Anti-ciliate drug target of cynanchum erectum glycoside C, application of anti-ciliate drug target and ciliate killing drug

Abstract

The invention provides an anti-ciliate drug target of cynanchum erectum glycoside C, application of the anti-ciliate drug target and a ciliate killing drug, and belongs to the technical field of parasite killing drugs. The potential target protein ABCE1 transporter of the cynanchum erectum glycoside C acting on the ichthyophthirius multifilis is obtained through a micromolecule immunoaffinity chromatography method, the ichthyophthirius multifilis resisting mechanism of the cynanchum erectum glycoside C is further clarified, and a new treatment target is provided for treatment of the white-spot disease and drug design. The method is of great significance to development of novel anti-ciliate natural drugs. A potential ichthyophthirius multifilis resisting drug is subjected to computer virtual screening on the basis of an ABCE1 transporter target and a TCMSP database, the natural compound pulsatilla saponin A3 capable of effectively killing ichthyophthirius is excavated, the compound and the ABCE1 transporter are high in butt-joint binding capacity, wide in source and easy to obtain, the effective concentration of killing ichthyophthirius multifilis larvae is 5 mg / L, and the compound can be applied to the field of medicine for killing ichthyophthirius multifilis larvae. The insecticidal effect is good.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for killing parasites, and particularly to a drug target for cynatratoside-C against ciliates, its application, and a drug for killing ciliates. Background Art

[0002] Parasitic ciliates are a type of parasites that can parasitize on the surface or inside of aquaculture organisms, causing diseases or deaths of the organisms, and pose great harm to the aquaculture industry. Ichthyophthirius multifiliis belongs to one of the ciliates and mainly parasitizes on the skin, gills, and fins of freshwater fish. White spots form at the parasitic sites, so the fish diseases caused by it are also known as "white spot disease". The white spot disease has a fast onset and a high lethality rate, but there is a lack of safe and efficient drugs globally, causing serious economic losses to the aquaculture industry every year. In 2022, it was listed as a class III animal disease in the announcements of the Ministry of Agriculture and Rural Affairs of China, and there is an urgent need to develop new safe and efficient prevention and control drugs.

[0003] Plant-derived active compounds are an important research object for screening drugs against Ichthyophthirius multifiliis at present, and a large number of natural active ingredients against Ichthyophthirius multifiliis have been disclosed. Among them, cynatratoside-C is the one with the best effect against Ichthyophthirius multifiliis among all the natural active ingredients reported so far. And experiments at different levels of toxicology, pharmacokinetics, and drug thermal degradation kinetics have all shown that cynatratoside-C is a safe and efficient natural active compound for preventing and controlling Ichthyophthirius multifiliis, with great development and application value, but the anti-Ichthyophthirius multifiliis target of cynatratoside-C is still unclear.

[0004] At present, the research on drug targets has received extensive attention. In the process of screening and discovering candidate new drugs, the application of drug targets has played an accelerating role. There are a large number of drugs acting on the same target in nature. As long as a new drug target is found, a batch of candidate drugs with similar effects can be found by using molecular virtual screening technology. Therefore, it is crucial to find the target that can kill Ichthyophthirius multifiliis, screen drugs for killing Ichthyophthirius multifiliis based on this target, accelerate the research and development of safe and efficient drugs against Ichthyophthirius multifiliis, and solve the problem of safe prevention and control of Ichthyophthirius multifiliis disease as soon as possible. Summary of the Invention

[0005] The purpose of the present invention is to provide a drug target for cynatratoside-C against ciliates, its application, and a drug for killing ciliates, to lay a foundation for the research on the molecular mechanism of natural products against Ichthyophthirius multifiliis, and to provide drug targets for screening more new, safe, and efficient anti-insect active ingredients with similar functions.

[0006] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The present invention provides a drug target for erectavoside C against ciliates, and the drug target is the ABCE1 transporter.

[0008] Preferably, the amino acid sequence of the ABCE1 transporter is as shown in SEQ ID NO.1.

[0009] Preferably, the ciliates include Ichthyophthirius multifiliis.

[0010] The present invention also provides the application of the ABCE1 transporter as a drug target in screening or preparing drugs for killing ciliates.

[0011] The present invention also provides a drug for killing ciliates, and the drug for killing ciliates targets the ABCE1 transporter.

[0012] Preferably, the drug for killing ciliates targeting the ABCE1 transporter includes pulchinenoside A3.

[0013] Preferably, the drug for killing ciliates further contains one or more pharmaceutically acceptable carriers or excipients.

[0014] The present invention also provides the application of pulchinenoside A3 in preparing drugs for killing ciliates.

[0015] The beneficial effects of the present invention compared with the prior art are as follows:

[0016] (1) The present invention obtains the potential target protein ABCE1 transporter of erectavoside C acting on Ichthyophthirius multifiliis by the method of small molecule immunoaffinity chromatography, further clarifies the anti-Ichthyophthirius multifiliis mechanism of erectavoside C, lays a foundation for the research on the molecular mechanism of natural products against Ichthyophthirius, and provides a drug target for screening more novel, safe and highly effective anti-insect active ingredients with similar functions, which is of great significance for the development of novel natural drugs against ciliates.

[0017] (2) Based on the ABCE1 transporter target and the TCMSP database, the present invention conducts computer virtual screening for potential drugs against Ichthyophthirius multifiliis, and discovers the natural compound pulchinenoside A3 that can effectively kill Ichthyophthirius. This compound has a strong docking and binding ability with the ABCE1 transporter, is widely sourced and easily obtained, and the effective concentration for killing the larvae of Ichthyophthirius multifiliis is 5 mg / L, with a good insecticidal effect. Description of the Drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 This is the protein detection result obtained from the pull-down experiment in Example 1 of the present invention, where M is the standard molecular weight protein Marker; 1 is the eluate of the control group; 2 is the affinity purification eluate;

[0020] Figure 2 This is the mass spectrometry identification result of ABCE1 in Example 1 of the present invention, where Match To represents the Uniprot accession number and protein name of the matched protein searched, and Matchedpeptides represents the matched sites;

[0021] Figure 3 This is the molecular docking result diagram of ABCE1 and cynanchoside C in Example 1 of the present invention, where A is the pymol visualization of the docking result, and B is the position of the amino acid residues where cynanchoside C binds to the ABCE1 protein;

[0022] Figure 4 This is the result diagram of the root mean square deviation (RMSD) value of the backbone atoms of the ABCE1 protein changing with time in Example 1 of the present invention. The black line represents the ABCE1 protein, and the red line represents the complex of ABCE1 and cynanchoside C;

[0023] Figure 5 This is the distribution diagram of the root mean square fluctuation (RMSF) value of the backbone atoms of the ABCE1 protein in the ABCE1-cynanchoside C complex in Example 1 of the present invention;

[0024] Figure 6 This is the western blotting detection result diagram after the incubation of the AAA domain and the RLI domain with biotin-cynanchoside C in Example 1 of the present invention, where M: standard molecular weight protein Marker; 1 is the recombinant AAA domain protein; 2 is the recombinant RLI domain protein;

[0025] Figure 7 This is the immunolocalization of the ABCE1 protein and biotin-cynanchoside C in the adults of Ichthyophthirius multifiliis in Example 1 of the present invention. Detailed implementation manners

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] In Example 1 of the present invention, the drug target of Ichthyophthirius multifida was screened by small molecule immunoaffinity chromatography, and the specific steps are as follows:

[0033] The host model was grass carp (Ctenopharyngodon idellus), weighing 22.6±3.5g, from Longfu Special Fish Fry Farm, Huadu District, Guangzhou. The parasite model was Ichthyopharyngodon multifleshed, from Huadiwan Flower, Bird, Fish and Insect Market, Guangzhou. The purity of the upright scutellarin C used in the experiment was ≥98%.

[0034] 1. Target screening and identification

[0035] (1) 100 μL streptavidin magnetic beads (Pierce TMStreptavidin Magnetic Beads) were incubated with 200 μL biotin-streptavidin C (800 μM) at room temperature for 30 min with rotation, and the supernatant was discarded. The control group was incubated with 200 μL biotin (800 μM) and repeated 3 times.

[0036] (2) The incubated magnetic beads and Ichthyophthirius punctatus total protein lysate were incubated with rotation at room temperature for 20 min, and the supernatant was discarded. This was repeated three times.

[0037] (3) Elute with 100 μL of 1 M NaCl eluent and collect the eluate for SDS-PAGE electrophoresis.

[0038] (4) The gel after electrophoresis was stained with silver and it was found that the eluate after incubation with biotin-upright scutellaria glycoside C and biotin had a differential band at 50-70 kDa ( Figure 1 ).

[0039] (5) The differential bands were cut out and commissioned to Beijing Liuhe BGI for protein profiling. A total of 4 proteins were detected, of which 1 protein had a molecular weight of 50-70 kDa and was identified as ABCE1 transporter ( Figure 2 ).

[0040] Molecular docking simulation of ABCE1 transporter and scutellaria baicalensis C was performed using LeDock ( Figure 3 ), the results showed that the binding energy of the complex was less than -1.2 kcal / mol, and its binding energy was effective. There were multiple polar hydrogen bonds and non-polar bonds between ABCE1 and upright scutellaria glycoside C, and the binding pocket was completely located in the RNaseL inhibitor domain (RNaseLInhibitor, RLI). The results of dynamic simulation MD, root mean square deviation (RMSD) value and root mean square fluctuation value (RMSF) analysis all showed that the conformation of ABCE1-upright scutellaria glycoside C complex was stable ( Figure 4 , Figure 5 ).

[0041] 2. In vitro binding verification of ABCE1 transporter and biotin-upright scutellaria glycoside C

[0042] (1) Full-length cloning, prokaryotic expression and affinity purification of the ABCE1 transporter to obtain purified recombinant protein:

[0043] 1) The full-length cDNA of ABCE1 gene (1924 bp) was obtained by RACE end cloning technology, and the TAA and TAG codons in the coding region were replaced with CAA and CAG codons.

[0044] (2) Synthesize the nucleotide sequences of the RLI domain (85 - 339 bp) and the ATPase domain (AAA, 378 - 532 bp) of the ABCE1 transporter respectively, and ligate them to the pGEX-4T1 and pET-30a vectors respectively. Use BL21(DE3) as the expression strain to construct recombinant strains.

[0045] (3) Induce the overexpression of the recombinant RLI domain and AAA domain proteins under IPTG induction, and purify them using magnetic beads carrying GST and His tags respectively to obtain purified recombinant proteins RLI and AAA with a purity ≥ 85%.

[0046] (2) Detect the in vitro binding ability of the RLI domain and the AAA domain to biotin-cynanchoside C by Western-blotting:

[0047] (1) Incubate 100 μL of streptavidin magnetic beads with 200 μL of biotin-cynanchoside C (800 μM) by rotation at room temperature for 30 min, then perform magnetic adsorption to discard the supernatant. Repeat 3 times.

[0048] (2) Incubate the purified AAA domain protein and RLI domain protein with the above magnetic beads by rotation at room temperature for 30 min respectively, then place the samples on a magnetic stand, perform magnetic adsorption on the magnetic beads, and discard the supernatant. Repeat three times.

[0049] (3) Add 100 μL of 1 M NaCl elution buffer for elution, collect the elution buffer respectively for SDS-PAGE electrophoresis, and then perform Western-blotting detection using a monoclonal mouse antibody against biotin labeled with HRP.

[0050] The results showed that: A distinct band was detected at approximately 70 kDa in the lane where the recombinant RLI domain protein was located ( Figure 6 ), indicating that the ABCE1 transporter has the in vitro binding ability to biotin-cynanchoside C, and the binding region is the RLI domain.

[0051] 3. Verification of the binding of the ABCE1 transporter to cynanchoside C in adult insects

[0052] (1) Preparation of polyclonal rabbit antibodies against the RLI domain:

[0053] (1) Antigen emulsification: Add PBS to dissolve the purified protein to make the protein solution concentration 1 mg / mL. For the first immunization, mix Freund's complete adjuvant with the same volume of the protein solution, and use a medical three-way valve to fully mix and emulsify the two to form a "water-in-oil" emulsion.

[0054] (2) Immunizing rabbits: ① Primary immunization: After the experimental rabbits had rested quietly for 5 - 7 days, the well-emulsified antigen was used to immunize the rabbits subcutaneously at multiple points on the neck, abdomen, and axilla. The immunization dose at each point did not exceed 100 μL, and the total immunization dose was 1 mg of protein antigen; ② Second immunization: The second immunization was carried out 14 days after the first immunization. The protein antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1, and was repeatedly pushed to be fully emulsified. Then it was used to immunize the rabbits subcutaneously at multiple points on the neck, abdomen, and axilla, with an immunization dose of 0.5 mg; ③ Third immunization: The third immunization of the rabbits was carried out 14 days after the second immunization, with the immunization method and dose the same as the second immunization; ④ One week after each immunization, 3 mL of blood was collected from the marginal ear vein using a blood collection needle. The blood collection tube was left standing at 37 °C for 1 h. After the serum was separated out, it was aliquoted into 1.5 mL centrifuge tubes, 500 μL in each tube, and stored at -80 °C for future use. Finally, through Elisa detection and calculation, the antibody titer of the rabbit serum after the third immunization was above 1:51200, and the antibody specificity was determined to be good by Western-blotting.

[0055] (2) Immunofluorescence co-localization assay to detect the interaction between ABCE1 transporter and biotin-cynanchoside C in Ichthyophthirius multifiliis:

[0056] (1) Drug treatment: Adult worms were treated with 0.25 mg / L biotin-cynanchoside C at room temperature for 30 min, then the supernatant was discarded. The adult worms were fixed with 5% paraformaldehyde for 15 min and washed 3 times with PBS.

[0057] (2) Cell membrane permeability treatment: Treated with 0.5% Triton-100 at room temperature for 15 min and washed 3 times with PBS.

[0058] (3) Blocking of irrelevant proteins: Blocked with 1% BSA at room temperature for 30 min and washed 3 times with PBS.

[0059] (4) Antibody incubation: Add the polyclonal antibody against the RLI domain of ABCE1 transporter diluted 5000 times with 1% BSA, incubate at 37 °C for 1 h, and wash 3 times with PBS. Add the FITC-labeled goat anti-rabbit secondary antibody diluted 5000 times with 1% BSA and the HRP-conjugated streptavidin diluted 5000 times with 1% BSA and incubate together, then observe under an LSM 880 laser confocal microscope, and excite green light and red light with argon laser and helium-neon laser respectively.

[0060] The results showed that the positions of ABCE1 transporter and biotin-erectuside C were observed by a laser confocal fluorescence microscope. The experimental group was the observation result after incubating the adults treated with biotin-erectuside C with a polyclonal antibody against the RLI domain and streptavidin conjugated with HRP; the first control group was the observation result after incubating the adults treated with biotin-erectuside C with rabbit negative serum and streptavidin conjugated with HRP; the second control group was the observation result after incubating the adults treated with biotin with a polyclonal antibody against the RLI domain and streptavidin conjugated with HRP( Figure 7 ). The red fluorescence represents the position of biotin-erectuside C, and the green fluorescence represents the position of the ABCE1 transporter. According to Figure 7 the display, the first control group showed that rabbit negative serum had no binding effect on both the ABCE1 transporter and biotin-erectuside C; the second control group showed that biotin had no binding effect on the ABCE1 transporter; the experimental group showed that the ABCE1 transporter and biotin-erectuside C were co-localized in the adult cells, showing a mixed orange-yellow color of red and green fluorescence. The above results indicated that there was an interaction between the ABCE1 transporter and biotin-erectuside C in the adult cells of Ichthyophthirius multifiliis, and it was the target protein.

[0061] (3) Indirect Elisa was used to detect the binding ability of biotin-erectuside C and erectuside C to the ABCE1 transporter:

[0062] 1) Optimal coating concentration of ABCE1: Using streptavidin conjugated with HRP and taking biotin-erectuside C as the antigen, the optimal coating concentration of ABCE1 was detected by Elisa. The purified RLI domain of the ABCE1 transporter was diluted to different concentrations with coating buffer, and the coating buffer without protein was used as a control. The RLI coating concentration when significant differences in OD 450 nm values appeared was 0.5 mg / L.

[0063] 2) Optimal coating concentration of biotin-erectuside C: Biotin-erectuside C was diluted to different concentrations with ultrapure water and incubated with 0.5 mg / L of RLI. The concentration of biotin-erectuside C when significant differences in OD 450 nm values appeared was 1 mg / L.

[0064] 3) Using the biotin antigen group as the negative control and the biotin-erectuside C antigen group as the experimental group, they were respectively mixed and incubated with erectuside C. 13 groups were set up, with three replicates in each group, and the OD 450nm Values. The concentrations of biotin-cynanchoside C and cynanchoside C in each group and OD 450 nm As shown in Table 1.

[0065] Table 1 Competitive binding of biotin-cynanchoside C and cynanchoside C detected by indirect ELISA

[0066]

[0067]

[0068] The results in Table 1 show that when biotin-cynanchoside C and cynanchoside C are co-incubated with the coated RLI domain of ABCE1 transporter, since both biotin-cynanchoside C and cynanchoside C can bind to ABCE1, they compete for the limited binding sites of ABCE1. When the concentration of cynanchoside C competing with biotin-cynanchoside C is higher, the chance of biotin-cynanchoside C binding to ABCE1 protein is less, and the proportion of ABCE1 binding to cynanchoside C is higher, and the OD 450nm value is lower. When the added concentration of cynanchoside C is 0.04 mg / L, cynanchoside C competes with biotin-cynanchoside C for the binding site of ABCE1, and the OD 450nm value decreases significantly. As the added concentration of cynanchoside C increases, the OD 450nm value becomes smaller, and there is no significant statistical difference in the OD 450nm values between the biotin-only group and the blank treatment group, indicating that ABCE1 does not bind to biotin but specifically binds to cynanchoside C, and ABCE1 protein is the target protein of cynanchoside C in the adults of Ichthyophthirius multifiliis.

[0069] Example 2

[0070] In Example 2 of the present invention, virtual screening of traditional Chinese medicine chemical components in the TCMSP database was carried out with ABCE1 as the target, and the anti-Ichthyophthirius multifiliis effect was tested. The specific steps are as follows:

[0071] 1. Using computer virtual screening technology to mine potential Chinese herbal medicine inhibitor molecules of ABCE1 transporter:

[0072] Taking the active pocket of cynanchum atratum Bunge C and ABCE1 transporter as the binding site, high-throughput screening of all compounds in the TCMSP database was completed through the Autodock Vina molecular docking program. Secondary screening was carried out by combining docking scores and the "ADME-Lipinski" rules, and then according to the interaction with ABCE1 transporter, potential small molecule compounds against Ichthyophthirius multifiliis were obtained.

[0073] The results showed that: targeting the ABCE1 transporter and using erectin C as the positive control, molecular docking was performed with all compounds in the TCMSP database. Compounds with the top 200 docking binding energies were selected and further screened by the "ADME-Lipinski" rule to obtain 10 compounds with potential anti-parasitic ciliate drug value. Among them, pulchinenoside A3 (MOL001989), smilax china (MOL003894), isohydnocarpic acid (MOL005555), and glycerol (MOL002600) were selected as references to detect the effect of these four compounds on killing Ichthyophthirius multifiliis. Their docking binding energies with the ABCE1 transporter were -9.6, -9.7, -10, and -11.1 kJ / mol respectively, and their affinity and interaction groups were superior to those of the original ligand erectin C.

[0074] The results showed that at 500 mg / L, all of the above four compounds could kill 100% of the adult Ichthyophthirius multifiliis within 4 h, and pulchinenoside A3 could still kill 100% of the adults when the concentration was reduced to 50 mg / L. Considering the economic cost and development value, pulchinenoside A3 was selected to further study its anti-Ichthyophthirius multifiliis drug effect.

[0075] 2. Killing effect of pulchinenoside A3 on infectious larvae of Ichthyophthirius multifiliis

[0076] (1) Preparation of pulchinenoside A3 drug: Weigh 0.002 g of pulchinenoside A3 drug powder with an electronic balance accurate to one hundred-thousandth. After dissolving it with 40 μL of ethanol, add 1960 μL of ultrapure water and mix well by pipetting along the wall to obtain a 1st-grade stock solution with a concentration of 1000 mg / L. Take 100 μL from the 1st-grade stock solution and add 900 μL of ultrapure water, then mix well by pipetting along the wall to prepare a 2nd-grade stock solution with a concentration of 100 mg / L. Subsequently, use the two-fold dilution method to obtain drug solutions with concentrations of 20, 10, 5, and 2.5 mg / L.

[0077] (2) Add 200 μL of an aqueous solution containing approximately 300 larvae of Ichthyophthirius multifiliis to a 96-well plate, and then add 200 μL of the above-prepared drug solutions with different concentrations. The control group only adds 200 μL of ultrapure water. Make the final drug solution concentrations be 10, 5, 2.5, 1.25, and 0 (control) mg / L in sequence. Set 3 parallels for each group and place them at room temperature (23 °C). Observe through the 10× objective lens of an optical microscope and record the time required for all larvae of Ichthyophthirius multifiliis in each group to die.

[0078] The results showed that the Pulsatilla saponin A3 solution with a concentration of 5 mg / L could completely kill the infective larvae of Ichthyophthirius multifiliis within 187.7 ± 13.1 min, and the Pulsatilla saponin A3 solution with a concentration of 10 mg / L could completely kill the infective larvae of Ichthyophthirius multifiliis within 118.7 ± 3.2 min. As the concentration increased, the time to kill the larvae became shorter (Table 2).

[0079] Table 2 Effect of Pulsatilla saponin A3 on killing the larvae of Ichthyophthirius multifiliis

[0080]

[0081]

[0082] 3. Killing effect of Pulsatilla saponin A3 on the cysts of Ichthyophthirius multifiliis

[0083] (1) The preparation method of the Pulsatilla saponin A3 solution was the same as in 2. The two-fold dilution method was used to finally obtain solutions with concentrations of 40, 20, 10, 5, and 2.5 mg / L.

[0084] (2) 500 μL of water containing approximately 30 cysts of Ichthyophthirius multifiliis was added to each well of a 24-well plate, and then 500 μL of the solution prepared in step (1) was added to the wells. As a control, only 500 μL of ultrapure water was added without the drug. The final concentrations of the solutions were 20, 10, 5, 2.5, 1.25, and 0 (control) mg / L. Three parallels were set for each group and placed at room temperature (23°C). Observation was carried out under a 4-fold magnification of an optical microscope, and the hatching rate of the cysts after 18 h was recorded, and then the average number of larvae hatched from each cyst was counted.

[0085] The results showed that the Pulsatilla saponin A3 solution with a concentration of 10 mg / L could kill 100% of the cysts of Ichthyophthirius multifiliis, completely preventing the hatching and release of the larvae from the cysts (Table 3). A concentration of 5 mg / L could significantly reduce the average number of larvae released from each surviving cyst.

[0086] Table 3 Effect of Pulsatilla saponin A3 on killing the cysts of Ichthyophthirius multifiliis

[0087]

[0088] 4. Killing effect of Pulsatilla saponin A3 on the adult Ichthyophthirius multifiliis

[0089] (1) The preparation method of the Pulsatilla saponin A3 solution was the same as in 2. The two-fold dilution method was used to obtain solutions with final concentrations of 40, 20, 10, 5, and 2.5 mg / L.

[0090] (2) Add 500 μL of water containing approximately 200 adult Ichthyophthirius multifiliis to each well of a 24-well plate, and then add 500 μL of the medicinal liquid prepared in step (1) to the wells. For the control group, add only 500 μL of ultrapure water without adding medicine. Make the final concentrations of the medicinal liquid be 20, 10, 5, 2.5, 1.25, and 0 (control) mg / L respectively. Set 3 parallels for each group and place them at room temperature (23 °C). Observe under a 4× objective lens of an optical microscope, record the time required for all adult Ichthyophthirius multifiliis in each group to die, record the formation of cysts in each group and the hatching rate at 18 h, and finally count the average number of larvae hatched from each cyst.

[0091] The results showed that the saponin A3 of Pulsatilla chinensis medicinal liquid at the concentrations of 20 and 10 mg / L could kill 100% of the adult Ichthyophthirius multifiliis within 212.3 ± 2.5 min and 238.7 ± 1.2 min respectively. Although the drug at the concentration of 5 mg / L did not completely kill the adults within 5 h and the killing rate was 97.0 ± 2.2%, it could completely prevent the larvae in the remaining cysts from hatching and releasing (Table 4).

[0092] Table 4 Effect of saponin A3 of Pulsatilla chinensis on killing adult Ichthyophthirius multifiliis

[0093]

[0094] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A drug target of scutellaria aviculare C against ciliates, characterized in that: The drug target is ABCE1 transporter protein.

2. The drug target of upright scutellaria glycoside C against ciliates according to claim 1, characterized in that: The amino acid sequence of the ABCE1 transporter is shown in SEQ ID NO.

1.

3. The drug target of the anti-ciliate glycoside C of claim 1, characterized in that: The ciliates include Ichthyophthirius multifida.

4. Application of ABCE1 transporter protein as a drug target in screening or preparing drugs for killing ciliates.

5. A drug for killing ciliates, characterized in that: The drug for killing ciliates takes the ABCE1 transporter as a target.

6. The drug for killing ciliates according to claim 5, characterized in that: Drugs that target the ABCE1 transporter and kill ciliates include pulsatilla saponin A3.

7. The drug for killing ciliates according to claim 5, characterized in that: The ciliate-killing drug also contains one or more pharmaceutically acceptable carriers or excipients.

8. Application of Pulsatilla saponin A3 in the preparation of drugs for killing ciliates.

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