancer, stomach cancer and lung cancer. 6 . The application according to claim 5 , wherein the antitumor drug is a drug that promotes tumor cell apoptosis. 7. The application according to claim 5, characterized in that the antitumor drug is a drug that inhibits tumor cell proliferation. 8. The application according to claim 7, wherein the drug for inhibiting tumor cell proliferation is a drug for arresting tumor cells in G2/M phase.      

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