An antisense nucleotide of a piR-bmo-796514 nucleotide and uses thereof

Abstract

The application belongs to the technical field of biology, and discloses a use of antisense nucleotide of piR-bmo-796514 nucleotide for preparing a medicine for inhibiting Bombyx mori nuclear polyhedrosis virus, the antisense nucleotide of the piR-bmo-796514 nucleotide is mainly used for inhibiting replication of the Bombyx mori nuclear polyhedrosis virus in ovary cells of the Bombyx mori, and a sequence of the piRNA nucleotide is shown as SEQ ID NO:1; in addition, the application also discloses the antisense nucleotide of the piR-bmo-796514 nucleotide, and an antisense nucleotide sequence is shown as SEQ ID NO:2.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an antisense nucleotide of piR-bmo-796514 nucleotide and use thereof. BACKGROUND

[0002] Silkworm is one of the raw materials of silk industry, and the silk produced by its larvae is used to make silk. Through long-term artificial selection, silkworm has become a high-quality silk producer and can produce high-quality silk. Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that seriously affects the health of silkworm and belongs to the family of double-stranded DNA baculovirus. After BmNPV infects silkworm larvae, virus particles replicate in cells and produce more virus particles, eventually leading to cell ulceration and causing the death of the host insect.

[0003] Chinese patent application 201410168991.4 discloses a method for inhibiting the proliferation of BmNPV virus in silkworm. Nano-titanium dioxide is configured into an additive solution with a concentration of 4.5-5.5 mg / L, and then the solution is sprayed on mulberry leaves according to a usage dose of 1.0 L solution per 100 kg mulberry leaves. After drying, the silkworms are fed, which can significantly inhibit the proliferation of BmNPV in silkworms.

[0004] This method utilizes the unique small size effect, surface effect, quantum size effect and macroscopic quantum tunneling effect of nano-oxide and combines the positive effect of titanium oxide on the regulation of immunity of silkworm to achieve the purpose of inhibiting the proliferation of BmNPV virus.

[0005] Chinese patent application 201510044561.6 discloses a piRNA antisense nucleotide pharmaceutical composition and its use. The sequence of the piRNA antisense nucleotide is 5'-CCUUGGCACAUGCGCAGAUU AUUUGUUUA-3', and the content of the piRNA antisense nucleotide in the pharmaceutical composition is 0.5-1 g.

[0006] The piRNA antisense nucleotide pharmaceutical composition in this method is mainly used for the preparation of drugs for treating myocardial infarction, myocardial ischemic injury and myocardial fibrosis, and achieves the therapeutic effect by inhibiting the apoptosis of myocardial cells.

[0007] In the two schemes, the beneficial effects of nano-oxide are combined with the positive effect of titanium oxide on the regulation of immunity or the principle that piRNA antisense nucleotide can inhibit the apoptosis of myocardial cells to achieve the therapeutic effect.

[0008] The problem to be solved by the present application is how to propose an active ingredient of a drug for inhibiting Bombyx mori nucleopolyhedrovirus. SUMMARY

[0009] The purpose of the present application is to provide a use of an antisense nucleotide of piR-bmo-796514 nucleotide as an active ingredient of a drug for inhibiting Bombyx mori nuclear polyhedrosis virus, which can effectively inhibit the expression of vp39 of Bombyx mori nuclear polyhedrosis virus and further inhibit the replication of Bombyx mori nuclear polyhedrosis virus.

[0010] To achieve the above-mentioned purpose, the present application discloses a use of an antisense nucleotide of piR-bmo-796514 nucleotide for preparing a drug for inhibiting Bombyx mori nuclear polyhedrosis virus.

[0011] Preferably, the piR-bmo-796514 nucleotide sequence is shown in SEQ ID NO: 1.

[0012] In addition, the present application also discloses an antisense nucleotide of piR-bmo-796514 nucleotide, which is designed for the piR-bmo-796514 nucleotide, and the antisense nucleotide sequence of the piR-bmo-796514 nucleotide is shown in SEQ ID NO: 2.

[0013] The present application has the following beneficial effects:

[0014] The present application provides a use of an antisense nucleotide of piR-bmo-796514 nucleotide for preparing a drug for inhibiting Bombyx mori nuclear polyhedrosis virus, and the piRNA has higher specificity and stability by inhibiting the expression of vp39 and thereby inhibiting the replication of the virus, compared with other small molecule RNAs. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The relative expression amount of piR-bmo-796514 after BmNPV infection is shown in the figure;

[0016] Figure 2 The relative expression amount of piR-bmo-567079 after BmNPV infection is shown in the figure;

[0017] Figure 3 The relative expression amount of piR-bmo-796514 nucleotide after transfection of piR-bmo-796514 nucleotide mimics is shown in the figure;

[0018] Figure 4 The relative expression amount of virus vp39 after transfection of piR-bmo-796514 nucleotide mimics is shown in the figure;

[0019] Figure 5Figure showing the relative expression of piR-bmo-567079 nucleotide after transfection of the antisense nucleotide of piR-bmo-567079 nucleotide;

[0020] Figure 6 Figure showing the relative expression of virus vp39 after transfection of the antisense nucleotide of piR-bmo-567079 nucleotide;

[0021] Figure 7 Figure showing the relative expression of piR-bmo-796514 nucleotide after transfection of the antisense nucleotide of piR-bmo-796514 nucleotide;

[0022] Figure 8 Figure showing the relative expression of virus vp39 after transfection of the antisense nucleotide of piR-bmo-796514 nucleotide;

[0023] Figure 9 Figure showing the relative expression of piR-bmo-567079 nucleotide after transfection of the antisense nucleotide of piR-bmo-567079 nucleotide;

[0024] Figure 10 Figure showing the relative expression of virus vp39 after transfection of the antisense nucleotide of piR-bmo-567079 nucleotide. DETAILED DESCRIPTION

[0025] In the description of the present application, it should be noted that, in the examples, if the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased on the market.

[0026] Commercial source information:

[0027] piRNA nucleotide and its antisense nucleotide: synthesized by Suzhou Jimabio Technology Co., Ltd.;

[0028] Fetal bovine serum: purchased from Gibco, USA;

[0029] HD transfection reagent: purchased from Promega, USA;

[0030] NucleoZOL lysis solution: purchased from Macherey Nagel, Germany;

[0031] Reverse transcription kit: purchased from TaKaRa, Japan.

[0032] Example 1

[0033] The piRNA nucleotides are screened preliminarily to select several piRNA nucleotides responding to BmNPV virus infection (the piRNA itself shows the change of expression fold with the infection of BmNPV virus), and the screening information of the specific piRNA nucleotides is shown in Table 1:

[0034] Table 1

[0035]

[0036] According to the data in Table 1, in the preliminary screening process, the piR-bmo-796514 with the highest expression fold and the piR-bmo-567079 with the positive trend of expression fold are selected for verification.

[0037] Example 2

[0038] Verification of the screened piR-bmo-796514 nucleotide and piR-bmo-567079 nucleotide

[0039] Step 1: The silkworm ovary cells (BmN) are inoculated into a 12-well cell culture plate and cultured overnight in a 28℃ incubator, and the cell density is 80%. The BmNPV-eGFP recombinant fluorescent reporter virus (multiplicity of infection is 1) is added, and after 1h incubation at 28℃, it is replaced with Grace's medium containing 10% fetal bovine serum. The cell samples at 24h and 48h after infection are collected respectively.

[0040] Step 2: 200μL of RNA extraction reagent NucleoZOL lysis solution is added to the harvested cell sample, and total RNA is extracted using the NucleoZOL hand instruction. The stem loop method is used to design the piRNA specific reverse transcription primer, and the RNA is reverse transcribed into cDNA using the reverse transcription kit.

[0041] Step 3: U6 is used as an internal reference to detect the transcription level of piR-bmo-796514 and piR-bmo-567079 after BmNPV infection by real-time fluorescent quantitative PCR. The PCR reaction system is configured according to the iTaq TM Universal GreenSupermix Kit reagents quantitative reagent instruction of the American Bio-Rad company. The qPCR detection is performed using the fluorescent quantitative PCR instrument (American Bio-Rad CFX TM Optics Module).

[0042] The results are as follows: Figure 1 、 Figure 2As shown, the expression of piR-bmo-796514 in the cells rose to about 2.1 at 24hpi when BmNPV infected, and the expression of piR-bmo-567079 was about 1.7; further, the expression of piR-bmo-796514 in the cells dropped to about 0.8 at 48hpi when BmNPV infected, and the expression of piR-bmo-567079 was about 1.3, both of which were not significant; this indicates that both piR-bmo-796514 and piR-bmo-567079 were significantly up-regulated in the early stage of BmNPV virus infection, and might play a role in the early stage of BmNPV virus infection.

[0043] Example 3

[0044] According to the two piRNA nucleotides verified in Example 2, the corresponding piRNA nucleotide mimics and piRNA antisense nucleotides were designed.

[0045] And the nucleotide sequences of the piRNA nucleotide mimics and piRNA antisense nucleotides are shown in Table 2:

[0046] Table 2

[0047]

[0048]

[0049] Example 4

[0050] Verification of the effect of piR-bmo-796514 nucleotide and piR-bmo-567079 nucleotide on BmNPV virus replication

[0051] The piRNA nucleotide mimics were transfected in the silkworm BmN cells, and the specific experimental steps were as follows:

[0052] The BmN cells were transfected with the piRNA nucleotide mimics (10 μg / well), and the cells were collected at 24 and 48h after transfection, and the RNA samples were prepared and extracted. The relative quantitative detection was used to detect the expression efficiency of the piRNA nucleotide mimics in the BmN cells, and the experimental results showed that the piRNA was significantly up-regulated in the BmN cells, and the results are shown in Figure 3 and Figure 5 .

[0053] BmN cells were then transfected with piRNA nucleotide mimicry (10 μg / well). 24 h post-transfection, cells were infected with BmNPV-eGFP recombinant fluorescent reporter virus (multiple of infection 1). After incubation at 28°C for 1 h, the medium was replaced with fresh Grace medium containing 10% fetal bovine serum. Cell samples were collected at 24 and 48 h post-transfection, and changes in the BmNPV viral gene vp39 were detected using relative quantification. The results are as follows: Figure 4 and Figure 6 .

[0054] Depend on Figure 3 , Figure 4 It was found that the relative expression level of piR-bmo-796514 nucleotides in the experimental group increased significantly 24 h and 48 h after transfection with the piR-bmo-796514 nucleotide mimic. However, observations... Figure 4 It can be seen that the relative expression level of the BmNPV virus gene vp39 did not change significantly at 24 hpi of infection, but showed a significant change after 48 hpi of infection, increasing from about 1.1 to about 1.3. It is speculated that piR-bmo-796514 may play a role in the later stage of BmNPV virus infection.

[0055] On the contrary Figure 5 , Figure 6 After 24 and 48 hours of transfection with the piR-bmo-567079 nucleotide mimic, the relative expression level of the piR-bmo-567079 nucleotide mimic in the experimental group also increased significantly. Although the relative expression level of the BmNPV virus gene vp39 also increased after 24 and 48 hours of BmNPV infection, it was not significant, fluctuating around 1.1 and showing no statistical significance.

[0056] In summary, from Figures 1-2 It can be seen that BmNPV virus infection of cells for 24 hpi can increase the relative expression levels of piR-bmo-796514 nucleotides and piR-bmo-567079 nucleotides. From Figures 3-6 It can be seen that when nucleotide mimics are added, the expression levels of piR-bmo-796514 nucleotide and piR-bmo-567079 nucleotide can also be increased. However, at 24 hpi post-infection, there was no significant change in the expression level of BmNPV virus vp39 in the cells of both. Moreover, at 48 hpi, only piR-bmo-796514 nucleotide mimics significantly upregulated the expression level of BmNPV virus vp39 gene in the cells.

[0057] Therefore, it can be concluded that even Figures 1-2The relative expression of piR-bmo-796514 and piR-bmo-567079 was significantly increased after BmNPV virus infection, but when the expression of the two was increased by using nucleotide mimics of the two, Figures 3-6 The relative expression of BmNPV virus vp39 gene showed different trends, which indicated that not all piRNAs with up-regulated expression during virus infection had a significant impact on the replication of BmNPV virus.

[0058] Example 5

[0059] Verification of the effect of antisense nucleotides of piRNA nucleotides promoting BmNPV replication

[0060] Transfect piRNA antisense nucleotides in BmN cells of Bombyx mori, and the specific experimental steps are as follows:

[0061] Transfect BmN cells with piRNA antisense nucleotides (10 μg / well). Collect cells at 24 h and 48 h after transfection, and prepare RNA samples. Use relative quantification to detect the expression efficiency of piRNA antisense nucleotides in BmN cells. The experimental results show that the expression of piRNA in BmN cells is significantly inhibited, and the results are shown in Figure 7 and Figure 9 .

[0062] Subsequently, transfect BmN cells with piRNA antisense nucleotides (10 μg / well). At 24 h after transfection, infect BmNPV-eGFP recombinant fluorescent reporter virus (multiplicity of infection is 1), and incubate at 28°C for 1 h, then replace with fresh Grace's medium containing 10% fetal bovine serum. Collect cell samples at 24 hpi and 48 hpi after transfection, respectively, and use relative quantification and absolute quantification to detect the changes of BmNPV virus gene vp39, and the results are shown in Figure 8 and Figure 10 .

[0063] From Figure 7 , Figure 8 It can be seen that in the control group, the relative expression of piR-bmo-796514 nucleotide in BmN cells decreased to about 0.2 after 24 h of transfection with piR-bmo-796514 antisense nucleotides, indicating that the antisense nucleotides can effectively inhibit the increase of piR-bmo-796514 nucleotide, and on the basis of the decrease of piR-bmo-796514 nucleotide, it can also be seen that BmNPV virus gene vp39 also showed significant changes, from 1.0 to about 0.8.

[0064] Further observation can know that, after 48h of transfection of BmN cells with antisense nucleotide of piR-bmo-796514, the relative expression amount of piR-bmo-796514 nucleotide is also reduced to 0.1, but the relative expression amount of BmNPV virus gene vp39 is increased from 0.8 to about 0.85, but compared with the control group, there is still a significant decrease.

[0065] Further observation Figure 9 、 Figure 10 After 24h and 48h of transfection of BmN cells with antisense nucleotide of piR-bmo-567079, the relative expression amount of piR-bmo-567079 nucleotide is reduced from 1.0 to about 0.02, but the relative expression amount of BmNPV virus gene vp39 does not correspondingly decrease. It is speculated that piR-bmo-567079 nucleotide does not participate in the replication of BmNPV virus.

[0066] In summary, from Figures 1-10 we find that when BmNPV virus is infected for 24hpi, the infection of the virus can significantly increase the relative expression amount of the two nucleotides; but observation Figures 3-6 can know that the mimics of piR-bmo-796514 can significantly promote the replication of BmNPV virus at 24hpi and 48hpi; further observation Figures 7-10 can know that the antisense nucleotide of piR-bmo-796514 can significantly inhibit the replication of BmNPV virus at 24hpi and 48hpi;

[0067] And observation Figure 4 and Figure 8 can see that the mimics of piR-bmo-796514 does not increase the expression amount of vp39 at 24h;

[0068] While Figure 8 the inhibitor (antisense nucleotide) of piR-bmo-796514 significantly inhibits the expression amount of vp39 at 24h; and its inhibition ability on vp39 is even higher than that at 48h; and Figure 4 reflects that the mimics of piR-bmo-796514 has more influence on vp39 at 48h. Obviously, the inhibition ability of the inhibitor of piR-bmo-796514 on vp39 at 24h is surprising.

[0069] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. The use of the antisense nucleotide of piR-bmo-796514 to prepare a drug for inhibiting silkworm nucleopolyhedrovirus; the sequence of the piR-bmo-796514 nucleotide is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that, The drug is used to inhibit the replication of silkworm nucleopolyhedrovirus in silkworm ovarian cells.

3. The use according to claim 1, characterized in that, The drug is a liquid or solid formulation containing an antisense nucleotide of piR-bmo-796514 nucleotide.

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