Acidithiobacillus ferrooxidans for efficiently leaching realgar

Abstract

The invention discloses a thiobacillus ferrooxidans domestication strategy which is more efficient compared with an existing strategy, and a novel acidophilic thiobacillus ferrooxidans Dom-BY3 (Acidithiobacillus ferrooxidans Dom-BY3) with the preservation number of CCTCC (China Center for Type Culture Collection) NO: M 20242585 is obtained through domestication and separation of the strategy. Compared with found acidophilic thiobacillus ferrooxidans, the expression level of realgar leaching related genes of the strain is remarkably improved, higher realgar leaching efficiency is achieved, and the leaching effect result is stable. The concentration of arsenic ions and the concentration of iron ions in a realgar microbiological leaching solution obtained by leaching realgar with the acidithiobacillus ferrooxidans Dom-BY3 are higher. In the aspect of drug effect, the realgar microbiological leaching solution not only has an obvious inhibition effect on HePG2 hepatoma cell lines and K562 leukemia cell lines, but also has a poison effect on rat myocardial cells H9C2 weaker than that of realgar microbiological leaching solutions obtained by leaching disclosed strains.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a method for taming a strain, in particular to a method for taming an acidophilic thiobacillus ferrooxidans strain. Technical Background

[0002] Bioleaching is a simple and effective biological method for extracting metals from minerals. Bioleaching can convert insoluble substances into soluble salts through acidophilic bacteria (such as At.ferrooxidans) to achieve the purpose of bioleaching. Applying bioleaching technology to the processing of realgar is an interdisciplinary study proposed by our laboratory. In recent years, realgar has achieved significant therapeutic effects in the treatment of blood system diseases and malignant tumors, which has attracted widespread attention. The traditional preparation methods of realgar include acid extraction and calcination, membrane separation, and mechanical grinding. However, the realgar processed by these methods has low water solubility, poor gastrointestinal absorption, high toxicity and low bioavailability (only 4% bioavailability in the human body), which seriously limits its clinical application. Based on the above problems, our laboratory cross-integrated this technology with the preparation of medicinal realgar, and used natural acidophilic ferrooxidans to perform microbial extraction of realgar. Studies have confirmed that the extract of realgar from At. ferrooxidans showed strong anti-cancer activity in both in vivo and in vitro experiments. Compared with traditional preparation methods, the biological extraction of realgar can significantly enhance the solubility and bioavailability of realgar, and this method is highly efficient, environmentally friendly and low-cost.

[0003] In the early stage of this experiment, the scheme of acclimation and leaching of Acidithiobacillus ferrooxidans At.f-BY3 was explored: 1.0g and 1.5g of water-flying realgar with a particle size of 180 mesh were used for acclimation and leaching experiments in 9K culture medium, and the leaching cycle was about 30 days. [1] The results showed that the domesticated bacteria At.f-BY3, which had been domesticated with realgar in the initial 0 to 15 days, had a high leaching rate for low concentrations of realgar and grew faster, but in the later stage, the growth of At.f-BY3 was inhibited in a high arsenic concentration environment. After At.f-BY3 was inoculated and activated in 9K culture medium, the iron content was gradually reduced from 7K to 1K, and then the bacterial solution with iron reduced to 1K was inoculated into 0K culture medium containing realgar powder suspension for domestication and leaching experiments. The results showed that the domesticated bacteria had a high leaching rate for low concentrations of realgar (0.5g realgar / 100mL), but when the realgar concentration was increased to 1.0g realgar / 100mL, the growth of the domesticated bacteria began to be inhibited. [4] .

[0004] The invention improves the domestication scheme. The domestication experiment is carried out in a 1K culture medium containing 0.4478 g of FeSO4·7H2O, and the concentration of realgar added is determined to be 0.5 g / 100 mL. During the domestication process, the pH value of the culture solution is adjusted to be stable at 1.8 with concentrated sulfuric acid. The culture solution is activated on a shaking table at 180 rpm and 30°C for 4 days. It is observed that the color of the culture solution changes from light yellow to light reddish brown, indicating that the growth state of the strain is normal and the domestication is completed.

[0005] The present invention optimizes the realgar domesticated microbial leaching scheme of acidithiobacillus ferroxidans strain, and adopts the above method to screen and obtain a domesticated strain (Acidithiobacillus ferroxidans Dom-BY-3), with a preservation number (CCTCCNO: M 20242585). The Dom-BY3 strain can extend the realgar leaching period to 68 days, significantly improve the arsenic concentration in the realgar microbial leaching solution, significantly enhance the anti-tumor activity, and have higher safety.

[0006] References

[0007] [1] Du Wenjing. Study on the microbial extraction of realgar and the antibacterial and pharmacological effects of its extract[D]. 2008.

[0008] [2] Haiyang. Study on the pharmacokinetics and antitumor efficacy of realgar FeSO4·7H2O yellow microbial extract[D]. Lanzhou University, 2014.

[0009] [3] Leng Feifan. Comparative study on the resistance of Thiobacillus ferrooxidans and Thiobacillus thiooxidans to inorganic arsenic compounds[D]. 2010.

[0010] [4] Liu Dongling. Study on the mechanism of down-regulation of ras overactivation in Caenorhabditis elegans by realgar transformation fluid[D]. 2013.

[0011] [5] Wang Xin. Reversal effect of realgar microbial transformation fluid on drug resistance of K562 / ADM cells and regulation of SirT1[D]. Lanzhou University, 2013. Summary of the invention

[0012] The invention discloses an acidithiobacillus ferroxidans domesticated Dom-BY3 (Acidithiobacillus ferroxidans Dom-BY-3), with a preservation number (CCTCC NO: M 20242585), a preservation time: November 18, 2024, a preservation institution: China Center for Type Culture Collection, an address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province; contact information: E-mail: cctcc@whu.edu.cn, telephone: 027-6875 4052.

[0013] The present invention also provides a realgar microbial extract obtained by extracting realgar with acidithiobacillus ferrooxidans Dom-BY3.

[0014] The invention also provides application of the realgar microorganism extract in preparing anti-tumor drugs.

[0015] Preferably, the tumor is liver cancer and leukemia.

[0016] Preferably, the leukemia is chronic myeloid leukemia.

[0017] The present invention also provides a domestication technology of acidithiobacillus ferroxidans BY-3, wherein the domestication steps are as follows:

[0018] S1: Prepare 9K culture medium: weigh (NH4)2SO43.00 g, K2HPO40.50 g, MgSO4·7H2O0.50 g, KCl0.10 g, Ca(NO3)20.01 g, FeSO4·7H2O44.78 g, stir and dissolve in 950 mL of distilled water, pipette 1 mL of concentrated sulfuric acid to adjust the pH to 1.8, make up to 1000 mL, and set aside.

[0019] S2: Take 10 mL of the bacterial liquid of Acidithiobacillus ferrooxidans At.f-BY3 and add it into 90 mL of the prepared 9K culture medium for activation.

[0020] S3: Weigh 0.5g of realgar and add it to 90mL of the prepared 9K medium. Take 10mL of the activated Thiobacillus ferrooxidans At.f-BY3 in S2 and add it to the 9K medium containing 0.5g of realgar. Leach it on a shaker at 180rpm and 30℃ for 24h until the liquid color turns reddish brown.

[0021] S4: Repeat step S3 twice to ensure the stability of the domesticated bacterial solution;

[0022] S5: Pipette 10mL of domesticated bacteria and inoculate into the prepared 9K medium. Continue domestication in a shaker at 180rpm, 30℃, and 24h in the culture flask. Then, the medium is reduced to 1K conditions by gradually reducing iron. The amount of FeSO4·7H2O is 0.4478g. The culture flask is 180rpm, 30℃, and shaker is used for 4d. Observe the color of the bacterial culture medium under 1K growth conditions, which changes from light yellow to light reddish brown, indicating that the growth state of the strain is normal and domestication is completed.

[0023] Beneficial effects of the present invention:

[0024] (1) Compared with the discovered acidithiobacillus ferroxidans, the acidithiobacillus ferroxidans Dom-BY3 (Acidithiobacillus ferroxidans Dom-BY3) disclosed in the present invention, with a preservation number (CCTCC NO: M 20242585), has the highest efficiency in leaching realgar, a higher leached arsenic ion concentration, and a higher expression level of genes related to realgar leaching;

[0025] (2) The Acidithiobacillus ferrooxidans domestication strategy disclosed in the present invention has better domestication effect on Acidithiobacillus ferrooxidans than the publicly disclosed domestication schemes;

[0026] (3) The realgar microbial extract obtained by extracting realgar from the bacteria Acidithiobacillus ferrooxidans Dom-BY3 had a significant inhibitory effect on the HePG2 liver cancer cell line and the K562 leukemia cell line, and the results were stable;

[0027] (4) The realgar microbial extract obtained by extracting realgar with the acidithiobacillus ferrooxidans Dom-BY3 had a weaker toxic effect on rat cardiomyocytes H9C2 than the realgar microbial extract obtained by extracting with the previously disclosed strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The expression level of COXB gene in the realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3

[0029] Figure 2 Expression level of COXC gene in realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3

[0030] Figure 3 Expression level of arsC gene in realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3 DETAILED DESCRIPTION

[0031] The following specific embodiments are provided to implement the technical solutions described in the present invention, but are not limited to these embodiments.

[0032] Example 1: Acclimation of Acidithiobacillus ferrooxidans

[0033] 1. Preparation of liquid culture medium:

[0034] Accurately weigh (NH4)2SO43.00 g, K2HPO40.50 g, MgSO4·7H2O0.50 g, KCl0.10 g, Ca(NO3)220.01 g, and FeSO4·7H2O44.78 g, stir and dissolve in 950 mL of distilled water, pipette 1 mL of concentrated sulfuric acid to adjust the pH to 1.8-2.0, and make up to 1 L.

[0035] 2. Domestication process of Dom-BY3 strain:

[0036] Acidophilic Thiobacillus ferrooxidans BY3 (Thiobacillus ferrooxidans BY3) was selected. Thiobacillus ferrooxidans BY3 was deposited in the China Center for Type Culture Collection on September 22, 2003, with a deposit number of CCTCC NO: M203071, and a deposited name: Acidophilic Thiobacillus ferrooxidans BY3 (Thiobacillus ferrooxidans BY3); realgar, sourced from Shimen Town, Hunan Province, China, was prepared by detoxification treatment according to the method in Part 1 of the Pharmacopoeia of the People's Republic of China (2020 Edition), and the realgar was ground until all the powder passed through a 200-mesh standard sample sieve, with a powder particle fineness of 74 μg, and was sterilized by irradiation for later use. Take 10 mL of the original At.f-BY3 strain and inoculate it into the prepared 9K liquid culture medium at an inoculation rate of 10%, ensuring that the pH value of the 9K liquid culture medium is 1.8, weigh 0.5 g of the prepared realgar and add it to 90 mL of the liquid 9K culture medium, absorb 10 mL of the enriched At.f-BY3 strain, and leaching it in a shaking bottle at 180 rpm and 30° C. for 24 hours until the liquid color turns reddish brown; take out 10 mL of the 9K domesticated bacterial solution domesticated with realgar, and repeat the above steps twice to ensure the stability of the domesticated bacterial solution; take out 10 mL of the 9K Dom-BY3 bacterial solution enriched for the third time and perform a step-by-step iron reduction leaching step: weigh 3.134 g, 2.239 g, 1.3434 g, and 0.4478 g of FeSO4·7H2O, respectively, and add them from 9K to 7K, 5K, 3K, and 1K in sequence to separate and obtain the domesticated bacteria of acidophilic ferrooxidans Dom-BY3.

[0037] Finally, a shaking leaching biotransformation step with a cycle of 68 days was carried out. Every 2 days, 1K culture medium accounting for 2% of the total volume of the culture bottle was added, and the pH value was adjusted to 1.8 with concentrated sulfuric acid. After 68 days, the domestication leaching liquid of the Dom-BY3 strain was completed, and the realgar microbial leaching liquid of acidophilic ferrothiobacillus Dom-BY3 was obtained.

[0038] Example 2 Evaluation of Realgar Microbial Extract from Domesticated Acidithiobacillus ferrooxidans Dom-BY3

[0039] 1. Preparation of realgar microbial extracts using different extraction schemes

[0040] (1) Preliminary Plan 1 [1]:Selected strains: Thiobacillus ferrooxidans (At.f-BY3) (CCTCC-M203071, collected from a waste mine in a mining area in Baiyin, Gansu; high-quality block realgar was purchased from the Yellow River Medicinal Materials Market in Lanzhou, the origin of which is Hunan, and it was qualified according to the "Pharmacopoeia of the People's Republic of China, 2008 Edition". The processing method is: the block of raw realgar is crushed and ground through a 200-mesh sieve, irradiated and extinguished, 2mL At.f-BY3 is inoculated into fresh 9K liquid culture medium, transferred 1 to 2 times and then domesticated and cultured, 1.5g of the above-mentioned realgar powder is added to obtain the domesticated strain, and the shaking table is leached at 160rpm for 30d.

[0041] (2) Preliminary Plan 2 [4] The strain selected was Thiobacillus ferrooxidans (At.f-BY3) (CCTCC-M203071), which was collected from an abandoned mine in a mining area in Baiyin, Gansu Province; realgar was purchased from Gansu Provincial Hospital of Traditional Chinese Medicine, the particle size of realgar powder was 180-200 mesh, the inoculation amount was 20%, and it was inoculated into fresh 9K liquid culture medium, 1.0 g of the above realgar powder was added, and the mixture was leached on a shaking table at 130 rpm for 30 days.

[0042] (3) Scheme of the present invention: The domestication scheme refers to the specific scheme in Example 1 to obtain the realgar microbial extract of the domesticated bacteria of Acidithiobacillus ferrooxidans Dom-BY3.

[0043] 2. The arsenic concentration in the sample was directly determined using an IRIS ER / S plasma emission spectrometer (produced by Thermo Fisher Scientific, Inc., USA).

[0044] The arsenic ion concentration of the At.f-BY3 domesticated strain without the leaching scheme was detected by the ICP method. 100 μL of the bacterial solution was taken, and 10 mL of 20% concentrated nitric acid was added for digestion. 5 mL of the digestion solution was taken for on-machine detection. The wavelength condition selected for the ICP chemical analyzer to detect the arsenic ion concentration was 193.696.

[0045]

[0046]

[0047] In the study of the extract of Dom-BY3 domesticated strain, we used an ICP chemical analyzer to measure its arsenic ion concentration. Compared with the results of the previous schemes 1 and 2, the arsenic ion concentration of the realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3 prepared by this method was significantly higher than the value measured by the previous laboratory scheme.

[0048] Arsenic ions are key components in the leachate, and accurate determination of their concentration is of great significance for evaluating the properties and quality of the leachate. This method can obtain higher arsenic and iron ion concentrations, and this result fully demonstrates the superiority of this method. From the perspective of arsenic ion concentration, a higher arsenic ion concentration means that this method can more effectively promote the release of arsenic ions from realgar during the leaching process. This may be because this method is more optimized in the cultivation of the Dom-BY3 domesticated strain or the control of leaching conditions, making the strain have stronger activity and arsenic leaching ability.

[0049] Example 3 Detection of stability of realgar extracted by Acidithiobacillus ferrooxidans Dom-BY3

[0050] The concentration of two batches of acidophilic ferrothiobacillus Dom-BY3 realgar microbial extracts prepared according to the scheme of the present invention was subjected to stability determination: Two batches of Dom-BY3 extracts were prepared before and after this experiment (the first batch was prepared in April 2023, and the second batch was prepared in December 2023), and the arsenic ion concentration was detected by ICP chemical analyzer. 100 μL of bacterial solution was taken, 10 mL of 20% concentrated nitric acid was added for digestion, 5 mL of digestion solution was taken for on-machine detection, and the wavelength condition for detecting arsenic ion concentration by ICP chemical analyzer was 193.696.

[0051]

[0052] As shown in the table above: The arsenic ion concentration of the two batches of leachate was measured. The results showed that the difference in the arsenic ion concentration between the two measurements was only 1, and the standard deviation was Arsenic ion is one of the important components in the leachate, and its concentration stability has a key influence on the properties and applications of the leachate. During the storage and use of the realgar microbial leachate of Acidithiobacillus ferrooxidans Dom-BY3, the arsenic ion concentration can be maintained at a relatively stable level, the chemical composition of the leachate has not changed significantly, and its performance and effects can also remain relatively consistent.

[0053] In summary, the fact that there was no difference in the arsenic ion concentrations between the two measurements strongly suggests that the Dom-BY3 strain has good stability.

[0054] Example 4: Determination of the expression level of genes related to the effect of strain leaching in the realgar microbial leaching solution of Acidithiobacillus ferrooxidans Dom-BY3 using real-time fluorescence qPCR technology

[0055] A control group and an experimental group were set up. The control group was A. Acidithiobacillus ferrooxidans At.f-BY3 culture solution; the experimental group was B. Acidithiobacillus ferrooxidans Dom-BY3 realgar microbial leaching solution. qPCR experiments were performed to determine the expression of metal ion genes that affect the leaching effect, and statistical analysis was performed.

[0056] (1) Extraction and concentration detection of total cell RNA

[0057] The instruments used in the RNA extraction process were sterilized by high-pressure steam at 121°C for 60 min and the total RNA was extracted using a bacterial total RNA extraction kit (RNAprep Pure Cell / Bacteria Kit, ) Extract total RNA from bacterial cells. The specific steps are as follows:

[0058] ①4℃, 12000rpm centrifugation for 2min to collect bacteria (the maximum amount of bacteria collected should not exceed 1×10 9 ), carefully remove all the culture supernatant, and all subsequent centrifugation steps are performed at room temperature (20-25°C); thoroughly resuspend the cells with 100 μL TE buffer containing lysozyme (final lysozyme concentration is 400 μg / mL), and incubate at room temperature for 3-5 min;

[0059] ② Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12000 rpm for 30-60 seconds, discard the waste liquid and put the adsorption column back into the collection tube; Preparation of DNase I working solution: take 10 μL of DNase I storage solution and put it into a new RNase-Free centrifuge tube, add 70 μL of RDD solution, and mix gently; add 80 μL of DNase I working solution to the center of the adsorption column CR3 and leave it at room temperature for 15 minutes;

[0060] ③ Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12000 rpm for 30-60 s, discard the waste liquid, and put the adsorption column back into the collection tube; add 500 μL of rinsing solution RW to the adsorption column CR3 (check whether ethanol has been added before use), leave at room temperature for 2 min, centrifuge at 12000 rpm for 30-60 s, discard the waste liquid, and put the adsorption column CR3 back into the collection tube;

[0061] ④ Centrifuge at 12000rpm for 2min, discard the waste liquid, and place the adsorption column CR3 at room temperature for several minutes to completely dry the adsorption. Transfer the adsorption column CR3 to a new RNase-Free centrifuge tube, and drop 30-100μL RNase-Free ddH2O in the middle of the adsorption membrane. Place it at room temperature for 2min, and centrifuge at 12000rpm for 2min to obtain total bacterial RNA;

[0062] ⑤Use NanoDrop 8 OD was measured by ND-1000 spectrophotometer (Gene Company Limited). 260 / OD 280 The purity of RNA was evaluated by the ratio, and the range of the samples was 1.75-2.1.

[0063] (2) Reverse transcription of the extracted total cell RNA into cDNA

[0064] ① Thaw the template RNA on ice; thaw 5×gDNA Buffer, FQ-RT Primer Mix, 10×Fast RTBuffer, and RNase-Free ddH2O at room temperature and quickly place on ice after thawing. Vortex and mix each solution before use, and centrifuge to collect the liquid remaining on the tube wall;

[0065] ② Prepare a mixture of 5×gDNA Buffer Total RNA 2μL, RNase-Free ddH2O to 10μL of genomic DNA removal system, mix thoroughly, centrifuge, incubate at 42℃ for 3min, and then place on ice;

[0066] ③ Prepare the mixed solution according to the reverse transcription reaction system below: 10×Fast RT Buffer 2μL, RT EnzymeMix 1μL, FQ-RT Primer Mix 2μL, and RNase-Free ddH2O to 10μL;

[0067] ④ Add the Mix in the transcription reaction to the reaction solution in the gDNA removal step and mix thoroughly; incubate at 42℃ for 15min; incubate at 95℃ for 3min and then place on ice. The obtained cDNA can be used for subsequent experiments or stored at -20℃.

[0068] (3) The primers used in the present invention were designed using Primer Premier 5.0 software for each gene and synthesized by Sangon Biotech Co., Ltd.

[0069]

[0070] (4) qPCR detection

[0071]

[0072] qPCR instrument: Life Technologies Holdings Pte Ltd; QuantStudio 5

[0073] The qPCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 45 s, annealing for 30 s, extension at 72°C for 30 s; extension at 72°C for 10 min, and 31 cycles.

[0074] like Figure 1 As shown, through the domestication scheme of the present invention, the cytochrome COXB gene is normally expressed in the realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3. COXB gene participates in the electron transport chain of At.f to promote Fe 2+ Oxidation, the encoded cytochrome C oxidase subunit II promotes the electron transfer process. In the bacterial leaching process, the normal expression of the COXB gene ensures the efficient operation of the electron transport chain, making Fe 2+ Can be continuously oxidized to provide energy for bacterial growth and metabolism.

[0075] like Figure 2 As shown in Figure 2, the cytochrome COXC gene is expressed normally in the realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3. COXC can assist in electron transfer and proton pump function, indirectly affecting Fe 2+ Oxidation; stabilize the structure of the enzyme and ensure Fe 2+ Normal operation of the oxidation system: If there is a problem with the COXC gene, resulting in abnormal structure or expression of subunit III, the overall function of cytochrome C oxidase may be impaired, thereby affecting Fe 2+ The normal operation of the oxidation system will eventually have an adverse effect on the growth and metabolism of Thiobacillus ferrooxidans.

[0076] like Figure 3 As shown, Acidithiobacillus ferrooxidans containing the arsC gene can resist the toxicity of arsenic by activating a series of arsenic resistance proteins during the leaching process, thereby affecting the arsenic content in the leachate. When the arsenic concentration in the environment is high, the expression of the arsC gene may increase to improve the bacteria's arsenic resistance, thereby affecting the arsenic concentration in the leachate. In the early stage of the laboratory, the arsC gene in the ars operon of Acidithiobacillus ferrooxidans was cloned by PCR amplification. Through sequencing, sequence alignment and cluster analysis of the cloned arsC gene, it was found that the arsC gene in the bacteria had a very high similarity with the arsC gene sequence reported in the GenBank database, confirming the high evolutionary conservation of the arsC gene. [3]The domestication scheme of the present invention further performs gene amplification detection on the arsC gene of the original bacteria At.f-BY3 and the domesticated strain Dom-BY3 by qPCR technology. The results show that the expression level of the arsC gene is improved, and the expression is compared with that of the undomesticated original strain At.f-BY3 and the statistical difference analysis is performed. The results show that the domesticated strain Dom-BY3 obtained by the scheme of the present invention is adapted to the growth environment, the expression amount of the arsC gene is significantly increased, the bacteria grows rapidly, the metabolism is vigorous, and it is beneficial to the leaching efficiency.

[0077] Example 5 Evaluation of the efficacy and stability of the realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3

[0078] 1. Detection of the inhibitory activity of the realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3 on HepG2 cell proliferation

[0079] HepG2 tumor cells in the logarithmic growth phase were cultured at about 10 5 100 μL of cell suspension was plated in each well of a 96-well plate at a density of cells / mL and cultured overnight. According to the results of the As ion concentration determination of the two batches of leaching solutions in Example 3, a dosing regimen was formulated with a maximum concentration of 8 μg / mL, and the drug was administered in a half-dilution. Eight parallel experiments were set for each drug concentration, and the treatments were performed for 24 h and 48 h, respectively. After the drug action time was over, 5 mg / mL of MTT was added to each well, and the cells were incubated at 37°C for another 4 h. The supernatant was discarded by the rapid plate flipping method, and 150 μL of DMSO was added to each well. The samples were detected on an enzyme reader, and the OD values ​​were measured. 570 IC50 50 .

[0080] The efficacy and stability of Dom-BY3 realgar microbial extract on HepG2 cells

[0081]

[0082] Two batches of acidophilic thiobacillus ferrooxidans Dom-BY3 realgar microbial extracts were independently prepared using the preparation process described in Example 2, and the MTT experimental method was used to test the inhibitory effects of the two batches of acidophilic thiobacillus ferrooxidans Dom-BY3 realgar microbial extracts on the activity of HepG2 cells. The experimental results showed that the two batches of acidophilic thiobacillus ferrooxidans Dom-BY3 realgar microbial extracts had the same tumor inhibitory activity, and the efficacy of inhibiting tumor cell activity was exactly the same. Therefore, the RTS obtained according to the preparation process of the present invention has a stable efficacy.

[0083] 2. Determination of the inhibitory activity of the realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3 on leukemia cells K562

[0084] The inhibitory effect of Dom-BY3 realgar extract on K562 leukemia cells was compared with that of realgar microbial extract prepared by a published method. [2][5]

[0085] Inhibitory effect of Dom-BY3 realgar microbial extract on K562 leukemia cells

[0086]

[0087] Effect of Dom-BY-3 Realgar Microbial Extract on IC of K562 Leukemia Cells 50 Significantly lower than the published IC value of realgar microbial extract 50 , the results can also prove that the Dom-BY-3 bacteria disclosed in the present invention has a better realgar extraction effect. For K562 leukemia cells, the anti-tumor activity of the realgar microbial extract of acidophilus ferrooxidans Dom-BY3 is obvious, and it has a significant inhibitory effect on the vitality of K562 leukemia cells. With the increase of the concentration of the realgar microbial extract of acidophilus ferrooxidans Dom-BY3, the survival rate of K562 cells gradually decreases.

[0088] 3. Safety evaluation of the realgar microbial extract of Acidithiobacillus ferrooxidans Dom-BY3 on normal rat cardiomyocytes H9C2 cells

[0089] The MTT cell viability test showed that compared with the realgar microbial extract prepared by the published method, the IC of Dom-BY-3 realgar extract on rat cardiomyocytes H9C2 was 50 It is larger, indicating that the safety of the realgar extract of Dom-BY-3 strain is better than that of the realgar microbial extract prepared by the disclosed method.

[0090] Safety evaluation of Dom-BY3 realgar microbial extract on normal H9C2 cells

[0091]

[0092] In summary, acidithiobacillus ferroxidans Dom-BY3 was obtained through domestication and screening through the optimization scheme in the present invention. Compared with the discovered acidithiobacillus ferroxidans, the expression level of realgar leaching-related genes of this strain was significantly improved, it had a higher realgar leaching efficiency, and the leaching effect results were stable.

[0093] The growth of this strain was not inhibited in the growth environment of 1K medium and 0.5g / 100mL realgar, and it was in good condition. After 68 days of leaching, the arsenic ion concentration of the Dom-BY3 realgar microbial extract was maintained at 322-323μg / mL, which was higher than the scheme explored in the early laboratory. Two batches of Dom-BY3 realgar microbial extracts were obtained by preparing different batches of the same acclimation scheme. The arsenic ion concentration range was still maintained between 322 and 323μg / mL, indicating that the strain had good stability.

[0094] Finally, in terms of efficacy evaluation, the two batches of Dom-BY3 realgar microbial extracts prepared by this scheme not only had a significant cell proliferation inhibitory effect on HePG2 cells, but also had stable efficacy without difference; the anti-tumor cell proliferation activity results on leukemia cells K562 showed that IC 50 The range is between 0.426±0.022 and 0.492±0.0465μg / mL, indicating that the anti-proliferation effect on leukemia cells is significantly improved compared with the published solution (1.00±0.177~2.25±0.18μg / mL); for rat cardiomyocytes H9C2, Dom-BY3 realgar microbial extract IC 50 Results for published leaching protocols for IC dosing 50 1.67 to 1.98 times, proving that it is less toxic and safer.

Claims

1. Acidithiobacillus ferroxidans Dom-BY-3, characterized in that: The acidophilic ferrothiobacillus Dom-BY3 has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is November 25, 2024, and the deposit number is CCTCC NO:M20242585.

2. Use of the acidithiobacillus ferrooxidans Dom-BY3 as claimed in claim 1 in leaching realgar.

3. A method for extracting realgar using the Acidithiobacillus ferrooxidans Dom-BY3 described in claim 1 to obtain a realgar microbial extract.

4. Use of the realgar microbial extract as claimed in claim 3 in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that The tumors are liver cancer and leukemia.

6. The use according to claim 5, characterized in that The leukemia is chronic myeloid leukemia.

7. A method for acclimating Acidithiobacillus ferrooxidans, characterized in that: The domestication technique steps are: S1: Prepare 9K culture medium: weigh (NH4)2SO43.00g, K2HPO40.50g, MgSO4·7H2O0.50g, KCl0.10g, Ca(NO3)20.01g, FeSO4·7H2O44.78g, stir and dissolve in 950mL distilled water, draw 1mL concentrated sulfuric acid to adjust the pH to 1.8, make up to 1000mL, and set aside. S2: Take 10 mL of the bacterial liquid of Acidithiobacillus ferrooxidans At.f-BY3 and add it into 90 mL of the prepared 9K culture medium for activation. S3: Weigh 0.5 g of realgar and add it to 90 mL of the prepared 9K medium. Take 10 mL of the activated acidithiobacillus ferrooxidans At.f-BY3 in S2 and add it to the 9K medium containing 0.5 g of realgar. Leach it on a shaker at 180 rpm and 30 °C for 24 h until the liquid color turns reddish brown. S4: Repeat step S3 twice to ensure the stability of the domesticated bacterial solution; S5: Pipette 10mL of domesticated bacteria and inoculate into the prepared 9K medium. Continue domestication in a shaker at 180rpm, 30℃, and 24h in the culture flask. Then, the medium is reduced to 1K conditions by gradually reducing iron. The amount of FeSO4·7H2O is 0.4478g. The culture flask is 180rpm, 30℃, and shaker is used for 4d. Observe the color of the bacterial culture medium under 1K growth conditions, which changes from light yellow to light reddish brown, indicating that the growth state of the strain is normal and domestication is completed.

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