An antisense nucleotide of piR-bmo-504830 nucleotide and its use

Abstract

This application belongs to the field of biotechnology and discloses an antisense nucleotide of piR-bmo-504830, designed from the piR-bmo-504830 nucleotide, the nucleotide sequence of which is shown in SEQ ID NO: 1. Furthermore, it discloses the use of the antisense nucleotide of piR-bmo-504830 in the preparation of a drug to inhibit the replication of silkworm nucleopolyhedrovirus. The application of the piR-bmo-504830 antisense nucleotide provided in this application in the preparation of a drug to inhibit the replication of silkworm nucleopolyhedrovirus provides a foundation for the prevention and control of silkworm nucleopolyhedrovirus.

Description

Technical Field

[0001] This application relates to the biological field, and more particularly to an antisense nucleotide of piR-bmo-504830 and its uses. Background Technology

[0002] piRNAs (PIWI-interacting RNAs), a class of long non-coding RNAs discovered in recent years, number over 40,000. They are primarily expressed in germ cells and function to regulate transposons and protect genome stability. PIWI proteins are proteins that specifically bind to piRNAs and are the most crucial proteins in the entire piRNA pathway. piRNAs and PIWI proteins combine to form PIWI-piRNA complexes, which, guided by PIWI proteins, target genes according to base complementarity rules to exert their biological functions. piRNAs can regulate transposon expression in a manner similar to an immune response. Endogenous retroviruses are retrotransposons within the host genome, and some endogenous retroviruses also possess infectious properties.

[0003] Silkworms are important economic insects and model organisms, while silkworm nucleopolyhedrovirus (BmNPV) is one of the most significant pathogens threatening sericulture. BmNPV is a DNA virus and is the second largest model baculovirus after the silver-striped moth nucleopolyhedrovirus. piRNA antisense nucleotides, by specifically binding to viral genomic RNA, can effectively induce RNA interference mechanisms, thereby inhibiting viral gene expression and replication. Compared to other small RNA molecules, piRNA exhibits higher specificity and stability.

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

[0005] This method inhibits BmNPV proliferation by reducing the expression of the BmSPP gene in silkworms.

[0006] Chinese patent application 201510065509.9 discloses a silkworm receptor expression enhancement protein BmREEPa gene, its recombinant expression vector, and its applications. It was found that the silkworm receptor expression enhancement protein BmREEPa gene is associated with BmNPV infection of silkworms. Therefore, interfering with the genes of the two splice variants BmREEPa-L and BmREEPa-S in the silkworm receptor expression enhancement protein BmREEPa gene can effectively inhibit BmNPV infection of BmN-SWU1 cells.

[0007] This method further inhibits the infection of BmN-SWU1 cells by BmNPV by interfering with the genes within the receptor-enhancing protein of silkworms.

[0008] Both approaches inhibit Bm NPV proliferation by suppressing or enhancing different expressed genes in silkworms.

[0009] The problem this proposal aims to solve is: how to develop a method to inhibit the infection of silkworm ovarian cells by silkworm nucleopolyhedrovirus. Summary of the Invention

[0010] The purpose of this application is to provide an antisense nucleotide of piR-bmo-504830 nucleotide and its application. This antisense nucleotide is designed for piR-bmo-504830 nucleotide and can effectively inhibit the expression of piR-bmo-504830 nucleotide, thereby inhibiting the replication of silkworm nucleopolyhedrovirus.

[0011] To achieve the above objectives, this application discloses an antisense nucleotide for piR-bmo-504830 nucleotide, which is designed for piR-bmo-504830 nucleotide, and the sequence of 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 shown in SEQ ID NO: 2.

[0013] In addition, this application also discloses the use of the antisense nucleotide of the above-described piR-bmo-504830 nucleotide in the preparation of a drug for inhibiting the replication of silkworm nucleopolyhedrovirus.

[0014] Preferably, the antisense nucleotide of the piR-bmo-504830 nucleotide inhibits the replication of silkworm nucleopolyhedrovirus by suppressing the expression of vp39.

[0015] Preferably, the drug is a drug that inhibits the replication of silkworm nucleopolyhedrovirus in silkworm ovarian cells.

[0016] The beneficial effects of this application are:

[0017] This application provides an antisense nucleotide of piR-bmo-504830 and its application. The antisense nucleotide of piR-bmo-504830 is designed by means of complementary base pairing rules, and the expression of piR-bmo-504830 is inhibited by the antisense nucleotide of piR-bmo-504830 to suppress the replication of silkworm nucleopolyhedrovirus, thereby reducing the harm of silkworm nucleopolyhedrovirus to silkworms. Attached Figure Description

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

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

[0020] Figure 3 This is a schematic diagram showing the relative expression levels of piR-bmo-504830 nucleotides after transfection with the piR-bmo-504830 mimic.

[0021] Figure 4 This is a schematic diagram showing the relative expression level of viral vp39 after transfection with the piR-bmo-504830 mimic.

[0022] Figure 5 This is a schematic diagram showing the relative expression levels of piR-bmo-553495 nucleotides after transfection with the piR-bmo-553495 mimic.

[0023] Figure 6 This is a schematic diagram showing the relative expression level of viral vp39 after transfection with the piR-bmo-553495 mimic.

[0024] Figure 7 This is a schematic diagram showing the relative expression levels of piR-bmo-504830 nucleotides after transfection with antisense nucleotides of piR-bmo-504830.

[0025] Figure 8 This is a schematic diagram showing the relative expression level of viral vp39 after transfection with antisense nucleotides of piR-bmo-504830.

[0026] Figure 9 This is a schematic diagram showing the relative expression levels of piR-bmo-553495 nucleotides after transfection with antisense nucleotides of piR-bmo-553495.

[0027] Figure 10 This diagram illustrates the relative expression level of viral vp39 after transfection with antisense nucleotides of piR-bmo-553495. Detailed Implementation

[0028] In the description of this application, it should be noted that, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[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 Corporation, Japan.

[0035] Example 1

[0036] Preliminary screening of piRNA nucleotides identified several piRNA nucleotides that respond to BmNPV virus infection (piRNA expression fold changes with BmNPV infection). Specific screening information for these piRNA nucleotides is shown in Table 1.

[0037] Table 1

[0038]

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

[0040] Example 2

[0041] The screened piR-bmo-504830 nucleotide and piR-bmo-553495 nucleotide were validated.

[0042] Step 1: Seed silkworm ovarian cells (BmN) into 12-well cell culture plates and incubate overnight at 28°C until the cell density reaches approximately 80%. Add BmNPV-eGFP recombinant fluorescent reporter virus (multiple of infection = 1), incubate at 28°C for 1 hour, then replace with Grace medium containing 10% fetal bovine serum. Collect cell samples at 24 hours and 48 hours post-infection.

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

[0044] Step 3: Using U6 as an internal control, the transcription levels of two piRNA nucleotides, piR-bmo-504830 and piR-bmo-553495, after BmNPV infection were detected by real-time quantitative PCR. This was performed according to the iTaq standard developed by Bio-Rad Laboratories. TM Universal The Green Supermix Kit reagents instructions are in place to prepare a real-time quantitative PCR reaction system. Use a real-time PCR instrument (Bio-Rad CFX, USA). TM Real-time quantitative PCR was performed using the Optics Module, and the results are as follows: Figure 1-2 As 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 24 hpi, increasing from 1.0 to approximately 1.55; further observation... Figure 2 It was found that after infection with BmNPV virus, the expression level of piR-bmo-553495 nucleotide in silkworm cells also showed a significant upward trend at 24 hpi, increasing from 1.0 to about 1.7. Compared with the two nucleotides, the expression level of piR-bmo-553495 nucleotide increased more within 24 hpi after BmNPV infection.

[0046] Further observation Figure 1 , Figure 2It was found that at 48 hpi of BmNPV infection, compared with 24 hpi infection, the upregulation trend of piR-bmo-504830 nucleotide expression was basically the same, while the relative expression of piR-bmo-553495 nucleotide decreased from 1.7 to about 0.5. It is speculated that during viral replication, the expression levels of piR-bmo-504830 and piR-bmo-553495 piRNAs may remain the same or decrease due to differences in their respective expression patterns. These results indicate that both piR-bmo-504830 and piR-bmo-553495 nucleotides may play important roles in the replication process of BmNPV.

[0047] Example 3

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

[0049] Furthermore, 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 nucleotide and piR-bmo-553495 nucleotide on BmNPV viral replication

[0054] The specific experimental steps for transfecting piRNA nucleotide mimics into silkworm BmN cells are as follows:

[0055] BmN cells were transfected with piRNA nucleotide mimicry (10 μg / well). Cells were collected at 24 h and 48 h post-transfection, and RNA samples were extracted and prepared. The expression efficiency of piRNA nucleotide mimicry in BmN cells was detected using relative quantification. The results showed that piRNA was significantly upregulated in BmN cells. Figure 3 and Figure 5 As shown.

[0056] 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 hpi and 48 hpi post-transfection, and changes in the viral gene vp39 were detected using a relative quantification method. The results are as follows: Figure 4 and Figure 6 .

[0057] Depend on Figure 3 , Figure 4 It was found that 24 hours after transfection with the piR-bmo-504830 nucleotide mimic, the relative expression level of piR-bmo-504830 nucleotide was significantly upregulated, but the expression of the vp39 gene did not change significantly at 24 hours post-infection. 48 hours after transfection, the relative expression level of piR-bmo-504830 nucleotide had reached approximately 600, while the relative expression level of the viral vp39 gene was significantly upregulated, increasing from 1.0 to approximately 1.5. This suggests that piR-bmo-504830 nucleotide may affect viral replication in the later stages of viral infection.

[0058] On the contrary Figure 5 , Figure 6 After 24 h and 48 h of transfection with the piR-bmo-553495 nucleotide mimic, the relative expression level of piR-bmo-553495 nucleotide was significantly upregulated. Meanwhile, the relative expression level of the Bm NPV virus gene vp39 did not change significantly at 24 hpi and 48 hpi of viral infection, fluctuating around 1.1.

[0059] In summary, from Figure 1-2 It can be seen that BmNPV virus infection of cells, without the addition of nucleotide mimics, increased the relative expression levels of both piR-bmo-504830 and piR-bmo-553495 nucleotides. Figure 3-6 It was observed that the expression levels of piR-bmo-504830 and piR-bmo-553495 nucleotides were significantly upregulated upon the addition of nucleotide mimics. However, at 24 hpi post-infection, the expression level of BmNPV virus vp39 in both types of cells did not show significant changes. Furthermore, at 48 hpi, only transfection with the piR-bmo-504830 nucleotide mimic significantly increased the expression level of the BmNPV virus vp39 gene in cells. Therefore, piR-bmo-504830 nucleotide promotes BmNPV virus replication, and this effect occurs in the later stages, while piR-bmo-553495 nucleotide has no significant effect on BmNPV virus replication.

[0060] Example 5

[0061] Verify the role of antisense nucleotides in piRNA nucleotides that promote BmNPV replication.

[0062] Transfecting silkworm BmN cells with piRNA antisense nucleotides, the specific experimental steps are as follows:

[0063] BmN cells were transfected with piRNA antisense nucleotides (10 μg / well). Cells were collected at 24 h and 48 h post-transfection, and RNA samples were prepared. The inhibitory efficiency of piRNA antisense nucleotides on piRNA nucleotides in BmN cells was detected by relative quantification. The results showed that the expression of piR-bmo-504830 nucleotide and piR-bmo-553495 nucleotide was significantly inhibited in BmN cells. The results are as follows: Figure 7 and Figure 9 As shown.

[0064] BmN cells were then transfected with piRNA antisense nucleotides (10 μg / well). 24 h post-transfection, cells were infected with BmNPV-eGFP recombinant fluorescent reporter virus (multiple of infection 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 h post-infection and 48 h post-infection, and changes in the viral gene vp39 were detected using relative quantification methods. The results are as follows: Figure 8 and Figure 10 As shown.

[0065] Depend on Figure 7 , Figure 8 It was found that after transfecting BmN cells with antisense nucleotides of piR-bmo-504830, the relative expression level of piR-bmo-504830 showed a significant decreasing trend at 24h and 48h, decreasing from 1.0 to 0.01. Furthermore, after transfecting BmN cells with antisense nucleotides of piR-bmo-504830 and infecting them with BmNPV virus for 24hpi and 48hpi, the relative expression level of the BmNPV virus vp39 gene showed a significant decreasing trend, decreasing from 1.0 to approximately 0.75.

[0066] Observe again Figure 9 , Figure 10 It was found that after transfecting BmN cells with the antisense nucleotide of piR-bmo-553495, the relative expression level of piR-bmo-553495 nucleotide also showed a significant decreasing trend at 24h and 48h, decreasing from about 1.0 to about 0.3. However, after BmN cells transfected with the antisense nucleotide of piR-bmo-553495 were infected with BmNPV virus for 24hpi, the relative expression level of the viral vp39 gene did not change, while at 48hpi, the relative expression level of the BmNPV virus vp39 gene showed a significant increasing trend, increasing from about 1.0 to about 1.6.

[0067] Furthermore, combining Table 2 and Figure 1-2It is evident that piRNA nucleotides that elicit a strong response to BmNPV infection do not necessarily maintain extremely high expression levels for extended periods after BmNPV infection.

[0068] On the other hand, observation Figure 1-10 It is evident that, firstly, the expression levels of piR-bmo-504830 nucleotide and piR-bmo-553495 nucleotide significantly increased within 24 hours after infection with BmNPV virus, with the increase in piR-bmo-553495 nucleotide being even more pronounced.

[0069] Further observation Figure 3-6 It is evident that when we attempted to increase the levels of both piR-bmo-504830 and piR-bmo-553495 mimics, they did not show a significant promoting trend in the expression level of the vp39 gene within 24 hours, meaning they did not significantly promote the infection or replication of 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 their corresponding nucleotides, the relative expression of vp39 in the piR-bmo-504830 inhibitor group showed a significant downward trend within 24 hours.

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

[0072] In summary, from Figure 1-10 In our study, we found that when infected with BmNPV virus for 24 hours post-infection, viral infection significantly increased the relative expression levels of two nucleotides; however, observation... Figure 3-6 It can be seen that the piR-bmo-504830 nucleotide mimic significantly promoted BmNPV viral replication at 48 hpi; further observation... Figure 7-10 It can be seen that the antisense nucleotides of piR-bmo-504830 can significantly inhibit the replication of BmNPV virus at both 24hpi and 48hpi.

[0073] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of the antisense nucleotide of piR-bmo-504830 nucleotide in the preparation of a drug for inhibiting the replication of silkworm nucleopolyhedrovirus, wherein the antisense nucleotide of piR-bmo-504830 nucleotide is designed for piR-bmo-504830 nucleotide, and the sequence of piR-bmo-504830 nucleotide is shown in SEQ ID NO: 1; The sequence of the antisense nucleotide of the piR-bmo-504830 nucleotide is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that, The antisense nucleotide of the piR-bmo-504830 nucleotide inhibits the replication of silkworm nucleopolyhedrovirus by suppressing the expression of vp39.

3. The use according to claim 1, characterized in that, The drug is a drug that inhibits the replication of silkworm nucleopolyhedrovirus in silkworm ovarian cells.

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