Antisense nucleotide of piR-bmo-504830 nucleotide and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses an antisense nucleotide of piR-bmo-504830, the antisense nucleotide of the piR-bmo-504830 is designed from the piR-bmo-504830 nucleotide, and the piR-bmo-504830 nucleotide sequence is shown as SEQ ID NO: 1; in addition, the invention also discloses an application of the antisense nucleotide of the piR-bmo-504830 in preparing a medicine for inhibiting the replication of the bombyx mori nuclear polyhedrosis virus. According to the application of the piR-bmo-504830 antisense nucleotide provided by the invention in preparing the medicine for inhibiting the replication of the bombyx mori nuclear polyhedrosis virus, a basis is provided for preventing and treating the bombyx mori nuclear polyhedrosis virus.

Description

Technical Field

[0001] The present application relates to the biological field, and in particular to an antisense nucleotide of piR-bmo-504830 nucleotide and its use. Background Art

[0002] piRNA (PIWI-interacting RNA) is a type of long non-coding RNA discovered in recent years. The total number of piRNAs exceeds 40,000. They are mainly expressed in germ cells and have the function of regulating transposons and protecting genome stability. PIWI protein is a protein that specifically binds to piRNA and is the most critical protein in the entire piRNA pathway; piRNA combines with PIWI protein to form a PIWI-piRNA complex. Under the guidance of PIWI protein, it targets the target gene through the complementary base pairing rules to exert biological functions. piRNA can regulate transposon expression in a manner similar to immune response strategies. Endogenous retroviruses are retrotransposons in the host genome, and some endogenous retroviruses also have infection characteristics.

[0003] Silkworm is an important economic insect and model organism, and Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the most important pathogenic microorganisms that endanger the silkworm industry. BmNPV is a DNA virus and is a model baculovirus second only to Autographa californica nuclear polyhedrosis virus. piRNA antisense nucleotides can effectively induce RNA interference mechanism by specifically binding to viral genomic RNA, thereby inhibiting viral gene expression and replication. Compared with other small molecule RNAs, piRNA has higher specificity and stability.

[0004] Chinese patent application 202311166959.8 discloses the application of the silkworm BmSPP gene in regulating the proliferation of BmNPV. By constructing a transient overexpression vector and a knockout vector containing the silkworm BmSPP gene, the application of the BmSPP gene in regulating the proliferation and virus resistance of BmNPV was discovered. The silkworm BmSPP gene can be used as a target gene for silkworm molecular breeding and used for the cultivation of silkworm varieties resistant to BmNPV.

[0005] The method inhibits the proliferation of BmNPV by reducing the expression of the silkworm BmSPP gene.

[0006] Chinese patent application 201510065509.9 discloses a silkworm receptor expression enhancing protein BmREEPa gene, its recombinant expression vector and application. It was found that the silkworm receptor expression enhancing protein BmREEPa gene is related to BmNPV infection of silkworm. Therefore, interfering with the genes of the two spliceosomes BmREE Pa-L and BmREEPa-S in the silkworm receptor expression enhancing protein BmREEPa gene can effectively inhibit BmNPV infection of BmN-SWU1 cells.

[0007] In this method, the gene within the receptor expression enhancing protein of the silkworm is interfered, thereby further inhibiting the infection of BmNPV to BmN-SWU1 cells.

[0008] In both schemes, the proliferation of Bm NPV is inhibited by suppressing or enhancing different expression genes in the silkworm.

[0009] The problem that this scheme needs to solve: How to propose a method that can inhibit the infection of silkworm ovarian cells by Bombyx mori nuclear polyhedrosis virus. Summary of the invention

[0010] The purpose of the present application is to provide an antisense nucleotide of piR-bmo-504830 nucleotide and its application. The antisense nucleotide is designed for piR-bmo-504830 nucleotide and can effectively inhibit the expression of piR-bmo-504830 nucleotide to achieve the inhibition of the replication of Bombyx mori nuclear polyhedrosis virus.

[0011] To achieve the above objectives, the present application discloses an antisense nucleotide of piR-bmo-504830 nucleotide. The antisense nucleotide of piR-bmo-504830 nucleotide is designed for piR-bmo-504830 nucleotide, and the sequence of the piR-bmo-504830 nucleotide is shown in SEQ ID NO: 1.

[0012] Preferably, the sequence of the antisense nucleotide of the piR-bmo-504830 nucleotide is as shown in SEQ ID NO:2.

[0013] In addition, the present application also discloses the use of the antisense nucleotide of the above-mentioned piR-bmo-504830 nucleotide for preparing a drug for inhibiting the replication of Bombyx mori nuclear polyhedrosis virus.

[0014] Preferably, the antisense nucleotide of the piR-bmo-504830 nucleotide inhibits the replication of Bombyx mori nuclear polyhedrosis virus by inhibiting the expression of vp39.

[0015] Preferably, the drug is a drug that inhibits the replication of Bombyx mori nuclear polyhedrosis virus in Bombyx mori ovarian cells.

[0016] The beneficial effects of this application are:

[0017] The present application provides an antisense nucleotide of piR-bmo-504830 nucleotide and its application. The antisense nucleotide of piR-bmo-504830 nucleotide is designed according to the base complementary pairing rules, and the expression of piR-bmo-504830 nucleotide is inhibited by the antisense nucleotide of piR-bmo-504830 nucleotide to achieve the inhibition of the replication of Bombyx mori nuclear polyhedrosis virus, thereby reducing the harm of Bombyx mori nuclear polyhedrosis virus to Bombyx mori. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the response of piR-bmo-504830 after infection with BmNPV;

[0019] Figure 2 This is a schematic diagram of the response of piR-bmo-553495 after infection with BmNPV;

[0020] Figure 3 It is a schematic diagram of the relative expression of piR-bmo-504830 nucleotide after transfection of piR-bmo-504830 mimics;

[0021] Figure 4 Schematic diagram of the relative expression of viral vp39 after transfection with piR-bmo-504830 mimics;

[0022] Figure 5 It is a schematic diagram of the relative expression of piR-bmo-553495 nucleotide after transfection of piR-bmo-553495 mimics;

[0023] Figure 6 Schematic diagram of the relative expression of viral vp39 after transfection with piR-bmo-553495 mimics;

[0024] Figure 7 It is a schematic diagram of the relative expression of piR-bmo-504830 nucleotide after transfection of the antisense nucleotide of piR-bmo-504830;

[0025] Figure 8 Schematic diagram of the relative expression of viral vp39 after transfection with the antisense nucleotide of piR-bmo-504830;

[0026] Fig. 9 It is a schematic diagram of the relative expression of piR-bmo-553495 nucleotide after transfection of the antisense nucleotide of piR-bmo-553495;

[0027] Fig.10 Schematic diagram of the relative expression level of viral vp39 after transfection with the antisense nucleotide of piR-bmo-553495. DETAILED DESCRIPTION

[0028] 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.

[0029] Product source information:

[0030] piR-bmo-504830 nucleotide and its antisense nucleotide: synthesized by Suzhou Jima Biotechnology Co., Ltd.;

[0031] Fetal bovine serum: purchased from Gibco, USA;

[0032] HD transfection reagent: purchased from Promega, USA;

[0033] NucleoZOL lysis buffer: purchased from Macherey Nagel, Germany;

[0034] Reverse transcription kit: purchased from TaKaRa, Japan.

[0035] Example 1

[0036] The piRNA nucleotides were initially screened and several piRNA nucleotides that responded to BmNPV virus infection (with the infection of BmNP V virus, piRNA itself showed a change in expression fold). The specific screening information of piRNA nucleotides is shown in Table 1:

[0037] Table 1

[0038]

[0039] As shown in Table 1, during the preliminary screening process, the expression multiples of piR-bmo-504830 and piR-bmo-553495 were higher than those of other piRNAs, so these two piRNAs were selected for verification.

[0040] Example 2

[0041] The screened piR-bmo-504830 nucleotide was validated with the piR-bmo-553495 nucleotide

[0042] Step 1: Bombyx mori ovary cells (BmN) were inoculated into a 12-well cell culture plate and cultured overnight in a 28°C incubator to a cell density of about 80%. BmNPV-eGFP recombinant fluorescent reporter virus (infection multiplicity of 1) was added, incubated at 28°C for 1 hour, and then replaced with Grace medium containing 10% fetal bovine serum, and cell samples were collected 24 hours and 48 hours after infection.

[0043] Step 2: Add 200 μL of RNA extraction reagent NucleoZOL lysis buffer to the collected cell sample, extract total RNA using the NucleoZOL handheld instruction manual, design piRNA-specific reverse transcription primers using the stem-loop method, and use a reverse transcription kit to reverse transcribe RNA into cDNA.

[0044] Step 3: Using U6 as an internal reference, the transcription levels of two piRNA nucleotides, piR-bmo-504830 and piR-bmo-553495, after infection with BmNPV were detected by real-time fluorescence quantitative PCR. TM Universal Green Supermix Kit reagents instructions for configuring the real-time fluorescence quantitative PCR reaction system. Use a fluorescence quantitative PCR instrument (CFX Bio-Rad, USA) TM Optics Module) for real-time fluorescence quantitative PCR detection, the results are as follows Figure 1-2 shown.

[0045] observe Figure 1 It can be seen that after infection with BmNPV virus, the expression level of piR-bmo-504830 nucleotide in silkworm cells showed a significant upward trend at 24hpi, rising from 1.0 to about 1.55; Figure 2 It can be seen that after infection with BmNPV virus, the expression level of piR-bmo-553495 nucleotide in silkworm cells also showed a significant upward trend at 24hpi, rising from 1.0 to about 1.7. Compared with the two nucleotides, the expression level of piR-bmo-553495 nucleotide increased more within 24hpi of infection with BmNPV virus.

[0046] Further observation Figure 1 , Figure 2It can be seen that when infected with BmNPV virus at 48hpi, compared with 24hpi, the expression of piR-bmo-504830 nucleotide has basically the same upward trend, while the relative expression of piR-bmo-553495 nucleotide decreases from 1.7 to about 0.5. It is speculated that during the replication of the virus, the expression of piR-bmo-504830 and piR-bmo-553495 piRNAs may remain the same or decrease due to differences in their respective expression patterns. From the above results, it can be seen that both piR-bmo-504830 nucleotides and piR-bmo-553495 nucleotides may play an important role in the replication of BmNPV virus.

[0047] Example 3

[0048] According to the two piRNA nucleotides verified in Example 2, corresponding piRNA nucleotide mimics and piRNA antisense nucleotides were designed.

[0049] The nucleotide sequences of piRNA nucleotide mimics and piRNA antisense nucleotides are shown in Table 2:

[0050] Table 2

[0051]

[0052] Example 4

[0053] Verification of the effects of piR-bmo-504830 nucleotides and piR-bmo-553495 nucleotides on BmNPV virus replication

[0054] The piRNA nucleotide mimics were transfected into Bombyx mori BmN cells. The specific experimental steps are as follows:

[0055] BmN cells were transfected with piRNA nucleotide mimics (10 μg / well), and the cells were collected at 24h and 48h 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.

[0056] 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 hpi and 48 hpi after transfection, and the changes of viral gene vp39 were detected by relative quantitative method. The results are shown in Figure 2. Figure 4 and Figure 6 .

[0057] Depend on Figure 3 , Figure 4 It can be seen that 24h after transfection with piR-bmo-504830 nucleotide mimics, the relative expression of piR-bmo-504830 nucleotide was significantly upregulated, but the expression of vp39 gene did not change significantly at 24hpi of viral infection; 48h after transfection, the relative expression of piR-bmo-504830 nucleotide had risen to about 600, and the relative expression of viral vp39 gene was significantly upregulated, from 1.0 to about 1.5. This indicates that piR-bmo-504830 nucleotide may affect viral replication in the late stage of viral infection.

[0058] On the other hand Figure 5 , Figure 6 , the relative expression level of piR-bmo-553495 nucleotide was significantly upregulated 24h and 48h after transfection with piR-bmo-553495 nucleotide mimics, while the relative expression level of Bm NPV viral gene vp39 did not change significantly when infected with the virus at 24hpi and 48hpi, fluctuating around 1.1.

[0059] In summary, from Figure 1-2 It can be seen that after BmNPV virus infects cells, without adding nucleotide mimics, the relative expression levels of piR-bmo-504830 nucleotide and piR-bmo-553495 nucleotide are increased. Figure 3-6 It can be seen that when nucleotide mimics were added, the expression of piR-bmo-504830 nucleotide and piR-bmo-553495 nucleotide was significantly upregulated, but at 24hpi after infection, the expression of BmNPV virus vp39 in both cells did not change significantly, and at 48hpi, only transfection of piR-bmo-504830 nucleotide mimic significantly increased the expression of BmNPV virus vp39 gene in cells. It can be seen that piR-bmo-504830 nucleotide has the effect of promoting BmNPV virus replication, and the action time is in the later stage, while piR-bmo-553495 nucleotide has no significant effect on the replication of BmNPV virus.

[0060] Example 5

[0061] Validation of the role of antisense nucleotides to piRNA nucleotides that promote BmNPV replication

[0062] The piRNA antisense nucleotides were transfected into Bombyx mori BmN cells. The specific experimental steps are as follows:

[0063] BmN cells were transfected with piRNA antisense nucleotides (10 μg / well). Cells were collected 24h and 48h after transfection, and RNA samples were prepared. Relative quantitative detection of the inhibitory efficiency of piRNA antisense nucleotides on piRNA nucleotides in BmN cells showed that the expression of piR-bmo-504830 nucleotide and piR-bmo-553495 nucleotide in BmN cells was significantly inhibited. Figure 7 and Fig. 9 shown.

[0064] 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 infection, and the changes of viral gene vp39 were detected by relative quantitative method. The results are shown in Figure 2. Figure 8 and Fig.10 shown.

[0065] Depend on Figure 7 , Figure 8 It can be seen that when the antisense nucleotide of piR-bmo-504830 nucleotide was used to transfect BmN cells, the relative expression of piR-bmo-504830 nucleotide showed a significant downward trend at 24h and 48h, from 1.0 to 0.01. When Bm N cells transfected with the antisense nucleotide of piR-bmo-504830 nucleotide were infected with BmNPV virus at 24hpi and 48hpi, the relative expression of BmNPV virus vp39 gene showed a significant downward trend, from 1.0 to about 0.75.

[0066] Observe again Fig. 9 , Fig.10 It can be seen that when the antisense nucleotide of piR-bmo-553495 nucleotide was used to transfect BmN cells, the relative expression of piR-bmo-553495 nucleotide also showed a significant downward trend at 24h and 48h, from 1.0 to about 0.3. However, when BmN cells transfected with piR-bmo-553495 antisense nucleotide were infected with BmNPV virus at 24hpi, the relative expression of the virus vp39 gene did not change, but at 48hpi, the relative expression of the BmNPV virus vp39 gene showed a significant upward trend, from 1.0 to about 1.6.

[0067] Furthermore, combining Table 2 and Figure 1-2It can be seen that piRNA nucleotides that produce a strong response to BmNPV virus infection are not necessarily able to maintain extremely high expression levels for a long time after infection with the BmNPV virus;

[0068] On the other hand, observation Figure 1-10 It can be seen that, firstly, after infection with BmNPV virus, the expression levels of piR-bmo-504830 nucleotide and piR-bmo-553495 nucleotide were significantly increased within 24 hours, and the increase trend of piR-bmo-553495 nucleotide was even more significant;

[0069] Further observations Figure 3-6 It can be seen that when we tried to use piR-bmo-504830 mimics and piR-bmo-553495 mimics to increase the content of the two, they did not produce a significant trend of promoting the expression of the vp39 gene within 24 hours, that is, they did not significantly promote the infection or replication of the BmNPV virus;

[0070] On the contrary Figure 7-10 When the piR-bmo-504830 inhibitor and the piR-bmo-553495 inhibitor inhibited the expression of the corresponding nucleotides of the two, the relative expression of vp39 in the piR-bmo-504830 inhibitor group showed a significant downward trend within 24 hours;

[0071] In summary, Figure 3-6 While it has been demonstrated that the expression levels of piR-bmo-504830 nucleotides and piR-bmo-553495 nucleotides did not show a significant correlation with the expression level of the vp39 gene, the addition of the piR-bmo-504830 inhibitor reduced the expression level of the vp39 gene, which was unexpected.

[0072] 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-504830 nucleotide analog can significantly promote BmNPV virus replication at 48hpi; further observation Figure 7-10 It can be seen that the antisense nucleotide of piR-bmo-504830 can significantly inhibit the replication of BmNPV virus at 24hpi and 48hpi.

[0073] 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. An antisense nucleotide of piR-bmo-504830 nucleotide, characterized in that The antisense nucleotide of the piR-bmo-504830 nucleotide is designed for the piR-bmo-504830 nucleotide, and the sequence of the piR-bmo-504830 nucleotide is shown in SEQ ID NO:

1.

2. The antisense nucleotide of piR-bmo-504830 nucleotide according to claim 1, characterized in that The sequence of the antisense nucleotide of the piR-bmo-504830 nucleotide is shown in SEQ ID NO:

2.

3. Use of the antisense nucleotide of the piR-bmo-504830 nucleotide as described in any one of claims 1 to 2 for preparing a drug for inhibiting the replication of Bombyx mori nuclear polyhedrosis virus.

4. The use according to claim 3, characterized in that The antisense nucleotide of the piR-bmo-504830 nucleotide inhibits the replication of the Bombyx mori nuclear polyhedrosis virus by inhibiting the expression of vp39.

5. The use according to claim 3, characterized in that: The drug is a drug for inhibiting the replication of Bombyx mori nuclear polyhedrosis virus in Bombyx mori ovarian cells.

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