Use of cholesterol-25-hydroxylase and its enzymatic product in the preparation of drugs against Bombyx mori nuclear polyhedrosis virus

Abstract

The present application belongs to the field of agricultural biology, and relates to the use of Bombyx mori cholesterol-25-hydroxylase and its enzymatic product in the preparation of drugs for resisting Bombyx mori nuclear polyhedrosis virus. The present application uses Bombyx mori nuclear polyhedrosis virus (BmNPV) to infect cells and Bombyx mori larvae to cause the expression of cholesterol-25-hydroxylase (BmCH25H) to be up-regulated, and the up-regulated BmCH25H can inhibit the replication of BmNPV, and it is found that the enzymatic product 25-hydroxycholesterol (25HC) of BmCH25H also has an inhibitory effect on the infection of BmNPV. The present application first proves that overexpression of BmCH25H and 25HC can both inhibit the infection of BmNPV through in vitro and in vivo experiments.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural biology, and particularly relates to the use of cholesterol-25-hydroxylase and its enzymatic product in the preparation of drugs against Bombyx mori nucleopolyhedrovirus. BACKGROUND

[0002] Cholesterol-25-hydroxylase (CH25H) is a 31.6 kDa multi-transmembrane endoplasmic reticulum (ER) associated enzyme, which mainly functions to catalyze the production of 25-hydroxycholesterol (25HC) from cholesterol, reducing the accumulation of cholesterol in the body. Studies have found that CH25H is a conservative interferon-stimulated gene (ISG) in mammals, with a broad spectrum of antiviral functions. CH25H mainly exerts an antiviral effect by converting cholesterol into 25HC through its hydroxylase activity, and 25HC is an endogenous hydroxysterol that maintains cholesterol homeostasis by inhibiting sterol regulatory element-binding proteins (SREBPs) and activating liver X receptors (LXRs). In addition, 25HC has also been shown to inhibit the replication of a variety of viruses, such as hepatitis C virus (HCV), murine herpesvirus 68 (MHV68), human immunodeficiency virus (HIV), Zika virus (ZIKV), and vesicular stomatitis virus (VSV).

[0003] Bombyx mori nucleopolyhedrovirus (BmNPV), a baculovirus, belongs to the genus nucleopolyhedrovirus of the family Baculoviridae, and its virions exhibit a rod-like morphology. It is one of the most well-studied baculovirus model species. Bombyx mori, as a lepidopteran insect with high economic benefits, is an important component of China's agricultural economy. Bombyx mori is susceptible to BmNPV, and BmNPV is the most serious virus in the production of Bombyx mori. After infection with BmNPV, Bombyx mori exhibits restlessness, crawls on the cocoon, exhibits a "upward movement", and shows a white body color, leading to the occurrence of blood-type pyemia, resulting in the death of Bombyx mori, and seriously affecting the yield and quality of cocoon, causing huge economic losses to the sericulture industry.

[0004] Therefore, it is of great significance to study the antiviral effect of cholesterol-25-hydroxylase (CH25H) and its enzymatic product 25-hydroxycholesterol (25HC) on Bombyx mori nucleopolyhedrovirus, and to use them to prepare drugs against Bombyx mori nucleopolyhedrovirus. SUMMARY

[0005] To solve the above problems, the present application aims to provide the application of cholesterol-25-hydroxylase (CH25H) or its enzymatic product 25-hydroxycholesterol (25HC) in the preparation of an anti-Bombyx mori nucleopolyhedrovirus drug, to provide a Bombyx mori nucleopolyhedrovirus inhibitor, and to provide a method for inhibiting BmNPV infection in Bombyx mori in vitro cells. The present application proves that cholesterol-25-hydroxylase (CH25H) and its enzymatic product 25-hydroxycholesterol (25HC) have good inhibitory effect on BmNPV through experiments.

[0006] The specific scheme adopted by the present application is as follows:

[0007] In the first aspect, the present application provides the application of any of the following in the preparation of an anti-Bombyx mori nucleopolyhedrovirus drug: a) cholesterol-25-hydroxylase;

[0008] b) a gene encoding cholesterol-25-hydroxylase;

[0009] c) a recombinant plasmid comprising b);

[0010] d) a recombinant bacteria comprising b);

[0011] e) the enzymatic product 25-hydroxycholesterol of cholesterol-25-hydroxylase.

[0012] In the second aspect, the present application provides a Bombyx mori nucleopolyhedrovirus inhibitor comprising cholesterol-25-hydroxylase or / and 25-hydroxycholesterol. Preferably, the inhibitor further comprises a pharmaceutically acceptable excipient or carrier.

[0013] In the third aspect, the present application provides a method for inhibiting Bombyx mori nucleopolyhedrovirus infection in Bombyx mori in vitro cells, which adopts the method shown in (a) or / and (b) as follows:

[0014] (a) first amplify the gene encoding BmCH25H, clone it into pIEX1 plasmid to construct an overexpression plasmid pIEX1-BmCH25H-V5 with a tag sequence, and then transfect BmN cells with the overexpression plasmid pIEX1-BmCH25H-V5 to induce the expression of BmCH25H in BmN cells;

[0015] (b) add exogenous 25-hydroxycholesterol to treat Bombyx mori in vitro cells.

[0016] Preferably, the Bombyx mori in vitro cells are Bombyx mori ovary cells, and the treatment concentration of the exogenous 25-hydroxycholesterol is 10 μM, and the treatment time is 24 h.

[0017] Beneficial effects: The present application uses silkworm nuclear polyhedrosis virus (BmNPV) to infect cells and silkworm larvae to cause the expression of cholesterol-25-hydroxylase (BmCH25H) to be up-regulated, and the up-regulated BmCH25H can inhibit the replication of BmNPV, and it is found that the enzymatic product 25-hydroxycholesterol (25HC) of BmCH25H also has an inhibitory effect on the infection of BmNPV. The present application first proves that overexpression of BmCH25H and 25HC can inhibit the infection of BmNPV through in vitro and in vivo experiments. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a result graph of the expression of BmCH25H caused by BmNPV infection of cells and silkworm larvae; wherein the expression of BmCH25H is significantly up-regulated after BmNPV infection of silkworm BmN cells for 24 hours (A); the expression of BmCH25H in the midgut tissue of silkworm is significantly up-regulated after BmNPV infection for 24 and 48 hours (B); the expression of BmCH25H in the hemocyte of silkworm is significantly up-regulated after BmNPV infection for 24 hours (C); the expression of BmCH25H in the fat body tissue of silkworm is significantly up-regulated after BmNPV infection for 24 hours (D).

[0019] Figure 2 is a result graph that overexpression of BmCH25H in silkworm BmN cells can inhibit the infection of BmNPV; wherein overexpression of BmCH25H and eGFP is successfully realized in silkworm BmN cells (A); real-time fluorescent quantitative PCR detection finds that overexpression of BmCH25H can inhibit the infection of BmNPV (B); virus titer detection finds that overexpression of BmCH25H can inhibit the infection of BmNPV (C).

[0020] Figure 3 is a result graph that addition of exogenous 25HC in silkworm BmN cells can inhibit the infection of BmNPV; wherein the concentration exploration of 25HC added in silkworm BmN cells (A); real-time fluorescent quantitative PCR detection finds that addition of 10 μM 25HC can inhibit the infection of BmNPV (B); virus titer detection finds that addition of 10 μM 25HC can inhibit the infection of BmNPV (C).

[0021] Figure 4 is a result graph that addition of exogenous 25HC in silkworm larvae can inhibit the infection of BmNPV; wherein real-time fluorescent quantitative PCR (relative quantification) detection finds that addition of 25HC can inhibit the infection of BmNPV (A); real-time fluorescent quantitative PCR (absolute quantification) detection finds that addition of 25HC can inhibit the infection of BmNPV (B). DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application.

[0023] Unless otherwise specified, the materials, reagents, etc. used below are commercially available, and the operations are conventional operations.

[0024] 1. Changes in BmCH25H transcription level after BmNPV infection.

[0025] The specific experimental process is as follows:

[0026] 1.1 The silkworm ovary cells (BmN) were inoculated into 24-well cell culture plates at a concentration of 50000 / well and cultured overnight in a 28°C incubator. BmNPV with a multiplicity of infection (MOI) of 1 was mixed with serum-free insect Grace basal medium and then added to the prepared 24-well cell culture plates, which were incubated at 28°C for 1 hour. Then, fresh Grace complete medium containing 10% FBS (fetal bovine serum, purchased from Gibco, USA) was added. Cell samples were collected at 24, 48 and 72 hours after infection.

[0027] 1.2 5 μL of BmNPV was injected into 5th instar larvae of the domesticated silkworm Bombyx mori by caudal injection. Tissue samples of the silkworm were collected at 24, 48, 72 and 96 hours after infection, and fat body, midgut and blood cells were collected respectively.

[0028] 1.3 100 μL of Trizol solution (RNA extraction reagent, purchased from Invitrogen, USA) was added to the harvested BmN cells and blood cells, and 1 mL of Trizol solution was added to the harvested silkworm fat body and midgut tissues for homogenization. Kit RNA fast 2000 (RNA extraction kit, purchased from Feijie, China) was used to extract total RNA from cells and tissues according to the manufacturer's instructions, and gDNA Eraser (reverse transcription kit, purchased from TaKaRa, Japan) was used to reverse transcribe the RNA into cDNA.

[0029] 1.4 The transcription level of BmCH25H after BmNPV infection was detected by real-time fluorescence quantitative PCR using RP49 as an internal reference. The PCR reaction system was prepared according to the iTaqTM Universal Green Supermix Kit reagents instruction manual of Bio-Rad, USA. PCR detection was performed using a fluorescence quantitative PCR instrument (CFX TM Optics Module, Bio-Rad, USA).

[0030] The results are as follows:Figure 1 The results showed that the transcription levels of BmCH25H in BmN cells and fat body, midgut and hemocytes of B. mori larvae were significantly up-regulated after BmNPV infection.

[0031] 2. Overexpression of BmCH25H in B. mori BmN cells can inhibit the infection of BmNPV.

[0032] The specific experimental process is as follows:

[0033] 2.1 The sequence encoding BmCH25H (nucleotide sequence as shown in SEQ ID NO: 1) was obtained by PCR amplification in BmN cells, and BmCH25H was cloned into pIEX1 plasmid by using Sac I and Sal I two restriction endonuclease enzyme cutting sites, and BmCH25H overexpression plasmid pIEX1-BmCH25H-V5 with V5-tag (tag sequence) was constructed.

[0034] 2.2 BmN cells were transfected with pIEX1-BmCH25H-V5 plasmid (0.5 μg / well), and cells transfected with pIEX1-eGFP were used as a control. Protein samples were collected and prepared at 24, 48, 72 and 96 hours after transfection. Mouse V5 antibody (purchased from Thermo, USA) and mouse anti-GFP (purchased from Biyun Tian, China) were used to detect the expression of BmCH25H-V5 protein and eGFP protein by western blot (WB), and α-tubulin was used as an internal reference.

[0035] 2.3 After 24 hours of transfection, BmN cells transfected with BmCH25H and eGFP were infected with 1 MOI of BmNPV virus, respectively. Total RNA and supernatant were collected at 24, 48 and 72 hours after infection, and real-time fluorescent quantitative PCR and virus titer (TCID 50 ) detection were used to detect the infection of BmNPV.

[0036] The results are shown in Figure 2 . The results showed that BmCH25H was successfully expressed in BmN cells after transfection of pIEX1-BmCH25H-V5 overexpression plasmid, with BmN cells transfected with pIEX1-eGFP recombinant plasmid as a control. It was found that overexpression of BmCH25H could significantly inhibit the infection of BmNPV by detecting the samples after infection of virus by real-time fluorescent quantitative PCR and virus titer.

[0037] 3. Addition of exogenous 25HC in B. mori BmN cells can inhibit the infection of BmNPV.

[0038] The specific experimental process is as follows:

[0039] 3.1 25HC powder (purchased from Sigma, USA) was diluted with absolute ethanol to a stock solution (50 mg / mL). The 25HC stock solution was diluted with Grace's medium (containing 10% FBS) to working concentrations of 4, 8, 10 and 15 μM. Then, BmN cells were treated with 25HC at concentrations of 4, 8, 10 and 15 μM for 24 hours. Cell toxicity was determined using Cell Counting Kit-8 dye (purchased from China's Jin Company) to determine the optimal concentration of 25HC.

[0040] 3.2 BmN cells were treated with 10 μM of 25HC and infected with 1 MOI of BmNPV virus 24 hours after treatment. BmN control cells were added with a final concentration of 0.0081% absolute ethanol. The supernatant was then replaced with Grace's medium (containing 10% FBS) supplemented with 10 μM 25HC. Total RNA and cell supernatant were collected at 24, 48 and 72 hours after infection, and BmNPV viral infection was detected using real-time fluorescent quantitative PCR and viral titer determination.

[0041] The results are shown in Figure 3 The results show that after adding exogenous 25HC in BmN cells, it is found that 25HC can significantly inhibit the infection of BmNPV in BmN cells by real-time fluorescent quantitative PCR and viral titer determination.

[0042] 4. Adding exogenous 25HC in silkworm larvae can inhibit the infection of BmNPV.

[0043] The specific experimental process is as follows:

[0044] 3.1 Five silkworm larvae were injected with 25HC (0.015 mg / larva) or absolute ethanol (control) (0.6 μL / larva), respectively. After 12 hours of treatment, the experimental group injected each larva with a mixture of BmNPV (10 7 PFU / mL) (5 μL / head) and 25HC (0.015 mg / head). The control larvae were injected with a mixture of BmNPV (5 μL / head) and absolute ethanol (0.6 μL / head).

[0045] 3.2 Fat body samples were collected at 24 and 48 hours after infection. At each sample time point, the fat bodies of three test silkworm larvae were mixed into one sample, with 3 replicates in each group. Total DNA and total RNA were extracted for real-time fluorescent quantitative PCR detection. Virus gene vp39 was used to detect viral mRNA abundance, and gp41 was used to quantify viral DNA load.

[0046] The results are shown in Figure 4The results showed that after adding exogenous 25HC in the silkworm larvae, through real-time fluorescence quantitative PCR and virus titer detection, it was found that 25HC could significantly inhibit the infection of BmNPV in silkworm larvae.

[0047] The experimental evidence is reliable, and cholesterol-25-hydroxylase (CH25H) and its enzymatic product 25-hydroxycholesterol (25HC) have good inhibitory effect on BmNPV.

[0048] In the above examples, the primers used are shown in Table 1.

[0049]

[0050] It should be noted that the above-mentioned embodiments should be understood as illustrative, rather than limiting the protection scope of the present application, and the protection scope of the present application is subject to the claims. For those skilled in the art, some non-essential improvements and adjustments of the present application without departing from the essence and scope of the present application still belong to the protection scope of the present application. SEQUENCE LISTING <110> South China Agricultural University <120> Use of cholesterol-25-hydroxylase and its enzymatic product in preparation of drug for resisting silkworm nucleopolyhedrosis virus <130> 1 <160> 15 <170> PatentIn version 3.3 <210> 1 <211> 1194 <212> DNA <213> BmN cells <400> 1 atgaaggcct ccgatacgtt gcacagaaac ggtaaaagcg agaatggatc caaaattaca 60 gagaaacacg aacaaaaaca taaagatcct ttgacggaag gcgtgaaatg gatcgaaaga 120 tacgccgaaa acttggaaaa tttcttcgag agactaccgg aattcatttc cacgtttata 180 GCGACTCTGG CGGTGTTTAC CTTTGGATCA ATTTTAAGAG GAGAGTGGGT CGTCATTCTA 240 GTCACAGCGC TGAAGCAAAT ACGGGACACA CGCAAACCAA AAATGCCTCA GTCGAGGAA 300 ATATTTCGAG CTGTTTACAT CTTAGAAACT TGAAAATGGA GAATTTCAGT ATCATATTCA TA 360 TTAGCTAATG TTGTTTCATT TGGATTTTAC TTTATCATCG GAGGATTTTT ACATTGGTAC 420 TTCTACGTGA AGAGAAGGCA CCTCGCCAGT GAATGGAAGA TCCAGCCGAA CAAATGGCTG 480 TCCCCTGAAG TTGGAACGCC ACGAAATCAT GATAGGAACC CTATCTCTCA TTATTACAAGT 540 TCATTCTCAG CTTTTTTAGC CTGTTACATC TTCAATGGGA ATCCGTGCAC AGTCTATTTT 600 CAGTTCGATG AGTACGGATG GATCTGGTTC TTCTTGCAGT TTCCAGTTAT TTTCACTTTA 660 ATTGATTACA CGACCTATAT TATGCAATCG TTATCACACA CTCCTTGGTT GTACAAGCAT 720 TTCCACAAGC TCCACCATAA GTACAAACAG CCTACAGCCT TTTCTGTAAC TGCCATCCAT 780 CCAGTAGAAA TTATGCATAT CCAACTAACG ATGTGTCTAC CTCTCTTCAC AATACTACAC 840 CATTGGGCAT CATTCTACGC TGTGGCCATT TACAATTACT ACCACGGCAT CATTGATCAT 900 tccggcatca attttaaggc ccagtggtgg cagccgtggc agcctgacgc cgaattccat 960 gatcagcatc acgaattttt ccattgcaac tttggcttca atatgtatct gtgggacaag 1020 tggcacggta cgatgaggaa gcagtacaac gtgtacaccg aagagacatt ccacggtgag 1080 gctccgtctg cagaaacggc cgaaggaaag gcgatattgg aagcgaaccc cgaacttatt 1140 gatagtatca acaaaccatt actgacaaag actgaaaccc aaaaaactaa atga 1194 <210> 2 <211> 56 <212> DNA <213> Artificial <400> 2 aaagagctcc atcatcacca ccatcacatg aaggcctccg atacgttgca cagaaa 56 <210> 3 <211> 73 <212> DNA <213> Artificial <400> 3 tttgtcgacc tacgtagaat cgagaccgag gagagggtta gggataggct tacctttagt 60 tttttgggtt tca 73 <210> 4 <211> 44 <212> DNA <213> Artificial <400> 4 aagttgcaat ggaaaaattc ctgctgctga tcatggaatt cggc 44 <210> 5 <211> 44 <212> DNA <213> Artificial <400> 5 gccgaattcc atgatcagca gcaggaattt ttccattgca actt 44 <210> 6 <211> 20 <212> DNA <213> Artificial <400> 6 aagtggcacg gtacgatgag 20 <210> 7 <211> 20 <212> DNA <213> Artificial <400> 7 taagttcggg gttcgcttcc 20 <210> 8 <211> 19 <212> DNA <213> Artificial <400> 8 ctaatgcccg tgggtatgg 19 <210> 9 <211> 19 <212> DNA <213> Artificial <400> 9 ttgatgaggt ggctgttgc 19 <210> 10 <211> 22 <212> DNA <213> Artificial <400> 10 caggcggttc aagggtcaat ac 22 <210> 11 <211> 19 <212> DNA <213> Artificial <400> 11 tgctgggctc tttccacga 19 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <400> 12 atgttgatgt gcggaaagc 19 <210> 13 <211> 16 <212> DNA <213> Artificial Sequence <400> 13 gtggcggaat cggtga 16 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <400> 14 taatacgact cactataggg ctcgccagtg aatggaagat 40 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <400> 15 taatacgact cactataggg ccactgggcc ttaaaattga 40

Claims

1. Use of any of the following in the preparation of a drug for resisting Bombyx mori nucleopolyhedrovirus: a) cholester-25-hydroxylase; b) a gene encoding cholester-25-hydroxylase; c) a recombinant plasmid comprising b); d) a recombinant bacterium comprising b); e) an enzymatic product of cholester-25-hydroxylase, 25-hydroxycholesterol; the cholester-25-hydroxylase and the gene encoding cholester-25-hydroxylase are both derived from Bombyx mori.

2. A method for inhibiting infection of Bombyx mori nucleopolyhedrovirus in Bombyx mori cells in vitro, characterized by, The following (a) or / and (b) is used: (a) first, a gene encoding cholester-25-hydroxylase BmCH25H is amplified and cloned into pIEX1 plasmid to construct an overexpression plasmid pIEX1-BmCH25H-V5 with a tag sequence; the overexpression plasmid pIEX1-BmCH25H-V5 is transfected into Bombyx mori ovary cells BmN, and BmCH25H is induced to express in the BmN cells; the nucleotide sequence of the BmCH25H gene is shown as SEQ ID NO: 1; (b) exogenous 25-hydroxycholesterol is added to treat Bombyx mori in vitro cells.

3. The method of claim 2, wherein: The Bombyx mori in vitro cells are Bombyx mori ovary cells.

4. The method of claim 2, wherein: In the (b), the treatment concentration of the exogenous 25-hydroxycholesterol is 10 μM, and the treatment time is 24 h.

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