Klebsiella sp.SXW12, exopolysaccharide EPS12 and application thereof

Abstract

The present invention discloses a Klebsiella sp.SXW12, and its deposit number is CCTCCNO:M 20221814. The present invention also discloses an extracellular polysaccharide EPS12, and the extracellular polysaccharide EPS12 is obtained by using the above-mentioned Klebsiella sp.SXW12. The present invention also discloses a production method of the above-mentioned extracellular polysaccharide EPS12. The present invention also discloses the use of the above-mentioned Klebsiella sp.SXW12 and the extracellular polysaccharide EPS12 in the preparation of anti-tumor drugs or health products. The culture medium required for culturing the Klebsiella sp.SXW12 of the present invention has clear components, the operation of culturing the strain is simple, and it is suitable for industrial production. The extracellular polysaccharide EPS12 has good anti-tumor activity.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Klebsiella sp.SXW12, exopolysaccharide EPS12 and applications thereof. Background Art

[0002] Cancer is the leading cause of high morbidity and mortality worldwide. Malignant tumors seriously endanger human health. Chemotherapy is one of the main methods of systemic treatment, but its side effects seriously affect the quality of life of patients. Currently, drug therapy using natural substances is considered an effective alternative to traditional therapies. Studies have shown that more and more natural macromolecular polysaccharides have been shown to have good anti-tumor activity. For example, polysaccharides extracted from wild Cordyceps sinensis can effectively inhibit the proliferation of H22 tumors in mice at doses of 100 mg / kg body weight and 300 mg / kg body weight, and have no toxic side effects compared with 5-FU, a commonly used drug for the treatment of liver cancer (Tan L, Liu S, Li X, He J, He L, Li Y, Yang C, Li Y, Hua Y, Guo J. The Large Molecular Weight Polysaccharide from Wild Cordyceps and Its Antitumor Activity on H22 Tumor-Bearing Mice. Molecules. 2023; 28(8): 3351.). SOWP, a water-soluble polysaccharide extracted from the root of Sanguisorba officinalis L., can effectively inhibit mouse sarcoma tumors in a dose-dependent manner at doses of 50, 100 and 200 mg / kg body weight (Cai Z, Li W, Wang H, Yan W, Zhou Y, Wang G, Cui J, Wang F. Anti-tumor and immunomodulating activities of a polysaccharide from the root of Sanguisorba officinalis L. Int J Biol Macromol. 2012; 51(4): 484-8). The polysaccharide DWPP-Is (10 mg / mL, 0.2 mL) extracted from Dendrobium warianum can effectively inhibit the proliferation of A549 tumors in mice, indicating that DWPP-Is has the potential to become a functional preparation for preventing lung cancer (Ye G, Li J, Zhang J, Liu H, Ye Q, Wang Z. Structural characterization and antitumor activity of a polysaccharide from Dendrobium warianum. Carbohydr Polym. 2021; 269: 118253).The extracellular succinoglycan Riclin secreted by soil Agrobacterium can significantly inhibit the growth of tumors in S180 tumor-bearing mice at doses of 10, 20 and 40 mg / kg body weight (Yang Y, Sun X, Zhao Y, Ge W, Ding Z, Liu J, Wang L, Xu X, Zhang J. Anti-tumor activity and immunogenicity of a succinoglycan riclin. Carbohydr Polym. 2021; 255: 117370).

[0003] However, the effective doses of natural polysaccharides reported so far for anti-tumor use are relatively high. Polysaccharides are macromolecular polymers. The higher the molecular weight, the higher the viscosity of the prepared preparation. If used on the human body in proportion, the dose is too high, limiting its further application as a biological agent. In addition, the separation cost of extracting high-purity polysaccharides from animals and plants is relatively high compared to microbial polysaccharides, and the operation is complicated, resulting in a reduced possibility of conversion into biological agents for clinical use. Therefore, it is of great significance to find economical and high-yield natural microbial polysaccharides that can effectively inhibit tumor proliferation at lower doses. Summary of the invention

[0004] Based on the technical problems existing in the background technology, the present invention proposes a Klebsiella sp. SXW12, an exopolysaccharide EPS12 and applications thereof. The components of the culture medium required for culturing the Klebsiella sp. SXW12 of the present invention are clear, the operation of culturing the strain is simple, and the strain is suitable for industrial production. The exopolysaccharide EPS12 has good anti-tumor activity.

[0005] The invention provides a Klebsiella sp.SXW12, whose deposit number is CCTCCNO:M20221814.

[0006] The inventor isolated a strain from pond sludge in Lujiang County, Hefei City, Anhui Province, which was identified as a Klebsiella sp. strain by molecular biology and named Klebsiella sp.SXW12. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on November 24, 2022, at Wuhan University, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with a deposit number of CCTCCNO:M 20221814.

[0007] The present invention also provides an extracellular polysaccharide EPS12, which is produced by using the above-mentioned Klebsiella sp.SXW12.

[0008] Preferably, the exopolysaccharide EPS12 consists of glucose, glucuronic acid and fucose in a molar ratio of 2:1:1.

[0009] Preferably, the structural formula of the extracellular polysaccharide EPS12 is as shown in formula (I):

[0010]

[0011] Wherein, n is an integer greater than 0.

[0012] The average molecular weight of the extracellular polysaccharide EPS12 is 90,000-100,000 Da, preferably 93,946 Da.

[0013] The present invention also proposes a method for producing the above-mentioned extracellular polysaccharide EPS12, comprising the following steps: taking the fermentation liquid of Klebsiella sp.SXW12, adding alkaline substances to mix, heat treatment, solid-liquid separation, taking the liquid and adding a precipitant, collecting the precipitate, and obtaining the extracellular polysaccharide EPS12.

[0014] Preferably, the alkaline substance is an inorganic base.

[0015] Preferably, the alkaline substance is sodium hydroxide.

[0016] Preferably, the precipitant is at least one of ethanol, acetone and isopropanol.

[0017] Preferably, the heat treatment method is a boiling water bath for 10-20 min.

[0018] Preferably, Klebsiella sp. SXW12 is fermented in a fermentation medium to obtain a fermentation broth.

[0019] Preferably, the formula of the fermentation medium is: 1L of water contains: KNO3 3.5-4.5g, CaCl2 0.03-0.04g, NaH2PO4 0.8-1.2g, ZnCl2 0.003-0.007g, MgSO4 0.15-0.25g, FeSO4 0.012-0.013g, MnSO40.003-0.005g, and sucrose 18-22g.

[0020] Preferably, the pH of the fermentation medium is 6.8-7.2.

[0021] Preferably, the fermentation temperature is 28-32°C and the fermentation time is 36-48h.

[0022] The Klebsiella sp.SXW12 is first cultured in a seed culture medium to obtain a seed solution, and then the seed solution is fermented in a fermentation medium to obtain a fermentation solution, wherein the seed culture medium and the fermentation medium have the same formula.

[0023] Preferably, the fermentation broth of Klebsiella sp.SXW12 is diluted with an equal volume of water, and then an alkaline substance is added and mixed.

[0024] Preferably, the concentration of the alkaline substance is 0.08-0.12 g / 100 mL.

[0025] Preferably, the volume ratio of the precipitant to the liquid is 2-3:1.

[0026] The above-mentioned extracellular polysaccharide EPS12 can be purified by removing proteins. The protein removal and purification process can be performed by the Sevag method, which specifically includes the following steps:

[0027] S1. Mix the extracellular polysaccharide EPS12 aqueous solution and the Sevag mixed solution, let it stand to separate into three layers, take the upper aqueous phase, centrifuge and take the supernatant;

[0028] S2. Replace the extracellular polysaccharide EPS12 aqueous solution with the supernatant, repeat the operation of S1 until there is no middle protein layer after standing, take the upper aqueous phase, centrifuge to take the supernatant, add a precipitant to the supernatant, collect the precipitate, and dry to obtain the extracellular polysaccharide EPS12.

[0029] Preferably, in the aqueous solution of exopolysaccharide EPS12, the concentration of exopolysaccharide EPS12 is 0.8-1.2 g / 100 mL;

[0030] Preferably, the Sevag mixed solution is a mixed solvent of chloroform and n-butanol; preferably, the volume ratio of chloroform to n-butanol is 3.5-4.5:1.

[0031] Preferably, the volume ratio of the extracellular polysaccharide EPS12 aqueous solution to the Sevag mixed solution is 1:3.5-4.5.

[0032] Preferably, the precipitant is at least one of ethanol, acetone and isopropanol; preferably, 2-3 times the volume of the precipitant is added to the supernatant.

[0033] The present invention also proposes the use of the Klebsiella sp.SXW12 and the extracellular polysaccharide EPS12 in the preparation of anti-tumor drugs.

[0034] Preferably, the tumor is a benign tumor or a malignant tumor.

[0035] Preferably, the tumor is lung cancer.

[0036] The water mentioned above can be sterile water, purified water, deionized water, etc.

[0037] Beneficial effects:

[0038] The present invention isolates a strain of Klebsiella sp.SXW12 from pond sludge in Lujiang County, Hefei City, Anhui Province, and finds that the strain can secrete exopolysaccharide EPS12. Studies have shown that the exopolysaccharide EPS12 can significantly inhibit the growth of lung cancer cells in mice at a relatively low concentration, indicating that the exopolysaccharide EPS12 has good anti-tumor activity.

[0039] The culture medium of Klebsiella sp.SXW12 of the present invention has simple and clear components, low cost, no pigment secretion during the growth process of the strain, and simple strain culture operation; and the shake flask fermentation yield of the extracellular polysaccharide EPS12 is 15 g / L, which has a high yield, a simple extraction and purification process, and low production cost, and is suitable for the industrial production of the extracellular polysaccharide EPS12.

[0040] The extracellular polysaccharide EPS12 of the present invention has good anti-tumor activity when used in mice at a dosage of 1, 2 and 4 mg / kg body weight. The extracellular polysaccharide EPS12 has a small dosage, is easily soluble, and can be prepared into an anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a photo of the plate colonies of Klebsiella sp.SXW12.

[0042] Figure 2 This is an ion chromatogram of component determination of extracellular polysaccharide EPS12, where a is a standard monosaccharide spectrum and b is a monosaccharide composition analysis spectrum of extracellular polysaccharide EPS12. Markings in the figure: Fuc is fucose, Rha is rhamnose, Ara is arabinose, Gal is galactose, Glc is glucose, Xyl is xylose, Man is mannose, Fru is fructose, Rib is ribose, GalUA is galacturonic acid, GlcUA is glucuronic acid, ManUA is mannuronic acid, and GulUA is guluronic acid.

[0043] Figure 3 is the one-dimensional NMR spectrum of the extracellular polysaccharide EPS12, where a is 1 H NMR, b is 13 CNMR.

[0044] Figure 4 This is the two-dimensional NMR spectrum of the extracellular polysaccharide EPS12, where Figure a is COSY, Figure b is HSQC, Figure c is HMBC, Figure d is TOCSY, and Figure e is NOESY.

[0045] Figure 5 This is the chemical structure diagram of the extracellular polysaccharide EPS12.

[0046] Figure 6This is the MTT graph of the effect of extracellular polysaccharide EPS12 on lung cancer cells in vitro.

[0047] Figure 7 The figures are analysis graphs of the antitumor activity of extracellular polysaccharide EPS12 on lung cancer cells in vivo, where a is the in vivo imaging results of mice in each group on days 3, 7, 10, and 14 after tumor inoculation, showing that the fluorescent area is the tumor, b is the statistical graph of the bioluminescence intensity of the fluorescent substance in the mouse tumor in figure a over time, c is a picture of the tumor peeled off from the mouse, and d is the mass statistics of the mouse tumor.

[0048] Figure 8 The results of the effect of extracellular polysaccharide EPS12 on mouse spleen, where a is the effect of extracellular polysaccharide EPS12 on mouse spleen index, b is the H&E stained section of mouse spleen, the black arrow indicates the white pulp, the red arrow shows the red pulp, magnification: 100×, scale width is 100μm.

[0049] Fig. 9 Figure 3 is the effect of extracellular polysaccharide EPS12 on T cells in mouse spleen, where a is the flow cytometry profile of CD4+T and CD8+T cells in spleen tissue, and b is the analysis and statistical results of Figure a.

[0050] Fig.10 Figure 2 is the effect of extracellular polysaccharide EPS12 on the expression of apoptosis-related genes in mouse spleen, where a is the relative expression of casp-3 gene, b is the relative expression of Bax gene, c is the relative expression of Bcl-2 gene; d is the relative expression of Bcl-xL gene. DETAILED DESCRIPTION

[0051] The technical solutions of the present invention are described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustration only and are not to be construed as limiting the scope of the present invention.

[0052] Example 1

[0053] 1. Screening and Isolation of Klebsiella sp.SXW12

[0054] Take pond sludge from Lujiang County, Hefei City, Anhui Province, mix 1g of sludge sample with 10mL of sterile physiological salt, take 1mL of the suspension and dilute it on a solid culture medium, culture it at 30℃ for 2-3 days, and after a single colony grows out, select the single colony with obvious sticky polysaccharide production on the colony surface, continue to streak and separate it on a new solid plate, culture it at 30℃, and after a single colony grows out, continue to streak and separate it on a new solid plate, and repeat this step until a single morphology colony grows on the solid culture medium.

[0055] The solid culture medium used above is the same, all containing: KNO3 1g, CaCl2 0.1g, NaH2PO41.0g, ZnCl2 0.005g, MgSO4 0.1g, FeSO4 0.0125g, MnSO4 0.004g, sucrose 20g, agar 15g in 1L water, with a pH of 7.0-8.0, and the culture medium is sterilized at 121°C for 20min.

[0056] 2. Identification of strains

[0057] The single strain obtained in step 1 was used for morphological characterization and species identification. The results are as follows:

[0058] (1) Morphological characteristics such as Figure 1 As shown, it can be seen that after the strain is cultured on a solid plate medium at 30°C for 2-3 days, the colonies are round and have a smooth surface. It can be observed that the strain secretes a large amount of viscous extracellular polysaccharides without producing pigments.

[0059] (2) Determine the 16S rRNA gene sequence of the above single strain, compare the 16S rRNA gene sequence of the strain with the gene sequence in the GenBank database for homology and analyze the results to determine the species of the bacteria at the molecular biological level.

[0060] After Blast comparison, the sequence similarity of 16S rRNA of Klebsiella sp.SXW12 strain was as high as 99.93% with Klebsiellavariicola strain AST, 99.93% with Klebsiella pneumoniae strainYJY5, 99.93% with Klebsiella sp.strain FX0039, and 99.86% with Klebsiellavariicola strain JM16. It belongs to the genus Klebsiella and is named Klebsiella sp.SXW12. It was deposited in the China Center for Type Culture Collection (CCTCC) on November 24, 2022. The preservation address is Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the preservation number is CCTCC NO:M20221814.

[0061] The gene sequence of 16S rRNA of Klebsiella sp.SXW12 is:

[0062]

[0063] 1381aagaagtagg tagcttaacc ttcggggaggg cgctacca

[0064] Example 2

[0065] A method for producing extracellular polysaccharide EPS12 comprises the following steps:

[0066] A single colony of Klebsiella sp.SXW12 was inoculated into a seed culture medium, and cultured at 30°C and 230 rpm for 24 hours to obtain a seed solution; the seed solution was added into a fermentation culture medium, and cultured at 30°C and 230 rpm for 48 hours to obtain a fermentation solution of Klebsiella sp.SXW12;

[0067] The seed culture medium and the fermentation culture medium are the same, and the formula is that 1L of sterile water contains: KNO3 4g, CaCl20.035g, NaH2PO4 1.0g, ZnCl2 0.005g, MgSO4 0.2g, FeSO4 0.0125g, MnSO4 0.004g, sucrose 20g, pH = 7.0, and the culture medium is sterilized at 121°C for 20min;

[0068] Take the fermentation broth of Klebsiella sp.SXW12, dilute it with an equal volume of deionized water, add sodium hydroxide and mix well to make the concentration of sodium hydroxide 0.1g / 100mL, then boil it in a boiling water bath for 10min, centrifuge it at 8000g for 10min, collect the supernatant, add 2 times the volume of ethanol to the supernatant and mix well, centrifuge it at 8000g for 10min, collect the precipitate, and dry it in an oven at 40°C to obtain the crude exopolysaccharide EPS12;

[0069] Take the crude exopolysaccharide EPS12 and prepare an exopolysaccharide EPS12 aqueous solution with a concentration of 1 g / 100 mL; mix the exopolysaccharide EPS12 aqueous solution with Sevag mixed solution (chloroform: n-butanol = 4:1 v / v) at a volume ratio of 1:4, shake vigorously for 20 minutes, and let the solution stand to separate into three layers (the middle layer is the denatured protein layer, the upper layer is the aqueous phase, and the lower layer is the organic phase), collect the upper aqueous phase, centrifuge at 8000g for 10 minutes, and collect the supernatant;

[0070] The supernatant was mixed with the Sevag mixture, and the above operation was repeated 3 times. The mixture was allowed to stand until there was no intermediate protein layer, and the upper aqueous phase was taken. The mixture was centrifuged at 8000 g for 10 min, and the supernatant was taken. Three times the volume of ethanol was added to the supernatant and mixed. The precipitate was collected by centrifugation at 8000 g for 10 min, and dried in an oven at 40°C to obtain the extracellular polysaccharide EPS12.

[0071] Example 3

[0072] The extracellular polysaccharide EPS12 prepared in Example 2 was analyzed for its structure, as follows:

[0073] (1) The average molecular weight of extracellular polysaccharide EPS12 was determined by gel permeation chromatography to be 93946 Da.

[0074] (2) The extracellular polysaccharide EPS12 was completely hydrolyzed with trifluoroacetic acid and then analyzed by ion chromatography. Figure 2 shown.

[0075] Figure 2 This is the ion chromatogram of the component determination of the extracellular polysaccharide EPS12, where a is the standard monosaccharide spectrum and b is the monosaccharide composition analysis spectrum of the extracellular polysaccharide EPS12.

[0076] Depend on Figure 2 It can be seen that the extracellular polysaccharide EPS12 is a heteropolysaccharide composed of glucose, glucuronic acid and fucose in a molar ratio of 2:1:1.

[0077] (3) The extracellular polysaccharide EPS12 and the reduced-exopolysaccharide EPS12 after reducing uronic acid (i.e., Reduced-EPS12) were subjected to methylation analysis. The results are shown in Table 1.

[0078] Table 1 Methylation analysis of exopolysaccharide EPS12

[0079]

[0080] Remark: a Partially methylated sugar alcohol acetates; b The results are expressed as molar ratios relative to 1,3-linked-Fucp residues; c Determined through comparative analysis of EPS12 and reduced-EPS12.

[0081] As shown in Table 1, the ratio of 1,4-linked Glcp in exopolysaccharide EPS12 and reduced exopolysaccharide EPS12 after uronic acid reduction is different, and the molar ratio of 1,4-linked Glcp in reduced exopolysaccharide EPS12 after uronic acid reduction increases from 4.23 to 5.97, indicating that there are about 2 molecules of 1,4-linked GlcAp glycoside in exopolysaccharide EPS12. Exopolysaccharide EPS12 is composed of 1,3-linked Fucp, 1,4-linked Fucp, 1,4-linked Glcp and 1,4-linkedGlcAp, and the molar ratio of each glycoside is consistent with the results of monosaccharide analysis.

[0082] (4) The exopolysaccharide EPS12 was partially hydrolyzed with trifluoroacetic acid, prepared with D2O to 50 mg / mL, and NMR analysis was performed using deuterated acetone as an internal standard. Figure 3-4 shown.

[0083] Figure 3 is the one-dimensional NMR spectrum of the extracellular polysaccharide EPS12, where a is 1 H NMR, b is 13 CNMR.

[0084] Figure 4 This is the two-dimensional NMR spectrum of the extracellular polysaccharide EPS12, where Figure a is COSY, Figure b is HSQC, Figure c is HMBC, Figure d is TOCSY, and Figure e is NOESY.

[0085] Depend on Figure 3-4 It can be seen that: 1 In H NMR, the chemical shift at δ5.3 ppm is the alpha anomeric proton, the signals at δ4.35, 4.38, and 4.45 ppm are the beta anomeric proton, and the chemical shifts at δ1.13 and 1.14 ppm are attributed to the –CH3 of fucose;

[0086] 13 In C NMR, there are 8 signals between δ98-103ppm, indicating that there are 8 anomeric carbons in the extracellular polysaccharide EPS12; in 13C NMR, the chemical shift δ15.43ppm is attributed to –CH3 of fucose, δ171.5 is attributed to –COOH of glucuronic acid and pyruvic acid, and the multiple peaks at δ30ppm and the peak at δ217ppm are the deuterated acetone peaks used for positioning;

[0087] Chemical shifts of glycoside protons were determined by COSY and TOCSY, combined with 1 H co-assignment; chemical shift assignments of glycosidic carbon signals are given by 13 C and HSQC, and the connection order of sugar residues in the exopolysaccharide EPS12 was determined by HMBC and NOESY. Based on the NMR results of the exopolysaccharide EPS12, combined with the above monosaccharide composition and methylation analysis, the chemical structure of the exopolysaccharide EPS12 was inferred and analyzed. Table 2 shows the chemical structure of the exopolysaccharide EPS12. 1 H NMR and 13 The chemical shifts of C NMR are summarized, and the bold parts are the substitution sites of sugar residues.

[0088] Table 2 Extracellular polysaccharide EPS12 1 H NMR and 13 C NMR chemical shift (ppm)

[0089]

[0090]

[0091] Based on the NMR results of the extracellular polysaccharide EPS12, combined with the above monosaccharide composition and methylation analysis, the chemical structure of the extracellular polysaccharide EPS12 was determined. Figure 5 It can be seen that the extracellular polysaccharide EPS12 is a new structure acidic heteropolysaccharide composed of glucose, glucuronic acid and fucose in a molar ratio of 2:1:1.

[0092] Example 4

[0093] The anti-tumor activity experiment of extracellular polysaccharide EPS12 is as follows:

[0094] Experimental methods:

[0095] (1) Analysis of the antitumor activity of extracellular polysaccharide EPS12 in vitro:

[0096] After culturing, lung cancer cells (LLC-LUC) were digested and counted to prepare 2×10 5 The single cell suspension of 100 μg / mL was inoculated into a 96-well cell culture plate, and 100 μL was inoculated into each well, and the cells were cultured overnight in a 37°C incubator with 5% CO2. The groups were set up as follows: control group (CK) and groups containing different concentrations of extracellular polysaccharide EPS12 (50, 100, 200 μg / mL), with 10 replicates in each group. The control group was given 50 μL of fresh culture medium, and other groups were given 50 μL of the corresponding concentration of extracellular polysaccharide EPS12, and cultured for 12 hours; then 10 μL of MTT was added to each well, and incubated at 37°C in the dark for 4 hours; the 96-well plate was removed, the supernatant was aspirated, 150 μL of DMSO was added to each well, and the purple crystals were fully dissolved in the dark for 20 minutes. The OD value was measured at a wavelength of 490 nm using an enzyme reader to calculate the cell inhibition rate.

[0097] (2) Analysis of the anti-tumor activity of extracellular polysaccharide EPS12 in vivo:

[0098] Establishment of mouse tumor model: After 6-8 week-old C57BL / 6 male mice were adaptively fed in SPF animal room for one week, 13 mice were set up in normal group (CK), and the other mice were inoculated with LLC-LUC cells (0.2 mL per mouse, 5×10 6cells / mouse); then they were randomly divided into 4 groups, with 13 mice in each group, namely the model group (Mod), the low-dose group (LE) with an extracellular polysaccharide EPS12 dose of 1 mg / kg body weight, the medium-dose group (ME) with a dose of 2 mg / kg body weight, and the high-dose group (HE) with a dose of 4 mg / kg body weight; starting from the day after LLC-LUC cells were inoculated, the corresponding groups of mice were intraperitoneally injected with the corresponding doses of extracellular polysaccharide EPS12 solution every day, and the control group was injected with normal saline, for a total of 14 days; on the 14th day, the mice were anesthetized with 2-3% isoflurane and killed by cervical dislocation, and the tumors and spleens of the mice were removed for use in the following experiments.

[0099] (3) In vivo imaging of mice:

[0100] On the 3rd, 7th, 10th and 14th days, the mice were anesthetized by isoflurane inhalation and intraperitoneally injected with luciferin (D-Luciferin, 150 mg / kg). The mice were tested on a machine 7 minutes after the injection. The in vivo imaging of the mice was performed to show the tumor size, and the corresponding bioluminescence intensity of the tumor was calculated.

[0101] (4) Effect of extracellular polysaccharide EPS12 on mouse spleen: The body weight of mice was recorded and the spleen weight of each group of mice was weighed. The spleen index was calculated according to spleen index = spleen mass ÷ mouse body weight.

[0102] (5) Histopathological analysis: The spleens of mice were collected for histopathological analysis. H&E staining was used to analyze the pathological changes of the spleens in different groups.

[0103] (6) Flow cytometry analysis: Mouse spleens were collected and single cell suspensions were prepared for flow cytometry antibody staining to analyze the proportion of relevant immune cells, as follows:

[0104] The method for preparing a single cell suspension from the spleen is as follows: fresh spleen is peeled off, cut into small pieces in 6 mL of tissue cell in vitro culture medium (PBS + 1% FBS), and then slowly ground into a cell suspension on a cell sieve, and the cell suspension is collected into a 2 mL centrifuge tube (4°C, 500g centrifugation for 5 min), the supernatant is removed, 1 mL of red blood cell lysis buffer (ACK) is added to resuspend the cells, ice bathed for 3-5 min, and then an equal volume of tissue cell in vitro culture medium is added to terminate the reaction, after 4°C, 500g centrifugation for 5 min, the supernatant is discarded, and 1 mL of tissue cell in vitro culture medium is added to obtain a single cell suspension of spleen cells;

[0105] Cell antibody staining: First, prepare the staining system: dilute the antibody with tissue cell in vitro culture medium, CD4 and CD8 antibodies at a dilution of 1:200; then count the single cell suspension, prepare 4×10 cells per sample 5After centrifugation at 4°C and 500g for 5 min, discard the supernatant, add 100 μL of staining system to resuspend the cells, stain for 20 min in an ice bath away from light, add 200 μL of tissue cell in vitro culture medium to terminate the reaction, mix well and add to the flow tube for flow cytometry detection.

[0106] (7) Effect of extracellular polysaccharide EPS12 on the expression of apoptosis-related genes in mouse spleen: Mouse spleen RNA was extracted and reverse transcribed into cDNA. Then, apoptosis genes were subjected to fluorescence quantitative PCR to detect their gene expression levels. The specific steps are as follows:

[0107] Extract RNA: Take about 20 mg of spleen tissue, add 600 μL Karral, homogenize, then add 120 μL chloroform to mix, oscillate for 15 seconds, let stand for 10 minutes, centrifuge at 4°C, 1100g for 15 minutes, discard the supernatant, add an equal volume of isopropanol, mix, let stand for 10 minutes, centrifuge at 4°C, 1100g for 10 minutes, discard the supernatant, add 1 mL 75% ethanol, wash the precipitate, centrifuge at 4°C, 8000g for 5 minutes, discard the supernatant, add 100 μL DEPC water to dissolve RNA.

[0108] Reverse transcription to cDNA: Mix 1μL 10mM dNTPs, 1μL Oligo dT, 8μL RNA solution, and 10μL DEPC water, place in a 65℃ warm bath for 5 minutes, and ice bath for 5 minutes; then add 4μL 5×First buffer, 2μL DTT, 1μL M-MLV reverse transcriptase, and 1μL DEPC water, mix, place in a 37℃ water bath for 50 minutes, and then place in a 70℃ warm bath for 15 minutes to obtain cDNA.

[0109] Fluorescence quantitative PCR: 10 μL of 2×SYBR Green master mix, 0.5 μL of Forward Primer, 0.5 μL of Reverse Primer, 5 μL of cDNA, and 4 μL of ddwater were mixed and placed in a PCR instrument. The amplification conditions were set as follows: 50°C, 2 min; then 95°C, 10 min; 95°C, 15 s, 60°C, 1 min, repeated 40 cycles, and finally 95°C, 15 s. β-actin was used as an internal reference, and the relative expression analysis method of the gene was 2- △△CT The primer sequences used for fluorescence quantitative PCR are shown in Table 3.

[0110] Table 3 Primer sequences used for fluorescence quantitative PCR

[0111]

[0112] Experimental results:

[0113] Figure 6 This is the MTT graph of the effect of extracellular polysaccharide EPS12 on lung cancer cells in vitro.

[0114] Depend on Figure 6 It can be seen that when the extracellular polysaccharide EPS12 is co-cultured with lung cancer cells in vitro, it has no effect on lung cancer cells, indicating that the extracellular polysaccharide EPS12 cannot directly inhibit the proliferation of lung cancer cells.

[0115] Figure 7 The figures are analysis graphs of the antitumor activity of extracellular polysaccharide EPS12 on lung cancer cells in vivo, where a is the in vivo imaging results of mice in each group on days 3, 7, 10, and 14 after tumor inoculation, showing that the fluorescent area is the tumor, b is the statistical graph of the bioluminescence intensity of the fluorescent substance in the mouse tumor in figure a over time, c is a picture of the tumor peeled off from the mouse, and d is the mass statistics of the mouse tumor.

[0116] Figure 7 The data in d are mean ± SEM, n = 11 or 13; *p < 0.05, **P < 0.01, ***P < 0.001 vs. model group (Mod).

[0117] Depend on Figure 7 ab shows that the volume and bioluminescence intensity of the tumors in the Mod group mice increased rapidly with time, while in the LE, ME, and HE groups using the extracellular polysaccharide EPS12, although the volume and bioluminescence intensity of the tumors also increased with time, the growth rate was significantly lower than that of the Mod group.

[0118] Depend on Figure 7 c It can be seen that the tumor volume of the Mod group is much larger than that of the LE, ME, and HE groups.

[0119] Depend on Figure 7 d shows that the average tumor mass in the Mod group was 572.89 mg, while the tumor mass in the LE, ME, and HE groups decreased to 123.26 mg, 147.73 mg, and 99.55 mg, respectively, indicating that the extracellular polysaccharide EPS12 can significantly inhibit the proliferation of lung cancer in mice. This indicates that the extracellular polysaccharide EPS12 has no inhibitory effect on the proliferation of lung cancer cells in vitro, but has a significant inhibitory effect in vivo.

[0120] Figure 8 The results of the effect of extracellular polysaccharide EPS12 on mouse spleen, where a is the effect of extracellular polysaccharide EPS12 on mouse spleen index, b is the H&E stained section of mouse spleen, the black arrow indicates the white pulp, the red arrow shows the red pulp, magnification: 100×, scale width is 100μm.

[0121] Figure 8The data in a are mean ± SEM, n = 11, *p < 0.05, **P < 0.01, ***P < 0.001 vs. model group (Mod), # p<0.05, ## P<0.01, ### P<0.001vs control group (CK).

[0122] Depend on Figure 8 aIt can be seen that the spleen index of the Mod group mice was not significantly different from that of the CK group, while the spleen index of the LE, ME, and HE groups using extracellular polysaccharide EPS12 was higher than that of the Mod group and the CK group, indicating that the treatment of extracellular polysaccharides activated the immune system of mice.

[0123] Depend on Figure 8 b It can be seen that part of the red pulp and white pulp structure of the spleen in the Mod group was destroyed, while in the LE, ME, and HE groups, the red pulp and white pulp were significantly increased. This shows that the extracellular polysaccharide EPS12 can stimulate the body to produce a strong immune response, suggesting that the extracellular polysaccharide EPS12 may inhibit tumor proliferation by improving the body's immune function.

[0124] Fig. 9 Figure 3 is the effect of extracellular polysaccharide EPS12 on T cells in mouse spleen, where a is the flow cytometry profile of CD4+T and CD8+T cells in spleen tissue, and b is the analysis and statistical results of Figure a.

[0125] Fig. 9 CD4 / CD8 double staining was used to display T lymphocyte populations. Fig. 9 The data are mean ± SEM, n = 3, *p < 0.05, **P < 0.01, ***P < 0.001 vs. model group (Mod).

[0126] Depend on Fig. 9 It can be seen that compared with Mod, the proportion of CD4 in the spleen tissue of LE, ME and HE groups using extracellular polysaccharide EPS12 was significantly increased, and all were statistically significant. The change in CD8 proportion was not statistically significant. This shows that extracellular polysaccharide EPS12 can significantly increase the proportion of T lymphocytes in the spleen, which again suggests that extracellular polysaccharide EPS12 may inhibit tumor proliferation by improving the body's immune function.

[0127] Fig.10 Figure 2 is the effect of extracellular polysaccharide EPS12 on the expression of apoptosis-related genes in mouse spleen, where a is the relative expression of casp-3 gene, b is the relative expression of Bax gene, c is the relative expression of Bcl-2 gene; d is the relative expression of Bcl-xL gene.

[0128] Fig.10The expression of related apoptosis genes in spleen tissue was detected by fluorescence quantitative PCR; Fig.10 The data are mean ± SEM, n = 3, *p < 0.05, **P < 0.01, ***P < 0.001 vs. model group (Mod), # p<0.05, ## P<0.01, ### P<0.001vs control group (CK).

[0129] Depend on Fig.10 It can be seen that Caspase-3 is involved in the execution of cell apoptosis. Compared with the Mod group, the LE, ME, and HE groups using extracellular polysaccharide EPS12 significantly downregulated the expression of Caspase-3 gene;

[0130] Bax is a pro-apoptotic gene. Compared with the CK group, the expression of Bax in the Mod group was increased, while the expression of Bax in the LE, ME, and HE groups was significantly lower than that in the Mod group.

[0131] Bcl-2 and Bcl-xL are both anti-apoptotic genes. Compared with the Mod group, the expression levels of Bcl-2 and Bcl-xL in the LE, ME, and HE groups were significantly increased.

[0132] In summary, the extracellular polysaccharide EPS12 effectively reduced the expression of the pro-apoptotic gene Bax in the spleen of mice, promoted the expression of the anti-apoptotic genes Bcl-2 and Bcl-xL, and reduced the expression of Caspase-3 that executes apoptosis, thereby inhibiting the apoptosis of spleen cells. According to the above results, the extracellular polysaccharide EPS12 inhibits the proliferation of tumors by inhibiting the apoptosis of spleen cells and improving the immune function of the body, rather than directly acting on the cancer cells themselves. Therefore, the tumor described in the present invention is not limited to lung cancer, but includes other benign or malignant tumors, such as osteosarcoma, colon cancer, gastric cancer, etc.

[0133] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A Klebsiella Klebsiella sp. SXW12, its deposit number is CCTCC NO: M 20221814.

2. A Klebsiella as claimed in claim 1 Klebsiella Application of sp. SXW12 in the preparation of drugs against lung cancer.

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