Antisense nucleotide of piR-bmo-796514 nucleotide and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses an application of antisense nucleotide of piR-bmo-796514 nucleotide in preparation of a medicine for inhibiting a 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 bombyx mori ovarian cells, and the sequence of piRN A nucleotide is shown as SEQ ID NO: 1; in addition, the invention also discloses an antisense nucleotide of the piR-bmo-796514 nucleotide, and the sequence of the antisense nucleotide is as shown in SEQ ID NO: 2.

Description

Technical Field

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

[0002] Silkworms are one of the raw materials for the silk industry, and the silk threads produced by their larvae are used to make silk. Through long-term artificial breeding, silkworms have become high-quality silk producers, capable of producing high-quality silk threads. Bombyx mori nuclear polyhedrosis virus (BmNPV) is a pathogen that seriously affects the health of silkworms and belongs to the double-stranded DNA baculoviridae family. After BmNPV infects silkworm larvae, the virus particles replicate in the cells and produce more virus particles, eventually leading to cell rot 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 silkworms. Nano-titanium dioxide is prepared into an additive solution with a concentration of 4.5 to 5.5 mg / L. The solution is then sprayed on mulberry leaves at a dosage of 1.0 L of solution per 100 kg of mulberry leaves. After drying, the solution is added to silkworms. This can significantly inhibit the proliferation of BmNPV in silkworms.

[0004] The method utilizes the unique small size effect, surface effect, quantum size effect and macroscopic quantum tunneling effect of nano-oxides and combines the positive effect of titanium oxide on silkworm immunity regulation to achieve the purpose of inhibiting the proliferation of Bm NPV 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 to 1 g.

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

[0007] In both schemes, the therapeutic effect is achieved by utilizing the beneficial effects of nano-oxides combined with the positive role of titanium oxide in regulating immunity or utilizing the principle that piRNA antisense nucleotides can inhibit the apoptosis of cardiomyocytes.

[0008] The problem to be solved in this scheme is: how to propose an active ingredient of a drug that can be used to inhibit Bombyx mori nuclear polyhedrosis virus. Summary of the invention

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

[0010] To achieve the above objectives, the present application discloses the 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 as shown in SEQ ID NO: 1.

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

[0013] Beneficial effects of this application:

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

[0015] Figure 1 This is a schematic diagram of the relative expression level of piR-bmo-796514 after infection with BmNPV;

[0016] Figure 2 This is a schematic diagram of the relative expression level of piR-bmo-567079 after infection with BmNPV;

[0017] Figure 3 It is a schematic diagram of the relative expression of piR-bmo-796514 nucleotide after transfection of piR-bmo-796514 nucleotide mimic;

[0018] Figure 4 Schematic diagram of the relative expression of viral vp39 after transfection with piR-bmo-796514 nucleotide mimic;

[0019] Figure 5It is a schematic diagram of the relative expression of piR-bmo-567079 nucleotide after transfection of piR-bmo-567079 nucleotide mimic;

[0020] Figure 6 Schematic diagram of the relative expression of viral vp39 after transfection with piR-bmo-567079 nucleotide mimics;

[0021] Figure 7 It is a schematic diagram of the relative expression of piR-bmo-796514 nucleotide after transfection of the antisense nucleotide of piR-bmo-796514 nucleotide;

[0022] Figure 8 Schematic diagram of the relative expression of viral vp39 after transfection with the antisense nucleotide of piR-bmo-796514 nucleotide;

[0023] Fig. 9 It is a schematic diagram of the relative expression amount of piR-bmo-567079 nucleotide after transfection of the antisense nucleotide of piR-bmo-567079 nucleotide;

[0024] Fig.10 Schematic diagram of the relative expression level of viral vp39 after transfection of the antisense nucleotide of piR-bmo-567079 nucleotide. DETAILED DESCRIPTION

[0025] In the description of this application, it should be noted that if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0026] Product source information:

[0027] piRNA nucleotides and their antisense nucleotides: synthesized by Suzhou Jima Biotechnology Co., Ltd.;

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

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

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

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

[0032] Example 1

[0033] The piRNA nucleotides were initially screened and several piRNA nucleotides that responded to BmNPV virus infection (with the infection of BmNPV virus, piRNA itself showed a change in expression fold). The specific screening information of piRNA nucleotides is shown in Table 1:

[0034] Table 1

[0035]

[0036] As can be seen from the data in Table 1, in the preliminary screening process, two piRNAs, piR-bmo-796514 with the highest expression fold and piR-bmo-567079 with a positive expression fold, were selected for verification.

[0037] Example 2

[0038] The screened piR-bmo-796514 nucleotide was validated with the piR-bmo-567079 nucleotide

[0039] Step 1: Inoculate Bombyx mori ovarian cells (BmN) into a 12-well cell culture plate and culture overnight in a 28°C incubator with a cell density of 80%. Add BmNPV-eGFP recombinant fluorescent reporter virus (infection multiplicity 1), incubate at 28°C for 1 hour, and then replace with Grace medium containing 10% fetal bovine serum. Collect cell samples 24h and 48h after infection.

[0040] Step 2: Add 200 μL of RNA extraction reagent NucleoZOL lysis buffer to the harvested cell sample, extract total RNA using the NucleoZOL handbook, design piRNA-specific reverse transcription primers using the stem-loop method, and use a reverse transcription kit to reverse transcribe RNA into cDNA.

[0041] Step 3: Using U6 as an internal reference, the transcription levels of piR-bmo-796514 and piR-bmo-567079 after infection with BmNPV were detected by real-time fluorescence quantitative PCR. TM Universal GreenSupermix Kit reagents instructions for PCR reaction system. Use fluorescent quantitative PCR instrument (CFX TM Optics Module) for qPCR detection.

[0042] The results are as follows Figure 1 , Figure 2As shown, when BmNPV was infected at 24hpi, the expression level of piR-bmo-796514 in the cells increased to around 2.1, while the expression level of piR-bmo-567079 was about 1.7; further, when BmNPV was infected at 48hpi, the expression level of piR-bmo-796514 in the cells decreased to around 0.8, while the expression level of piR-bmo-567079 was about 1.3, both of which were not significant; this indicates that piR-bmo-796514 and piR-bmo-567079 are significantly upregulated in the early stage of BmNPV virus infection, and may 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, corresponding piRNA nucleotide mimics and piRNA antisense nucleotides were designed.

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

[0046] Table 2

[0047]

[0048]

[0049] Example 4

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

[0051] The piRNA nucleotide mimics were transfected into Bombyx mori BmN cells. The specific experimental steps are as follows:

[0052] BmN cells were transfected with piRNA nucleotide mimics (10 μg / well), and the cells were collected at 24 and 48 hours after transfection, and RNA samples were extracted and prepared. The expression efficiency of piRNA nucleotide mimics in BmN cells was detected by relative quantitative analysis. The experimental results showed that piRNA was significantly upregulated in BmN cells. Figure 3 and Figure 5 shown.

[0053] Subsequently, BmN cells were transfected with piRNA nucleotide mimics (10 μg / well) and infected with BmNPV-eGFP recombinant fluorescent reporter virus (infection multiplicity 1) 24 h after transfection. 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 hpi after transfection, and the changes of BmNPV viral gene vp39 were detected by relative quantitative method. The results are shown in Figure 2. Figure 4 and Figure 6 .

[0054] Depend on Figure 3 , Figure 4 It can be seen that the relative expression of piR-bmo-796514 nucleotide in the experimental group increased significantly 24h and 48h after transfection with piR-bmo-796514 nucleotide mimics. Figure 4 It can be seen that at 24hpi of infection, the relative expression level of the BmNPV virus gene vp39 did not undergo obvious changes, but showed significant changes after 48hpi 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 other hand Figure 5 , Figure 6 , 24h and 48h after transfection with piR-bmo-567079 nucleotide mimics, the relative expression of piR-bmo-567079 nucleotide mimics in the experimental group also increased significantly. At 24hpi and 48hpi of BmNPV virus infection, the relative expression of BmNPV virus gene vp39 also increased, but it was not obvious, fluctuating around 1.1, and there was no significance.

[0056] In summary, from Figure 1-2 It can be seen that when BmNPV virus infects cells at 24hpi, it can increase the relative expression levels of piR-bmo-796514 nucleotide and piR-bmo-567079 nucleotide. Figure 3-6 It can be seen that when nucleotide analogs are added, the expression levels of piR-bmo-796514 nucleotide and piR-bmo-567079 nucleotide can also be increased, but at 24hpi after infection, there is no obvious change in the expression level of BmNPV virus vp39 in both cells, and at 48hpi, only the piR-bmo-796514 nucleotide analog significantly upregulates the expression level of BmNPV virus vp39 gene in the cells.

[0057] From this we can see that even if Figure 1-2After BmNPV infection, the relative expression levels of piR-bmo-796514 and piR-bmo-567079 were significantly increased compared with the control group. However, when their nucleotide mimics were used to increase their expression levels, Figure 3-6 In the experiment, the relative expression of BmNPV vp39 gene showed a different trend of change. It can be seen that not all piRNAs whose expression is upregulated during viral infection have a significant effect on the replication of BmNPV virus.

[0058] Example 5

[0059] Validation of the role of antisense nucleotides to piRNA nucleotides that promote BmNPV replication

[0060] The piRNA antisense nucleotides were transfected into Bombyx mori BmN cells. The specific experimental steps are as follows:

[0061] BmN cells were transfected with piRNA antisense nucleotides (10 μg / well). Cells were collected 24h and 48h after transfection, and RNA samples were prepared. The expression efficiency of piRNA antisense nucleotides in BmN cells was detected by relative quantitative analysis. The experimental results showed that the expression of piRNA in BmN cells was significantly inhibited. Figure 7 and Fig. 9 shown.

[0062] Subsequently, BmN cells were transfected with piRNA antisense nucleotides (10 μg / well). 24 h after transfection, BmNPV-eGFP recombinant fluorescent reporter virus was infected (infection multiplicity was 1), incubated at 28°C for 1 h, and then replaced with fresh Grace medium containing 10% fetal bovine serum. Cell samples were collected at 24 hpi and 48 hpi after transfection, and the changes of BmNPV viral gene vp39 were detected by relative quantitative and absolute quantitative methods. The results are shown in Figure 8 and Fig.10 .

[0063] from Figure 7 , Figure 8 It can be seen that in the control group, 24 hours after transfection of piR-bmo-796514 antisense nucleotide, the relative expression level of piR-bmo-796514 nucleotide in BmN cells had dropped to around 0.2, indicating that the antisense nucleotide can effectively inhibit the growth of piR-bmo-796514 nucleotide, and based on the reduction of piR-bmo-796514 nucleotide, it can also be seen that the BmNPV viral gene vp39 has also undergone significant changes, dropping from 1.0 to around 0.8.

[0064] Further observation showed that 48 hours after BmN cells were transfected with piR-bmo-796514 antisense nucleotide, the relative expression level of piR-bmo-796514 nucleotide also dropped to 0.1, but the relative expression level of BmNPV viral gene vp39 increased from 0.8 to about 0.85, but it was still significantly decreased compared with the control group.

[0065] Observe again Fig. 9 , Fig.10 , 24 and 48 hours after BmN cells were transfected with piR-bmo-567079 antisense nucleotide, the relative expression of piR-bmo-567079 nucleotide decreased from 1.0 to about 0.02, but the relative expression of BmNPV viral gene vp39 did not decrease accordingly. It is speculated that piR-bmo-567079 nucleotide is not involved in the replication of BmNPV virus.

[0066] In summary, from Figure 1-10 In this study, we found that when infected with BmNPV virus at 24 hpi, virus infection could significantly increase the relative expression levels of the two nucleotides; however, Figure 3-6 It can be seen that the piR-bmo-796514 nucleotide mimic can significantly promote BmNPV virus replication at 24hpi and 48hpi; further observation Figure 7-10 It can be seen that the antisense nucleotide of piR-bmo-796514 can significantly inhibit the replication of BmNPV virus at 24hpi and 48hpi;

[0067] And, observe Figure 4 and Figure 8 It can be seen that the mimic of piR-bmo-796514 did not increase the expression of vp39 when used for 24 h;

[0068] and Figure 8 However, the inhibitor (antisense nucleotide) of piR-bmo-796514 significantly inhibited the expression of vp39 when used for 24 hours, and its inhibitory ability on vp39 was even higher than that at 48 hours. Figure 4 This reflects that the mimic of piR-bmo-796514 has a greater impact on vp39 when used for 48 hours. Obviously, the inhibitory ability of piR-bmo-796514 on vp39 achieved when used for 24 hours is surprising.

[0069] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of antisense nucleotides of piR-bmo-796514 nucleotides for preparing drugs for inhibiting Bombyx mori nuclear polyhedrosis virus.

2. The use according to claim 1, characterized in that The drug is used for inhibiting the replication of Bombyx mori nuclear polyhedrosis virus in Bombyx mori ovarian cells.

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

4. The use according to claim 1, characterized in that The nucleotide sequence of piR-bmo-796514 is shown in SEQ ID NO:

1.

5. The use according to claim 1, characterized in that: The antisense nucleotide of the piR-bmo-796514 nucleotide inhibits the replication of the Bombyx mori nuclear polyhedrosis virus by inhibiting the expression of vp39.

6. An antisense nucleotide of piR-bmo-796514 nucleotide, characterized in that The antisense nucleotide of the piR-bmo-796514 nucleotide 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.

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