CN106047751A - Nocardiopsis, separation method of active metabolites of Nocardiopsis and application of Nocardiopsis - Google Patents

Abstract

A Nocardiopsis Actinomyces strain and its active metabolite separation method and application. The strains involved in the present invention are Nocardiopsis Actinomyces NX032 , Nocardiopsis sp . NX032, which are preserved in China Typical Culture Culture Collection Center, the strain preservation number is CCTCC NO: M 2015361. The invention also includes the separation and application of the active metabolites of the nocardioid actinomycetes. The active metabolite of the nocardioid actinomycetes of the invention has broad-spectrum anti-tumor activity.

Description

Isolation method and application of a nocardioid actinomycetes and its active metabolites

technical field

The present invention relates to a separation method and application of a strain of Nocardiopsis actinomyces and its active metabolites, in particular to a separation method and application of a high-efficiency anti-tumor wild-type strain (Nocardiopsis sp.NX032) and its active metabolites .

Background technique

Nocardiopsis (Nocardiopsis) is a genus under the Nocardiopsis family of Actinomycetes, Actinomycetes, Streptocystis suborder, and was effectively described by Meyer in 1976. It is a class of high G+C content Gram-positive aerobic bacteria. The main characteristics of the strains of this genus: Gram-positive, aerobic, moderate temperature, chemoorganotrophic type; the hyphae in the base are well developed, long and multi-branched, and will break into rods and spheres. The aerial hyphae are well developed, long and medium branched, straight or Z-shaped, and all break into rod-shaped spores of different lengths. The surface of the spore is smooth, the cell wall contains meso-DAP, no characteristic sugar, amycolic acid, and the dominant menaquinone is MK-10 (H2, H4, H6) or MK-9 (H4, H6). Fatty acid 3a type, phospholipid PⅢ type, the (G+C) mol% of DNA is 70%-76%. Through our tracking, we found that by January 2014, the effective published species of this genus had reached 53. Nocardiae are distributed in a variety of environments, showing the characteristics of the dominant bacterial group in moderate and high-salt environments.

From the birth of penicillin to the beginning of the 21st century, there were about 22,500 antibiotics produced by microorganisms, of which about 8,600 were from fungi, accounting for 38%, and about 13,900 were from bacteria, accounting for 62%. Among the antibiotics derived from bacteria, more than 10,000 species were produced by actinomycetes, accounting for 73%, and about 3,800 species of active compounds were produced by other bacteria, accounting for 27%. Therefore, actinomycetes have always been the main source of human discovery of effective antibiotics. However, since the chances of discovering new antibacterial drugs from Streptomyces have been decreasing in recent decades, the research strategy of finding new antibacterial drugs from rare actinomycetes as a bacterial excretion has gradually become the focus of research. In a narrow sense, rare actinomycetes refer to actinomycetes not belonging to the genus Streptomyces. When conventional isolation methods are used, their bacterial yield is much lower than that of Streptomyces. Some antibacterial compounds produced by rare actinomycetes have become drugs and are widely used clinically, such as gentamicin, erythromycin, vancomycin, rifampicin and so on.

In recent years, the discovery of secondary metabolites of this genus has progressed rapidly, and many new antibiotics have been discovered. For example, Shen Yuemao's research group at Xiamen University isolated Nocardiopsis Nocardiopsis A00203 (98% similarity with DSM44442), and isolated 3 new 3,6-disubstituted 2-pyrones from its fermentation broth ( α-pyrone) derivatives, respectively named Norcardiatones AC. MTT cytotoxic activity test showed that Norcardiatones A had weak cytotoxic activity to Hela cells, and Norcardiatones B and C had no cytotoxic activity; Australian Robert J. Nocardiopsis sp. (CMB-M0232), nocardiopsins A and nocardiopsins B were isolated from the fermentation broth of this strain in 2010, and nocardiopsins C and nocardiopsins D were isolated from the metabolites of this strain by the research group in 2013. These four compounds are all polyketide macrolide compounds. Nocardiopsins A and nocardiopsins B have no antifungal, antibacterial and cytotoxic activity. They are consistent with the structure-activity relationship of immunosuppressant FK506 and rapamycin. Can bind to immunophilin FKBP12 at a low molar concentration; William Fenical's research group isolated the strain Nocafdiopsis lucentensis (CNR-712) from a saline-alkaline pond on the Bahamas in northern Latin America, and isolated 5 strains from its fermentation broth Peptide compounds: Lucentamycins AE, compounds Lucentamycins A and Lucentamycins B have obvious cytotoxicity against human colon cancer HTC-116, with IC ₅₀ values of 0.20 μM and 11 μM, respectively, and compounds Lucentamycins C and Lucentamycins D when the concentration reaches 150 μM , and had no obvious cytotoxicity to colon cancer cells. However, the antitumor activity spectrum of these substances is relatively narrow, and it is of great significance to find new antitumor active substances in the screened strains.

At present, research on the biological activity of secondary metabolites of Nocardioides mainly focuses on three aspects: anti-tumor and anti-cancer active compounds; anti-inflammatory and anti-infective compounds; non-cytotoxic and non-bacteriostatic active compounds. Although people have made progress in the discovery of new species of Nocardioides and the research on secondary metabolites, the number of compounds obtained is still not considerable, and the research on the mechanism of action and biosynthesis of new compounds is still lacking.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a separation method and application of a strain of Nocardioides actinomycetes and its active metabolites with broad-spectrum anti-tumor activity and good activity.

The technical scheme that the present invention solves its technical problem adopts is:

The Nocardiopsis Actinomycetes of the present invention is Nocardiopsis Actinomyces NX032 (Nocardiopsis sp.NX032), which was preserved in the China Center for Type Culture Collection (CCTCC for short, address) on June 8, 2015. : Wuhan University, Wuhan, China), the strain preservation number is CCTCC NO: M 2015361.

Isolation and identification of Nocardiopsis sp.NX032 (Nocardiopsis sp.NX032) of the present invention: using the Gaoshi No. 1 medium screening method, directly isolate an actinomycete from a soil sample collected from a paddy field in Hunan, and obtain an actinomycete through Colony morphology observation, Gram staining, physiological and biochemical characteristics, and 16S rRNA gene homology analysis identified the strain as Nocardiopsis genus and named it Nocardiopsis sp. NX032 (Nocardiopsis sp.NX032).

Antitumor Biological Activity:

Ferment and culture Nocardioides NX032 in shake flasks (preferably 300ml shake flasks) for 10-12 days, centrifuge, collect supernatant, and conduct tumor cytotoxicity experiments.

Separation and purification of anti-tumor protein: place Nocardioides NX032 in the fermentation medium, shake culture at 28-30°C, 160-170rpm/min for 10-12 days, then 8000-12000rpm/min Centrifuge for 15-20min (it is best to repeat the centrifugation for more than 3 times), collect the fermentation supernatant, and use different concentration gradients of ammonium sulfate to precipitate the protein; then further separate and purify on the protein purifier, collect the components with biological activity, and obtain metabolite.

When separating, the gel column used on the protein purification instrument is preferably a Superdex 75 Sephadex column.

Separation and purification of anti-tumor small molecules: place Nocardioides NX032 in the fermentation medium, shake culture at 28-30°C, 160-170rpm/min for 10-12 days, then 8000-12000rpm/min Min centrifuge for 15-20min, collect the fermentation supernatant; soak the macroporous adsorption resin DM301 in methanol for 12h-18h, wash it with sterile water until it is odorless, and the pH value reaches neutral, then mix the macroporous adsorption resin with the fermentation supernatant Mix at a volume ratio of 1:2.5-1:3, let stand at 4°C for 24h-30h, collect 100% methanol analysis solution, and freeze-dry to obtain a crude extract.

Conditions for further separation and purification with high performance liquid chromatography: column model: ZORBAX SB-C189.4×150mm 5um, mobile phase: water, acetonitrile, flow rate: 1mL/min, sample volume: 20μL, operating method: 0- 10min water: acetonitrile 60%-40%, 10-13min water: acetonitrile 0-100%.

At present, chromatography technology has been widely used in the separation and analysis of bacterial metabolites. For example, Robert J. Capon et al. obtained a new 3, The 6-disubstituted a-pyrone compound Nocardiopyrone A, Johannes F. Imhoff's research group obtained four new 2,5 from Nocardiopsis strain HB383 by liquid chromatography - Disubstituted y-pyrone derivatives: Nocapyrones A-D. Although chromatographic techniques are frequently used, the separation conditions are also different due to the different bacterial species involved and the different active substances obtained.

The metabolites of Actinomycetes pseudocardioides NX032 obtained by the above separation and purification method are identified by LC-MS/MS anti-tumor activity, which proves that they have good anti-tumor activity.

The present invention obtains small molecules and proteins with anti-tumor activity on the basis of improved separation and purification, and its active substance has broad-spectrum anti-tumor activity. Melanoma cells) and Hela (human cervical cancer cells) have cytotoxicity, and the activity is good, and the toxicity to normal cells is low. And the anti-tumor mechanism was studied, and it was found that the ATP synthasesubunit a isolated from Nocardioides actinomycetes had anti-tumor activity.

The research object of the present invention is the actinomycete of the genus Nocardiella, which is widely distributed in various environments, and especially shows the characteristics of dominant bacterial groups in moderate and high salinity environments. Although the exploration of actinomycetes producing new active secondary metabolites in high-salt environment is less, many new active secondary metabolites have been found in high-salt actinomycetes, indicating that actinomycetes in this ecological environment It has great potential in generating new active compounds.

The active product of Nocardia pseudocardiae will become a potential target for future natural product research because of its unique structure, diversity, and secondary metabolites with good biological activity. In particular, the Nocardia spp. strains collected and isolated in saline-alkaline and marine environments can not only produce new compounds, but also have a large number. They are rare strains for the development of natural products derived from microorganisms. In the present invention, a strain of Nocardioides actinomyces was screened through soil, and proteins and small molecular compounds with broad-spectrum anti-tumor activity were separated by AKTA Purifier10 and high-performance liquid chromatography, and its anti-tumor mechanism was preliminarily studied. It is helpful to enrich the natural products of anti-tumor activity of Nocardioides, provide a theoretical basis for exploring the mechanism of anti-tumor action, provide new materials for innovative drug research, and provide strain resources for obtaining compounds with novel structures.

Description of drawings

Figure 1 is a morphological characteristic diagram of strain Nocardiopsis sp.NX032 isolated and purified on TSB medium;

Figure 2 is a Gram staining morphological characteristic diagram of strain Nocardiopsis sp.NX032 after isolation and purification;

Figure 3 is a scanning electron microscope morphological characteristic diagram of strain Nocardiopsis sp.NX032 after isolation and purification;

Fig. 4 is a growth curve diagram of Actinomyces pseudonocardia NX032 strain;

Figure 5 is a graph showing the effect of Nocardiopsis sp.NX032 fermentation supernatant on the activity of different tumor cells; in the figure a, e are Hep-3B (human liver cancer cells), b, f are B16 (mouse melanoma cells), c, g is Hela (human cervical cancer cells), d, h are HUVEC (human umbilical vein endothelial cells: normal cells), where a, b, c, d are experimental groups, e, f, g, h are control groups;

Figure 6 is a graph showing the effects of sterile fermentation supernatants treated at different temperatures on the toxicity of B16 cells (a-e in the figure are fermentation supernatants treated at 40°C, 60°C, 80°C, 90°C, and 100°C respectively on The influence diagram of B16; f is the influence diagram that the fermentation supernatant without heat treatment acts on B16);

Figure 7 is a graph showing the toxicity of sterile fermentation supernatants treated at different pH levels on B16 cells (a-f in the figure shows that the fermentation supernatants are treated with pH 2, 4, 6, 7, 10, and 12, respectively, and act on B16);

Figure 8 is a graph showing the toxic influence of the precipitate collected from the aseptic fermentation supernatant after different concentrations of ammonium sulfate precipitation on B16 cells (A in the figure is a control group, B is 30% ammonium sulfate precipitation protein, and C is 50% ammonium sulfate precipitation protein, D is 80% ammonium sulfate precipitated protein);

Fig. 9 is the separation and purification figure of AKTA Purifier10 to ammonium sulfate crude extract;

Figure 10 is a graph showing anti-tumor activity of anti-tumor protein component A on B16 cells;

Fig. 11 is the purity detection chart of the SDS-PAGE of ammonium sulfate crude extract A by AKTA Purifier10;

Figure 12 is an MTT detection diagram of the activity of active component A separated and purified by AKTA Purifier 10 on Hep-3B, Hela, B16 and HUVEC cells;

Figure 13 is the separation diagram of the fermentation supernatant on the Agilent 1290 Infinity after the preliminary purification of the macroporous adsorption resin DM301;

Fig. 14 is each unimodal anti-tumor activity assay figure obtained by separation;

Figure 15 is the MTT assay diagram of the activity of Agilent 1290Infinity separation active fraction Peak1 on Hela, B16, Hep-3B and HUVEC cells;

Fig. 16 is a diagram of mass spectrometry identification results of anti-tumor protein substance A;

Figure 17 is a diagram of the mass spectrometric identification results of the anti-tumor small molecule Peak1;

Explanation of Microorganism Preservation

Nocardiopsis sp. NX032 (Nocardiopsis sp.NX032), the strain was preserved in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, Wuhan, China) on June 8, 2015, and the strain preservation number is CCTCC NO: M 2015361.

detailed description

The present invention will be described in further detail below in conjunction with specific examples.

① The Nocardiopsis sp.NX032 of the present embodiment, which was preserved on June 8, 2015 at the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China), The strain preservation number is CCTCC NO: M 2015361.

2. The separation process of Actinomyces pseudonocardiii NX032 bacterial strain of the present embodiment: collect soil samples from the paddy field in Hunan, get 1g of soil samples, put into the Erlenmeyer flask that glass ball is housed, add the sterile water of 99ml, vibrate 20min. The treated soil suspension was serially diluted 10 times, diluted 10 ³ times, 10 ⁴ times, 10 ⁵ times respectively, and finally formulated into 10 ^-3 , 10 ^-4 , 10 ^-5 dilution suspensions. Aspirate 100 μL on Gaoshi No. 1 plate, spread evenly, place it upside down in a constant temperature incubator at 28-30°C for 6 days, repeat purification, and finally pick out actinomycete colonies on each plate and inoculate into Gaoshi No. No. slant, cultured at 28-30°C for 6 days, routine smear, phenolic fuchsin staining and microscopic examination. The single colonies of purified actinomycetes were selected and placed in an Erlenmeyer flask containing 30 mL sterile seed medium (TSB), placed on a shaker at 28-30° C., and cultured at 280 r/min for 4 days. Mix with 50% glycerol and store in -80°C refrigerator for a long time.

③Observation by optical microscope and scanning electron microscope:

Specific implementation process: Take 1.5 mL of Nocardiopsis sp. NX032 (Nocardiopsis sp. NX032) cultured for 2 days, centrifuge at 10,000 rpm/min for 3 min, remove the supernatant, wash twice with double distilled water, and wash with 200 μL double The bacterial cells were resuspended in distilled water, and 10 μL of the bacterial liquid was dropped on the center of the glass slide. The Gram staining method was used to stain the Nocardioides actinomyces NX032, and the morphology of the bacterial cells was observed under a 100 times oil microscope. At the same time, it was diluted to 10 ^-4 suspension, spread on the TSB plate, and observed the colony morphology on the TSB plate. Take 1.5mL of the bacterial solution, centrifuge at 10000rpm/min for 3min, remove the supernatant, wash 8-10 times with PBS buffer, fix with glutaraldehyde at 4°C overnight, and use volume concentrations of 30%, 50%, 60%, and 70% respectively , 80%, 90%, 95%, and 100% ethanol were washed every 5 minutes, and 50 μL of bacterial solution was applied to a cover glass, and the morphology of the bacterial cells was observed under a scanning electron microscope.

Figure 1, Figure 2 and Figure 3 are the morphological characteristic diagrams of strain Nocardiopsis sp.NX032 after isolation and purification. Figure 1 is a graph of the colony morphology on the TSB plate. Fig. 2 is an optical microscope picture, and the results show that the strain is long, medium branched, straight or zigzag. Figure 3 is a scanning electron microscope image.

④ Homologous sequence analysis of Nocardiopsis sp.NX032 strain 16S rRNA gene:

The specific implementation process: Pick a single colony from the TSB plate, transfer it to 50mL TSB liquid medium, 28-30°C, 160rpm/min, shake culture for 2 days, use the bacterial genome DNA extraction kit, and follow the operation steps Whole genome extraction. Universal primers (Bf-F, AGAGTTTGATCCTGGCTCAG; Bf-R, ACGGCTACCTTGTTACGACTT) were designed according to the bacterial 16S rRNA gene sequence and synthesized by Sangon (Shanghai) Biotechnology Co., Ltd.

Perform 16S rRNA gene amplification according to the following reaction system and reaction conditions:

Reaction system (20 μL): sterile double distilled water 12 μL; 5XBuffer 4 μL; dNTP 1.6 μL; Bf-R (10 μM) 0.6 μL; Bf-F (10 μM) 0.6 μL; genome template 1 μL; Primer star DNA Polymerase 0.2 μL;

Reaction program: pre-denaturation at 94°C for 4min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 90s, 30 cycles, extension at 72°C for 10min.

The PCR product was purified by a multifunctional DNA purification and recovery kit, and sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing together with appropriate primers. Sequencing results showed that the 16S rRNA gene sequence length of the isolated strain Nocardiopsis sp.NX032 was 1524bp. The measured 16S rRNA gene sequence of Nocardiopsis sp.NX032 was compared with Blast in the US National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov). The homology analysis of the 16S rRNA gene sequences of different strains showed that the strain belonged to the genus Nocardiopsis Actinomyces, and was related to Nocardiopsis sp.AF-333(FJ481931.1) and Nocardiopsis sp.FXJ6.077 respectively. (GU002080.1)、Streptomyces sp.Ahbb4KM214828.1、Nocardiopsis dassonvillei subsp.dassonvilleistrain NRRL B-16366(AY999914.2)、Nocardiopsis sp.AM8(AM236241.1)、Nocardiopsissp.An26AM039886.1、Nocardiopsis sp.87H32-3EU196476 .1 and so on have the highest similarity, both are 99%. According to the results obtained by BLAST, it was speculated that the strain belonged to Nocardiopsis sp. subspecies, named Nocardiopsis.sp NX032, and a phylogenetic tree was constructed (Replications=1000, Bootstrap value was taken as a percentage).

⑤Determination of growth curve of Nocardiopsis sp.NX032:

TSB medium: 5.0g sodium chloride, 17g tryptone, 3g soybean peptone, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, add water to 1L, pH 7.3;

Specific implementation process: Pick a single colony from the TSB plate, transfer to 30mL TSB liquid medium, 28-30°C, 160rpm/min, shake culture for 2 days, transfer to 50mL TSB medium at 2%, 28 -30°C, 160rpm/min, shaking culture, sampling every 6h, and measuring OD ₆₀₀ value.

The growth curve of Nocardiopsis sp.NX032 strain is shown in Figure 4. The growth lag period of Nocardiopsis sp.NX032 is about 24 hours, the exponential growth period is 30-48 hours, the stable period is 48-60 hours, and the decline period is after 60 hours. The results of the growth curve test show that the growth cycle of the Nocardiopsis sp.NX032 strain is basically consistent with the growth cycle of the known Nocardiopsis actinomyces. At the same time, referring to its growth cycle, it can be known that the strain has the best activity in the exponential phase, so this time period is selected Inoculate the fermentation medium.

⑥Determination of antitumor activity of Nocardiopsis sp.NX032 strain:

Specific implementation process: take the Nocardiopsis sp.NX032 bacterial strain 10-12 day fermentation liquid (the acquisition method of the 10-12 day fermentation liquid is consistent with the bacterial strain culture method when measuring the growth curve) in a refrigerated centrifuge (10000rpm) centrifugal 15mim, cross 0.22 μm filter membrane to obtain sterile fermentation supernatant. Spread mouse melanoma cells B16 in 96-well plates at ¹⁰ cells/well (100 μL) and place them in a 37°C, 5% CO ₂ constant temperature incubator for static culture for 24 hours to allow the cells to adhere to the wall. grow. Add 2, 5, 10, 15 μL of sterile fermentation supernatant, and 10 μL of sterile fermentation supernatant after treatment at 40°C, 60°C, 80°C, 90°C, 100°C for 1 hour; The supernatants were treated with pH gradients of 2, 4, 6, 7, 10, and 12 for 1 hour, then the pH was adjusted to neutral, and 10 μL of each was added for antitumor experiments. A control group is also set up (the control is CK, plus the fermentation supernatant of uninoculated strains cultured for 10-12 days). Temperature treatment and pH treatment 10-12 day fermentation supernatant are two independent and non-interfering experiments. The control group and the aseptic fermentation supernatant were respectively set up 3 times, and cultured for 24 hours. Observe the changes of tumor cells with an inverted microscope. The same experiment was performed on Hep-3B (human liver cancer cells), Hela (human cervical cancer cells), and HUVEC (human umbilical vein endothelial cells: normal cells).

Figure 5 shows the effects of 5 μL sterile fermentation supernatant on Hep-3B (human liver cancer cells), B16 (mouse melanoma cells), Hela (human cervical cancer cells), and HUVEC (human umbilical vein endothelial cells: normal cells) cells Toxicity impact diagram, in which a and e are Hep-3B (human liver cancer cells), b and f are B16 (mouse melanoma cells), c and g are Hela (human cervical cancer cells), d and h are HUVEC (Human umbilical vein endothelial cells: normal cells), where a, b, c, and d are the experimental groups, and e, f, g, and h are the control groups.

Figure 6 is a graph showing the effects of sterile fermentation supernatants treated at different temperatures on the toxicity of B16 cells (a-e in the figure are fermentation supernatants treated at 40°C, 60°C, 80°C, 90°C, and 100°C respectively on The influence diagram of B16; f is the influence diagram of the unheated fermentation supernatant acting on B16).

Figure 7 is a graph showing the toxicity of sterile fermentation supernatants after different pH treatments on B16 cells (a-f in the figure shows the effects of fermentation supernatants on B16 after being treated with pH 2, 4, 6, 7, 10, and 12 respectively picture).

The results of the study showed that Nocardiopsis sp.NX032 fermentation supernatant could make Hep-3B (human liver cancer cells), B16 (mouse melanoma cells), Hela (human cervical cancer cells), HUVEC ( Human umbilical vein endothelial cells: normal cells) The cells shrunk and became round (see Figure 5). There is no significant difference in the toxicity of the sterile fermentation supernatants treated at different temperatures to tumor cells, all of which have strong cytotoxicity, and can cause the B16 cells to shrink and round to lyse (see Figure 6).

Compared with the activity of the antitumor active substance under neutral conditions, the activity of the antitumor active substance under strong acid treatment is almost lost; the activity of the antitumor active substance after strong alkali treatment is the same as that of strong acid treatment, and its activity is reduced (see Fig. 7); thus it can be speculated that the antitumor active substance in the fermentation supernatant is a substance resistant to high temperature but not resistant to acid and alkali.

⑦AKTA Purifier10 separation active component A anti-tumor experiment and 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl bromide tetrazolium blue tetrazolium (MTT) determination:

Preparation of active component A1: Collect Nocardiopsis sp.NX032 10-12 day fermentation supernatant at 12,000rpm, add solid ammonium sulfate to 500ml supernatant (add slowly, add while stirring), to the mass of ammonium sulfate in the mixture The concentration was 30%, and it was left standing at 4° C. for 4 hours, and the precipitate was collected by centrifugation at 9000-12000 rpm. The supernatant was added to solid ammonium sulfate until the mass concentration of ammonium sulfate in the mixture was 50%, and the precipitate was collected by centrifugation. Then solid ammonium sulfate was added to the supernatant until the mass concentration of ammonium sulfate in the mixture was 80%, and the precipitate was collected by centrifugation. The collected 30%, 50% and 80% ammonium sulfate precipitates were redissolved in water, and the salt ions were removed with a dialysis bag with a cut-off molecular weight of 3.5KD to obtain crude protein. Utilize the Superdex75 dextran gel chromatographic column to separate the crude protein on the protein purification instrument, (separation conditions: mobile phase: water, flow rate: 1mL/min, sample volume: 1mL, operation method: 100% water, operation Time: 30min), the active component A obtained was collected; and its purity was detected by SDS-PAGE.

Specific implementation process: Hep-3B (human liver cancer cells), Hela (human cervical cancer cells), B16 (mouse melanoma cells) and HUVEC (human umbilical vein endothelial cells: normal cells) cells were respectively used at ¹⁰⁴ /per The number of wells was inoculated in a 96-well plate, placed in a 37°C, 5% CO ₂ cell constant temperature incubator and cultivated to allow the cells to grow on the wall, and cultivated for 24 hours; adding different concentrations of active component A at room temperature, and continuing to cultivate for 24 hours (each group Set 3 repetitions); the control group continued to culture for 24 h without adding active component A; aspirated the supernatant, added 90 μL of fresh RPMI-1640 medium, then added 10 μL of MTT reagent, and continued to culture for 4 h; aspirated the supernatant, and added 110 μL of Formazan solution, placed for 10 minutes, shaking at intervals to fully dissolve the crystals (MTT reagent and Formazan solution are easy to decompose when exposed to light, the above steps are protected from light), placed on an enzyme-linked immunoassay instrument to measure the absorbance value of each well at 490nm , and calculate cell viability.

After the collected sample A was freeze-concentrated, 12% gel concentration was used for SDS-PAGE detection. Observe the protein band size.

Figure 8 is a graph showing the toxic influence of the precipitate collected from the aseptic fermentation supernatant after different concentrations of ammonium sulfate precipitation on B16 cells (A in the figure is a control group, B is the protein precipitated by 30% ammonium sulfate, and C is 50% ammonium sulfate Ammonium sulfate precipitated protein, D is 80% ammonium sulfate precipitated protein);

Figure 9 AKTA Purifier10 separation and purification of ammonium sulfate crude extract;

Fig. 10 is the anti-tumor activity assay figure of anti-tumor protein component A on B16 cells; (A is the morphological figure of B16 cells after 24 hours of cultivation without adding active component A to the control CK; B is adding active component A and culturing Morphology of B16 cells after 24h)

Fig. 11 SDS-PAGE purity detection of ammonium sulfate crude extract A by AKTA Purifier10.

Figure 12 MTT assay of the activity of the active fraction A separated and purified by AKTA Purifier 10 on Hep-3B, Hela, B16 and HUVEC cells. MTT results showed that active component A was active on Hela, B16, Hep-3B and HUVEC cells, and when the concentration reached 105.584 μg/mL, the inhibition rates of the three tumor cells were all at or close to 50%, while the normal cells The inhibitory rate of <RTI ID=0.0>is</RTI> less than 25%, where the activity was the most toxic to B16 cells (see Figure 12). The formula for calculating the cell inhibition rate is as follows:

Inhibition rate = [(ODCK-OD zeroing)-(OD sample-OD zeroing)]/(ODCK-OD zeroing)

ODCK: normal cultured cells;

OD zero adjustment: only add medium without adding cells;

OD sample: normally cultured cells plus sample processing.

⑧ Chromatographic separation of anti-tumor small molecule active substances of Nocardiopsis sp.NX032 and MTT detection of active substances:

Specific implementation process: Collect Nocardiopsis sp.NX032 10-12 day fermentation supernatant at 12000rpm/min, activate DM301 macroporous adsorption resin with 100% methanol overnight, and wash the activated resin with sterile water until it is odorless. The macroporous adsorption resin and the fermentation supernatant were mixed at a volume ratio of 1:3, left standing at 4°C for 24 hours, and the 100% methanol analysis solution was collected, centrifuged at 12000rpm/min, passed through a 0.22μm filter membrane, and analyzed by high performance liquid chromatography (Agilent 1290Infinity ) to further separate anti-tumor active substances (column: ZORBAX SB-C18 9.4×150mm 5μm, mobile phase: water, acetonitrile, flow rate: 1mL/min, sample volume: 20μL, operation method: 0-10min water: acetonitrile 60%- 40%, 10-12min acetonitrile 100%.

Figure 13 is the separation of the aqueous phase on the Agilent 1290 Infinity after the fermentation supernatant was passed through the DM301 macroporous adsorption resin; Figure 14 is the activity measurement of the prepared single peak, and the Peak1 and Peak2 components were separated on the Agilent 1290 Infinity After each single peak was freeze-dried by a freeze-drying concentrator, take 20 μL of ddH ₂ O to redissolve, centrifuge at 13,000 rpm for 30 minutes, pass through a 0.22 μm membrane to obtain a sterile collection of each single peak, take 10 μL of each to act on B16 cells for 24 hours, and observe the cell form.

Figure 15 is the MTT assay of the activity of Agilent 1290 Infinity isolated active fraction Peak1 on Hela, B16, Hep-3B and HUVEC cells. MTT results showed that the active component Peak1 was active on Hela, B16, Hep-3B and HUVEC cells, and when 72.536 μL Peak1 was added to 100 μL cell culture medium, the inhibition rates of the three tumor cells were more than 50%, while the normal The inhibition rate of the cells was maintained at about 25%.

⑨LC-MS/MS identification of Nocardiopsis.sp NX032 anti-tumor active substance:

Specific implementation process: Anti-tumor protein in-gel enzymolysis mass spectrometry identification: prepare a sterile 1.5mEP tube, and cut the target band with a sharp sterile needle. Add 280 μL of 100 mM NH ₄ HCO ₃ and 120 μL of 30% acetonitrile to decolorize, place at room temperature until the gel is colorless and transparent, pour off the supernatant, and lyophilize; add 90 μL of 100 mM NH ₄ HCO ₃ , 10 μL of 100 mM DTT, incubate at 56°C for 30 minutes, and reduce the protein; Remove the supernatant, add 100μL 100% ACN, suck it off after 5min; add 70μL 100mM NH ₄ HCO ₃ , 30μL 200mMIAA, and keep in the dark for 20min; remove the supernatant, add 100μL 100mM NH ₄ HCO ₃ , room temperature for 15min; 100 μL of acetonitrile, sucked off after 5 min, and freeze-dried; after freeze-drying, add 5 μL of 10 ng Trypsin solution and place in a refrigerator at 4 °C for 30-60 min to fully expand the gel pieces; then add an appropriate amount of 50 mM ammonium bicarbonate buffer (without Trypsin), pH 7.8-8.0. React at 37°C for about 20 hours; collect the proteolysis solution into a new centrifuge tube, add 100 μL 60% acetonitrile/0.1% trifluoroacetic acid to the original tube, ultrasonicate 3 times, each time for 15 minutes, aspirate the solution before adding secondary solution. Combined and freeze-dried, they were stored in a -80°C refrigerator for later use. The results are shown in Figure 16. The protein was identified as ATP synthase subunit a by mass spectrometry.

Mass spectrometric identification of anti-tumor small molecules: The anti-tumor small molecules collected from Aglient 1290 were lyophilized and concentrated, and 100 μL was taken for LC-MS/MS identification. The detection ion source is a positive ion, and the mass spectrometry results show that the molecular weight of Peak 1 is 158.03KDa (see Figure 17).

Claims (6)

1. A Nocardioid Actinomycetes, characterized in that it is Nocardioid Actinomyces NX032, Nocardiopsis sp . NX032, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2015361. 2. as claimed in claim 1, the method for isolating active metabolites of Pseudo-Nocardiae actinomycetes, is characterized in that, comprises the following steps: Put Nocardioides actinomyces NX032 in the fermentation medium, shake culture at 28-30℃, 160-170rpm/min for 10-12 days, centrifuge at 8000-12000rpm/min for 15-20min, and collect the fermented The supernatant was passed through a 0.22 μm filter membrane to obtain a sterile fermentation supernatant; preliminary separation was carried out on a protein purification instrument to collect biologically active components; further separation and purification was carried out by a high-performance liquid chromatography to obtain active metabolites. 3. the method for separating active metabolites of Nocardioides actinomycetes according to claim 2, characterized in that, during preliminary separation, the gel column used on the protein purification instrument is a Superdex 75 Sephadex column. 4. according to claim 2 or the separation method of Nocardioides actinomycetes active metabolite described in 3, it is characterized in that, during further separation and purification, used chromatographic column is C18 reverse-phase chromatography in the described high performance liquid chromatograph column. 5. the application of Nocardioid actinomycetes in anti-tumor as claimed in claim 1. 6. The anti-tumor application of the active metabolite of Nocardioides actinomycetes isolated according to any one of claims 2-4.