Use of transporter protein tspos and agonists, inhibitors thereof in n. sphaerica related drugs

Abstract

The application discloses application of a transport protein TSPO, a nucleic acid or gene coding the transport protein TSPO, a recombinant vector or a recombinant strain thereof in affecting multiplication of a microsporidium of Bombyx mori. By adding the recombinant vector or an agonist of the transport protein TSPO in a medicine, multiplication of the microsporidium of Bombyx mori can be effectively inhibited, after adding an overexpression vector of the transport protein TSPO gene in ovary cells of the Bombyx mori, the genome copy number of the microsporidium of Bombyx mori can be reduced by about 80%; multiplication of the microsporidium can also be promoted by adding siRNA or an inhibitor of the transport protein TSPO in the medicine, and the transport protein TSPO can be used as a synergist of a microsporidium pesticide.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and particularly to the use of a transporter protein TSPO and its agonists and inhibitors in the preparation of drugs related to silkworm microsporidia. Background Technology

[0002] Microsporidia ( Microsporidia Microsporidia muscarinii are obligate intracellular parasitic unicellular eukaryotes with a wide host range, capable of infecting almost all animals from vertebrates to invertebrates. Microsporidia muscarinii is an example of this. Nosema bombycis Microsporidiasis, caused by microsporidia, is one of the major diseases of silkworms. The pathogen infects silkworms through both oral and embryo-seed transmission, spreading throughout the host and causing damage to most tissues and organs, including the midgut, silk glands, and Malpighian tubules, ultimately leading to host death. This results in significant economic losses for the sericulture industry. Furthermore, due to its embryo-seed transmission characteristic, it is currently the only legally mandated quarantine object in silkworm seed production. During evolution, some organelles of microsporidia have undergone deformation and degeneration. For example, mitochondria in silkworm microsporidia have changed into a two-membrane spindle-shaped remnant, retaining only partial mitochondrial function. Therefore, they need to utilize energy and substances produced by the host's mitochondria to meet their own needs. Through proteomics, comparative genomics, and transcriptome sequencing, studies have revealed a complex interaction between silkworm microsporidia and the silkworm. Infection with silkworm microsporidia induces a series of changes in silkworm energy metabolism, amino acid metabolism, immune response, and apoptosis.

[0003] Transporter protein 18kDa ( Translocator protein 18kDa, TSPO 18kDa TSPO is a conserved protein located on the outer mitochondrial membrane, closely related to the regulation of mitochondrial homeostasis and involved in various physiological functions such as apoptosis, immune response, cell growth and proliferation, damage and aging, and cholesterol synthesis. Given the dependence of silkworm microsporidia on the silkworm and the important function of the transport protein TSPO in mitochondria, it is a worthwhile direction to explore the use of TSPO to inversely regulate host mitochondrial function and thereby inhibit the replication of silkworm microsporidia in the host. Therefore, studying the inhibitory effect of the transport protein TSPO on silkworm microsporidia and developing anti-silkworm microsporidia drugs using it as a target is of great significance. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of a transport protein TSPO, a nucleic acid or gene encoding the transport protein TSPO, and its recombinant vector or recombinant strain in influencing the proliferation of silkworm microsporidia.

[0005] Another technical problem to be solved by the present invention is to provide an application of the transporter protein TSPO in the preparation of drugs against silkworm microsporidia.

[0006] Another technical problem to be solved by the present invention is to provide an application of nucleic acid or gene in the preparation of anti-bombyx mori microsporidia drugs.

[0007] Another technical problem to be solved by the present invention is to provide an expression vector or recombinant bacteria for the preparation of drugs against silkworm microsporidia.

[0008] Another technical problem to be solved by the present invention is to provide a method for inhibiting the infection of silkworm microsporidia in vitro.

[0009] Another technical problem to be solved by the present invention is to provide a method or substance for reducing the expression level or activity of the transport protein TSPO and its application in drugs that promote the proliferation of silkworm microsporidia.

[0010] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the application of transport protein TSPO, nucleic acid or gene encoding transport protein TSPO, and recombinant vector or recombinant strain in influencing the proliferation of silkworm microsporidia.

[0011] This invention discovers that when silkworm larvae are infected with silkworm microsporidia, the expression of the transport protein TSPO is upregulated, and the upregulated TSPO can inhibit the replication of silkworm microsporidia. Therefore, the transport protein TSPO, its gene, recombinant vector or recombinant strain can be used in applications related to the proliferation of silkworm microsporidia.

[0012] This invention also provides the application of the transporter protein TSPO in the preparation of an anti-bombyx mori microsporidium drug, the drug containing the transporter protein TSPO and / or its agonist, the amino acid sequence of the transporter protein TSPO being shown in SEQ ID NO: 2.

[0013] Furthermore, the agonist is FGIN-1-27.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients or carriers.

[0015] The present invention also includes the application of a nucleic acid or gene in an anti-bombyx mori microsporidia drug, wherein the drug contains a gene encoded by the transporter protein TSPO, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0016] The present invention also includes the application of an expression vector or recombinant bacteria in the preparation of a drug against silkworm microsporidia, wherein the expression vector or recombinant bacteria contains nucleic acid or gene as shown in SEQ ID NO: 1.

[0017] The present invention also includes a method for inhibiting the in vitro cell infection of silkworm microsporidia, employing the method shown in (a) or / and (b) below: (a) First, the BmTSPO gene is amplified and cloned into a plasmid to construct an overexpression plasmid; the overexpression plasmid is transfected into cells to express BmTSPO in the cells; (b) Treat silkworm cells with exogenous small molecule BmTSPO agonist.

[0018] Furthermore, the plasmid may be, but is not limited to, piZ / V5-His-mcherry.

[0019] Furthermore, the transfected cells may be, but are not limited to, silkworm BmN ovarian cells.

[0020] Furthermore, the agonist is FGIN-1-27.

[0021] Furthermore, the agonist is FGIN-1-27, and the treatment concentration of FGIN-1-27 is 1-100 μM.

[0022] The present invention also includes a method or substance for reducing the expression level or activity of the transport protein TSPO in the application of a drug for promoting the proliferation of silkworm microsporidia, wherein the method for reducing the expression level of the transport protein TSPO is knocking down the transport protein TSPO gene, and the substance for reducing the activity of the transport protein TSPO is a transport protein TSPO inhibitor.

[0023] Furthermore, the method for knocking down the TSPO transporter gene involves transfecting double-stranded interfering RNA into cells using liposome Lipo8000.

[0024] Furthermore, the double-stranded interfering RNA sequence is shown below: siRNA1-F:5'-GCAAUUCCCUUUGACUCUUTT-3' (SEQ ID No: 9) siRNA1-R:5'-AAGAGUCAAAGGGAAUUGCTT-3' (SEQ ID No: 10) siRNA2-F:5'-GCUGCGCAAUAAGCUUCUATT-3' (SEQ ID No: 11) siRNA2-R:5'-UAGAAGCUUAUUGCGCAGCTT-3' (SEQ ID No: 12) siRNA3-F:5'-GCUACCUGCCUAACUGCUATT-3'(SEQ ID No: 13) siRNA3-R:5'-UAGCAGUUAGGCAGGUAGCTT-3' (SEQ ID No: 14) Furthermore, the TSPO transporter inhibitor is PK11195.

[0025] Furthermore, the treatment concentration of PK11195 is 1-100 μM.

[0026] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The present invention provides the application of the transport protein TSPO, its gene, recombinant vector, or recombinant strain in influencing the proliferation of silkworm microsporidia. By adding the transport protein TSPO to the drug, its recombinant vector or its agonist can effectively inhibit the proliferation of silkworm microsporidia. After adding an overexpression vector of the transport protein TSPO gene into silkworm ovarian cells, the copy number of the silkworm microsporidia genome can be reduced by about 80%. Alternatively, the proliferation of microsporidia can be promoted by adding siRNA or inhibitors of the transport protein TSPO to the drug, serving as a synergist for microsporidia pesticides. Attached Figure Description

[0027] Figure 1 The graph shows the changes in BmTSPO expression levels in the midgut of silkworms at different time points after infection with *Microsporidium*. Figure 2 The images show the results of inhibiting the proliferation of silkworm microsporidia after overexpressing BmTSPO in silkworm BmN cells. Figure A is a fluorescence microscope image of BmN cells 48 hours after transfection, Figure B is the quantitative PCR detection result of BmTSPO expression 48 hours after transfection, and Figure C is the quantitative PCR detection result of the silkworm microsporidia genome copy number 72 hours after transfection in BmN cells. Figure 3 The image shows the results of quantitative PCR detection of the genome copy number of silkworm microsporidia after incubation of silkworm BmN cells with different concentrations of FGIN-1-27. DMSO is the negative control group, FG1 is 1 μM, FG10 is 10 μM, FG50 is 50 μM, and FG100 is 100 μM. Figure 4 Figure A shows the results of quantitative PCR detection of BmTSPO expression after siRNA transfection and Figure B shows the results of quantitative PCR detection of the genome copy number of silkworm microsporidia after siRNA transfection. Figure 5The image shows the results of quantitative PCR detection of the genome copy number of silkworm microsporidia after incubation of BmN cells with different concentrations of PK11195. DMSO is the negative control group, PK1 is 1 μM, PK10 is 10 μM, PK50 is 50 μM, and PK100 is 100 μM. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0029] Materials: P50 silkworms from the fifth instar onwards (preserved and provided by the Silkworm Physiology and Pathology Research Laboratory of the Sericulture Research Institute, Chinese Academy of Agricultural Sciences), silkworm microsporidia spore suspension (10... 9 (pcs / mL) Reagents: RNAiso plus kit (R401-01), ChamQ SYBR qPCR Master Mix (High ROX Premixed) kit (Q341-02), HiScript Q RT SuperMix for qPCR (+gDNA wiper) kit (R123-01), all purchased from Nanjing Novizan Biotechnology Co., Ltd.; FGIN-1-27 activator (T22782), PK11195 inhibitor (T29258), all purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0030] Example 1: Changes in BmTSPO expression in silkworms infected with *Bombyx mori* Take the silkworm microsporidia spore suspension purified by the Percoll method (10 9 (each cell / mL), diluted with double-distilled water to 10. 7 The sample was prepared by soaking fresh mulberry leaves for 10 minutes, drying them, and then feeding 90 P50 silkworms (5th instar) to the feed. The midgut of the silkworms was collected at 0h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, and 180h. The midgut of 3 silkworms was used as one tube, and the collection was repeated for 3 tubes.

[0031] Total RNA was extracted from samples at different time points using the RNAiso plus kit. cDNA was obtained using the HiScript QRT SuperMix for qPCR reverse transcription kit. The expression of BmTSPO was quantitatively detected using the ChamQ SYBR qPCR Master Mix kit, with the silkworm GAPDH gene as an internal control. The quantitative PCR primers used are as follows: qPCR-BmTSPO-F: 5'-CCTGACGGAGACTCCTGGTA-3' (SEQ ID NO: 3) qPCR-BmTSPO-R: 5'-AGCCTCCACCGTCTCTGTAT-3' (SEQ ID NO: 4) The results are as follows Figure 1 As shown, 12 hours after infection with *Bombyx mori*, the expression level of BmTSPO began to increase significantly, increasing 1.65 times compared to normal silkworms from the 5th instar. With increasing time, the infection of *Bombyx mori* in silkworms intensified, and the expression level of BmTSPO gradually increased, peaking at 144 hours. It decreased slightly at 168 and 180 hours, but remained above normal levels. These results indicate that the transcriptional level of BmTSPO was significantly upregulated after infection with *Bombyx mori*.

[0032] Example 2: Method for inhibiting the infection of silkworm microsporidia in in vitro cells The gene sequence encoding BmTSPO was obtained by PCR amplification in silkworm ovary cells (BmN cells, preserved and provided by the Silkworm Physiology and Pathology Research Laboratory of the Sericulture Institute, Chinese Academy of Agricultural Sciences). Its nucleotide sequence is shown in SEQ ID NO: 1. BmTSPO was cloned into the piZ / V5-His-mcherry vector (preserved and provided by the Silkworm Physiology and Pathology Research Laboratory of the Sericulture Institute, Chinese Academy of Agricultural Sciences) using EcoRI and XhoI restriction enzymes, resulting in the mcherry-TSPO fusion expression plasmid pIZV5-BmTSPO-mcherry. Primers are as follows: pIZV5-BmTSPO-mcherry-F:5'-CGGGATCCatgagagtctgggtcttaatagt-3' (SEQ IDNO: 5) pIZV5-BmTSPO-mcherry-R: 5'-CCG CTC GAGTttaattaagatgccaaagagta-3' (SEQ ID NO: 6) BmN cells were cultured overnight in 12-well plates, and then transfected into BmN cells with 1 μg pIZV5-BmTSPO-mcherry plasmid according to the Entranster™-H4000 transfection reagent instructions.

[0033] Forty-eight hours after transfection, germinated silkworm microsporidia spores treated with 0.1 M KOH were added at a spore-to-cell ratio of 5:1. Samples were collected after 72 hours. DNA and RNA were extracted from the samples using a DNA / RNA Isolation Kit. cDNA was synthesized using PrimeScript™ RT Master Mix (Perfect Real Time). The change in the relative copy number of Nbβ-tubulin was detected using a real-time quantitative PCR instrument (ABI 7300). Primers are as follows: qPCR-Nbβ- tubulin -F:5'-TTCCCTTCCCTAGACTTCACTTC-3' (SEQ ID NO:7) qPCR-Nbβ- tubulin -R: 5'-CAGCAGCCACAGTCAAATACC-3' (SEQ ID NO: 8) The results are as follows Figure 2 As shown, the fusion expression of BmTSPO with the red fluorescent protein mcherry was successfully achieved in BmN cells using the piZ / V5-His-mcherry vector. Figure 2 A. Quantitative PCR analysis revealed that compared to the control group (piZ / V5-His-mcherry empty plasmid), the expression level of BmTSPO in the pIZV5-BmTSPO-mcherry transfected group was significantly increased. Figure 2 B. Quantitative PCR was used to detect the relative copy number of Nbβ-tubulin from *Bombyx mori* microsporidia* in total DNA. The results showed that, compared to the control group, BmN cells transfected with the BmTSPO gene overexpression plasmid exhibited an approximately 80% reduction in the relative copy number of Nbβ-tubulin in *Bombyx mori* microsporidia after infection. Figure 2 C. The results showed that BmTSPO overexpression could inhibit the proliferation of silkworm microsporidia.

[0034] Example 3: Method for inhibiting the infection of silkworm microsporidia in in vitro cells by the TSPO agonist FGIN-1-27 FGIN-1-27 powder was dissolved in DMSO to prepare a 1M stock solution. When incubating BmN cells, the stock solution was added to the BmN cell culture medium (TC-100 insect culture medium, prepared by adding 0.1% penicillin-streptomycin mixture and 10% fetal calf serum), and the final concentrations of FGIN-1-27 in the culture medium were adjusted to 1 μM, 10 μM, 50 μM, and 100 μM, respectively; DMSO incubation of BmN cells served as a negative control. After 48 h of incubation, budding spores of *Bombyx mori* treated with 0.1 M KOH were added at a spore-to-cell ratio of 5:1. Samples were collected after 72 h, and DNA and RNA were extracted using a DNA / RNA Isolation Kit. cDNA was synthesized using HiScript QRT SuperMix for qPCR. Changes in the relative copy number of Nbβ-tubulin were detected using a real-time quantitative PCR instrument (ABI 7300).

[0035] The results are as follows Figure 3 As shown, after treating BmN cells with the agonist FGIN-1-27, the copy number of the microsporidia genome in BmN cells infected with silkworms was significantly reduced 72 hours after infection compared with the control group. This demonstrates that the agonist FGIN-1-27 can inhibit the replication of silkworm microsporidia in BmN cells, and also indicates that BmTSPO has an antimicrosporidia effect.

[0036] Example 4: Knockdown of BmTSPO promotes the proliferation of silkworm microsporidia. Three siRNAs (siRNA1, siRNA2, and siRNA3) for TSPO were designed and synthesized, with the following interference sequences: siRNA1-F:5'-GCAAUUCCCUUUGACUCUUTT-3' (SEQ ID No: 9) siRNA1-R:5'-AAGAGUCAAAGGGAAUUGCTT-3' (SEQ ID No: 10) siRNA2-F:5'-GCUGCGCAAUAAGCUUCUATT-3' (SEQ ID No: 11) siRNA2-R:5'-UAGAAGCUUAUUGCGCAGCTT-3' (SEQ ID No: 12) siRNA3-F:5'-GCUACCUGCCUAACUGCUATT-3'(SEQ ID No: 13) siRNA3-R:5'-UAGCAGUUAGGCAGGUAGCTT-3' (SEQ ID No: 14) Three pairs of double-stranded interfering RNAs (DRNAs) were transfected into BmN cells using liposome Lipo8000. After 48 h, the transcriptional level of the BmTSPO gene was detected by quantitative PCR to analyze the interference effect of the DRNAs. Quantitative results showed that, compared with the control group, the relative transcriptional level of the BmTSPO gene was significantly downregulated in all three experimental groups, indicating that the three designed DRNAs could interfere with the BmTSPO gene with significant effects. Figure 4 A. Forty-eight hours after siRNA transfection, budding spores of *Bombyx mori* treated with 0.1% MKOH were added, with a spore-to-cell ratio of 5:1. Seventy-two hours after infection, the relative genome copy number of *Bombyx mori* was detected by quantitative PCR. The results showed that BmTSPO knockdown significantly increased the genome copy number of *Bombyx mori*, such as... Figure 4 B indicates that knocking down BmTSPO can promote the proliferation of microsporidia.

[0037] Example 5: TSPO inhibitor PK11195 promotes the proliferation of silkworm microsporidia. PK11195 powder was dissolved in DMSO to prepare a 1M stock solution. This stock solution was added to the BmN cell culture medium (TC-100 insect culture medium, prepared by adding 0.1% penicillin-streptomycin mixture and 10% fetal calf serum) to achieve final PK11195 concentrations of 1 μM, 10 μM, 50 μM, and 100 μM, respectively. DMSO incubation of BmN cells served as a negative control. Samples were collected after 72 h of incubation, and DNA and RNA were extracted using a DNA / RNA Isolation Kit. cDNA was synthesized using HiScript QRT SuperMix for qPCR. Changes in the relative copy number of Nbβ-tubulin were detected using a real-time quantitative PCR instrument (ABI 7300).

[0038] The results are as follows Figure 5 As shown, after treating BmN cells with the inhibitor PK11195, the copy number of the microsporidia genome in BmN cells 72 hours after infection was significantly increased compared with the control group. It was found that the inhibitor PK11195 can promote the replication of silkworm microsporidia in BmN cells, which also indicates that BmTSPO has an anti-microsporidia effect. At the same time, it shows that the small molecule PK11195 can be used as a synergist in the development of microsporidia pesticides.

Claims

1. Use of overexpression of the product of the transporter protein BmTSPO gene or a method for inhibiting the proliferation of Nosema bombycis, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that: The product is a recombinant vector or recombinant bacterial strain.

3. The application of the transport protein BmTSPO agonist in the preparation of drugs against silkworm microsporidia, characterized in that: The agonist is FGIN-1-27.

4. The application of the transporter protein BmTSPO agonist according to claim 3 in the preparation of drugs against silkworm microsporidia, characterized in that: The drug also includes pharmaceutically acceptable excipients or carriers.

5. The application of a product overexpressing the transporter protein BmTSPO gene in the preparation of a drug to inhibit the proliferation of silkworm microsporidia, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

6. The application according to claim 5, characterized in that, The product is a recombinant vector or recombinant bacterial strain.

7. A method for inhibiting the in vitro cell infection of silkworm microsporidia, characterized in that: Use the methods shown in (a) and / or (b) below: (a) First, the BmTSPO gene was amplified, and the nucleotide sequence of the gene is shown in SEQ ID NO: 1; it was cloned into a plasmid to construct an overexpression plasmid; the overexpression plasmid was transfected into cells to express BmTSPO in the cells; (b) Treat silkworm cells with an exogenous small molecule BmTSPO agonist, wherein the agonist is FGIN-1-27.

8. The method for inhibiting the in vitro cell infection of silkworm microsporidia according to claim 7, characterized in that: The treatment concentration of FGIN-1-27 is 1-100 μM.

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