A cytokine-like protein of Bombyx mori and its uses

Abstract

This application belongs to the field of biotechnology and provides the use of a cytokine-like protein of Bombyx mori as an active ingredient of a drug for inhibiting Bombyx mori nucleopolyhedrovirus. The amino acid sequence of the cytokine-like protein of Bombyx mori is shown as SEQ ID NO: 1, and this protein can inhibit the replication of Bombyx mori nucleopolyhedrovirus. In addition, this application also provides a cytokine-like protein of Bombyx mori, which is obtained by amplifying a cytokine-like plasmid of Bombyx mori with a PCR primer set to obtain a cytokine-like amplification product of Bombyx mori, then double-digesting the cytokine-like amplification product and the cytokine-like plasmid of Bombyx mori with restriction enzymes respectively, purifying the digested products and then ligating them with T4 ligase, transforming Escherichia coli DH5α competent cells, screening positive clones, and extracting plasmids.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and particularly to a cytokine-like protein of Bombyx mori and its uses. Background Art

[0002] Bombyx mori nucleopolyhedrovirus (BmNPV-eGFP) is a typical circular double-stranded DNA baculovirus. As an important viral pathogen of Bombyx mori, the septicemia it causes is one of the most common and severe infectious diseases in sericulture production, causing extremely serious harm to the sericulture industry and resulting in serious economic losses to sericulture production every year. However, at present, no effective treatment for BmNPV-eGFP has been found.

[0003] Chinese Patent Application No. 202410339412.1 discloses an expression method of the antiviral gene Vago protein of Procambarus clarkii, which includes the following steps: cloning the antiviral gene Vago of Procambarus clarkii; constructing a recombinant plasmid pET28a-Vago using the prokaryotic expression vector pET28a, and then inducing expression with 16 degrees of IPTG; the target protein can be found in the supernatant to complete the protein expression of Vago of Procambarus clarkii; injecting the expressed and purified Vago protein and white spot syndrome virus WSSV into Procambarus clarkii successively, and then culturing in a specific temperature environment; extracting the blood of Procambarus clarkii to detect the expression changes of major immune genes.

[0004] In this method, the antiviral mechanism of Procambarus clarkii is deeply explored, and the antiviral function of Vago is used to prevent and treat viral diseases of Procambarus clarkii. This scheme extracts the antiviral gene Vago from Procambarus clarkii and uses it for the prevention and treatment of viral diseases of Procambarus clarkii.

[0005] Chinese Patent Application No. 202311457695.1 discloses an application of glutathione in the preparation of drugs for preventing or treating Bombyx mori nucleopolyhedrovirus disease. By using a drug containing glutathione with an effective concentration of 5 mM to 10 mM, the glutathione against Bombyx mori nucleopolyhedrovirus can be reasonably utilized to inhibit its replication in cells, and it can play a therapeutic role when mixed in the food of Bombyx mori for consumption during infection; it can also be fed by adding glutathione 24 hours before virus infection to inhibit the proliferation of BmNPV-eGFP and play a preventive role against infection.

[0006] This method shows that the small peptide glutathione of Bombyx mori can effectively inhibit the replication of Bombyx mori nucleopolyhedrovirus (BmNPV-eGFP) in Bombyx mori cells. Therefore, glutathione can be used to inhibit the replication of this virus in Bombyx mori cells and in the body of Bombyx mori through appropriate concentration and drug addition time.

[0007] Problems to be solved by this solution: How to propose a new way to inhibit the replication of Bombyx mori nucleopolyhedrovirus in Bombyx mori. Summary of the Invention

[0008] The object of the present invention is to provide a Bombyx mori cytokine-like protein capable of effectively inhibiting the replication of Bombyx mori nucleopolyhedrovirus in Bombyx mori, and this protein inhibits the replication of Bombyx mori nucleopolyhedrovirus through the Jak-STAT signaling pathway.

[0009] To achieve the above object, the present application discloses the use of a Bombyx mori cytokine-like protein as an active ingredient of a drug for inhibiting Bombyx mori nucleopolyhedrovirus.

[0010] Preferably, the amino acid sequence of the Bombyx mori cytokine-like protein is as shown in SEQ ID NO: 1;

[0011] The coding nucleotide sequence of the Bombyx mori cytokine-like protein is as shown in SEQ ID NO: 2.

[0012] Preferably, the Bombyx mori cytokine-like protein inhibits the replication of Bombyx mori nucleopolyhedrovirus through the Jak-STAT signaling pathway.

[0013] In addition, a Bombyx mori cytokine-like protein is also disclosed, which is obtained by amplifying a Bombyx mori cytokine-like plasmid with a PCR primer set to obtain a Bombyx mori cytokine-like amplification product, then double-digesting the Bombyx mori cytokine-like amplification product and the Bombyx mori cytokine-like plasmid with endonucleases respectively, purifying the digested products and then ligating them with T4 ligase, transforming Escherichia coli DH5α competent cells, screening positive clones, and extracting plasmids.

[0014] Preferably, the PCR primer set includes an upstream primer F1 and a downstream primer R1;

[0015] The sequence of the upstream primer F1 is as shown in SEQ ID NO: 3;

[0016] The sequence of the downstream primer R1 is as shown in SEQ ID NO: 4.

[0017] Preferably, the endonucleases are respectively the nucleic endonuclease BamHI enzyme for the Bombyx mori cytokine-like amplification product and the restriction endonuclease SacI enzyme for the Bombyx mori cytokine-like plasmid.

[0018] Advantages of the present application:

[0019] The present application provides a cytokine-like protein of silkworm that can effectively inhibit the replication of Bombyx mori nucleopolyhedrovirus in silkworms. This protein inhibits the replication of Bombyx mori nucleopolyhedrovirus through the Jak-STAT signaling pathway, and this protein can also provide a theoretical reference for the development of drugs against BmNPV-eGFP. Description of the Drawings

[0020] Figure 1 It is the expression results of cytokine-like in silkworm cells after BmNPV-eGFP infects silkworm BmN cells for 24h, 48h, and 72h;

[0021] Figure 2 It is the expression quantity results of cytokine-like in silkworm tissues after BmNPV-eGFP infects silkworm tissues for 72h;

[0022] Figure 3 It is the result of signal peptide prediction for the protein using the NovoPro website, where SP (Sec / SPI) is the signal peptide type, CS is the signal peptide cleavage site, and OTHER is other signal peptides;

[0023] Figure 4 It is the protein result of detecting cytokine-like in silkworms by Western Blot. The left part of the bands is the result of detecting cytokine expression in the culture medium supernatant; the right part of the bands is the result of detecting cytokine expression in the cell precipitate;

[0024] Figure 5 It is the result of detecting the expression quantity of vp39 after the overexpression vector BmLOC101736094 is transfected into BmN cells and then infects BmNPV-eGFP at 36h and 48h;

[0025] Figure 6 It is the result of detecting protein expression by SDS-PAGE. Among them, M is the protein Marker, 1 and 2 are empty vectors, ①②③ represent different positive strains, 3, 7, and 11 are uninduced bacterial solutions, 4, 8, and 12 are induced bacterial solutions, 5, 9, and 13 are precipitates of induced bacterial solutions, and 6, 10, and 14 are supernatants of induced bacterial solutions;

[0026] Figure 7 It is the result of purifying the protein by SDS-PAGE. Each lane is respectively: M is the protein Marker, 1 is the original bacterial solution, 2 is the supernatant flow-through solution after the bacterial solution is broken, FT1 is the 20mM imidazole washing solution, FT2 is the 50mM imidazole washing solution, FT3 is the 100mM imidazole washing solution, and E1~E6 are the 250mM imidazole elution solutions;

[0027] Figure 8Results of verifying the purified protein by Western Blot, where each lane is as follows: M is the protein Marker, 1 is the protein solution before renaturation, and 2 is the protein solution after renaturation;

[0028] Figure 9 Cytotoxicity results after treating BmN cells with silkworm cytokine-like proteins at concentrations of 0.6 μg / mL, 1.8 μg / mL, and 3 μg / mL respectively;

[0029] Figure 10 For knocking out the cell line STAT KO Results of the expression level of vp39 at 48 h and 72 h after adding BmNPV-eGFP;

[0030] Figure 11 Inhibitory results of the replication of the vp39 gene of BmNPV-eGFP by 0.6 μg / mL of silkworm cytokine-like protein;

[0031] Figure 12 For knocking out the cell line STAT KO Results of the expression level of vp39 of the BmNPV-eGFP gene after adding silkworm cytokine-like protein. Detailed implementation methods

[0032] In the description of this application, it should be noted that for those not specifying specific conditions in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0033] The sources of the reagents are shown in Table 1:

[0034] Table 1

[0035]

[0036]

[0037] Example 1

[0038] Changes in the transcriptional level of silkworm cytokine-like in BmN cells after BmNPV-eGFP infection. The specific experimental procedure is as follows:

[0039] (1) Inoculate silkworm ovarian cells (BmN) into a 12-well cell culture plate and incubate overnight in an incubator at 28°C. Add the BmNPV-eGFP recombinant fluorescent reporter virus (multiplicity of infection is 5) to the culture plate. After incubating at 28°C for 1 h, replace it with fresh Grace medium containing 10% FBS. Collect cell samples at 24 h, 48 h, and 72 h after infection respectively.

[0040] (2) By the method of injecting through the caudal legs, 5 μL of the recombinant fluorescent reporter virus BmNPV-eGFP was injected into the larvae of the "Dazao" variety of silkworms at the 5th instar stage. The silkworm tissue samples were collected 3 days after infection: fat body, midgut, hemocytes, head, silk gland, Malpighian tubules, trachea, and epidermis.

[0041] (3) 100 μL of Trizol lysis solution (RNA extraction reagent) was added to the harvested cells, and 1 mL of Trizol solution was added to the silkworm tissues for homogenization. Total RNA was extracted from both cells and tissues using the Kit RNA fast 2000 (RNA extraction kit) according to the manufacturer's instructions, and the RNA was reverse-transcribed into cDNA using the kit of gDNA Eraser (reverse transcription kit).

[0042] (4) Using TIF4A as an internal reference, the transcriptional levels of cytokine-like in silkworm cells after infection with BmNPV-eGFP were detected by real-time fluorescence quantitative PCR. The PCR reaction system was configured according to the instructions of the iTaqTM Universal SY Green SupermixKit reagents of Bio-Rad Company, USA. qPCR detection was performed using a fluorescence quantitative PCR instrument (CFXTMOptics Module of Bio-Rad Company, USA).

[0043] The results are as Figure 1 shown. Within 24 hpi, 48 hpi, and 72 hpi after infection with the BmNPV-eGFP virus, the expression level of the silkworm cytokine BmLOC101736094 in silkworm cells increased significantly at a multiple rate, and it was found through Figure 2 that the expression level of the silkworm cytokine BmLOC101736094 was most significantly up-regulated in the three major tissues of silkworm larvae, namely the midgut, hemocytes, and Malpighian tubules.

[0044] Example 2

[0045] Prove that BmLOC101736094 is a type of secreted protein and can play an antiviral role at the protein level

[0046] (1) The silkworm ovary cells (BmN) were inoculated into a 12-well cell culture plate and cultured overnight in an incubator at 28 °C. The constructed overexpression vector pIEX-BmLOC101736094 was transfected into BmN cells at a concentration of 1000 ng per well, and the Western Blot cell samples were collected at 24 h, 48 h, 72 h, and 96 h after transfection respectively.

[0047] (2) The harvested cells were added with 80 μL of RIPA cell lysate and 2 μL of PMSF protease inhibitor. After ice-bathing for 30 min, 20 μL of 5× loading Buffer was added to prepare protein samples for subsequent Western Blot.

[0048] (3) Similarly, silkworm ovary cells (BmN) were inoculated into a 12-well cell culture plate and cultured overnight in an incubator at 28 °C. The constructed overexpression vector pIEX-BmLOC101736094 was transfected into BmN cells at a concentration of 1000 ng per well. After 24 h, 1 MOI of BmNPV-eGFP virus was added for infection, and then RNA cell samples were collected at 36 h and 48 h.

[0049] (4) 100 μL of Trizol lysate (RNA extraction reagent) was added to the harvested cells, and 1 mL of Trizol solution was added to the silkworm tissues for homogenization. Total RNA was extracted from both cells and tissues using Kit RNA fast 2000 (RNA extraction kit) according to the manufacturer's instructions, and the RNA was reverse-transcribed into cDNA using the gDNA Eraser (reverse transcription kit).

[0050] (5) Using TIF4A as an internal reference, the transcriptional level of cytokine-like in silkworm cells after infection with BmNPV-eGFP was detected by real-time fluorescence quantitative PCR. The PCR reaction system was prepared according to the instructions of the iTaqTM Universal SY Green SupermixKit reagents from Bio-Rad Company, USA. qPCR detection was performed using a fluorescence quantitative PCR instrument (CFXTM Optics Module from Bio-Rad Company, USA).

[0051] The results were as Figure 3 shown. BmLOC101736094 contains a signal sequence of 17 amino acids at the 5' end, indicating that BmLOC101736094 is a secreted protein containing a signal peptide.

[0052] As Figure 4As shown, after the constructed overexpression vector pIEX-BmLOC101736094-his was transfected into cells, the intracellular reference antibody GAPDH was used for detection. Clear and bright bands could be detected from the cell pellet, while no bands were detected in the culture medium supernatant, thus proving that this sample was the culture medium supernatant of the cells. Then, the expression of the target protein could be detected in both the culture medium supernatant and the pellet of the cells using the tag antibody anti-his. It can be seen from this that the sample of the culture medium supernatant of the cells was correct and the expression of the target protein BmL OC101736094 could be detected. Therefore, it was indicated that the silkworm cytokine BmLOC101736094 was a type of secreted protein;

[0053] As Figure 5 shown, when the silkworm ovarian cells transfected with the BmLOC101736094 secreted protein were infected with the BmNPV-eGFP virus, compared with the control group, at 36 hpi after virus infection, the expression level of vp39 in the BmNPV-eGFP virus decreased from 1.0 to around 0.6, showing a significant change. It can be seen from this that the BmLOC101736094 secreted protein could inhibit the replication of the BmNPV-eGFP virus. And further observing at 48 hpi, the expression level of vp39 in the BmNPV-eGFP virus could further decrease from 0.6 to around 0.5, and the inhibitory effect of the BmLOC101736094 secreted protein could be further enhanced.

[0054] Example 3

[0055] Codon optimization of the silkworm cytokine-like gene and construction of the expression vector

[0056] Obtain the open reading frame sequence of the cytokine-like gene of Bombyx mori (Gene ID: 101736094) from NCBI. First, perform bioinformatics analysis on this sequence. The sequence contains a 17-amino acid signal peptide at the 5' end. Optimize the codons of this gene sequence, add a BamHI restriction site at the 5' end, add a SacI restriction site at the 3' end, remove the signal peptide sequence MTMLAFVLVGLITTISA, and add a 6*His tag at the C-terminus to obtain the codon-optimized Bombyx mori cytokine-like gene. Its amino acid sequence is as follows: MTMLAFVLVGLITTISAGPSNDSFKDQYCTDPRTMQKHAAYSEWADAYSCTRHRCQPGGRNLAIYTVGCKRVEAPESAIECEEVVEDTNMQFPFCCTRLRCLVVVRGEVWTRVLGQPWETLPAAPWSHMYKMKKPPPGDTSFLNKETEQKGPVYELSEQDAPKHEQVLRSEKRTTEDPNCKEAVLRSAPTPDIVIALSTNNDEKEPKRDIERRKRVHEIPDEERNEPENQDVEELPASTDKPLGNSDEAYNSNTDAMTKKPKTKIQNQVTWTEIPPNQWTERDPSMDAENVKDVQEEKKEVKSSNLQALVDAIGTRMRDIETVVQKMSQKVQQVKPEDAVASSEKRSDEGKRHERKEHEKNYDELKKPSPDGDHYKRFVVSSHSKYLHSADGVTEKPLYIEDNTNGYYSDASNSVIRRVNVPKEPSPTYMAPVETRRKSIHPVPENSEEVGKKRKKHSHKKKGKGKSHKKHRKDEKRRRYNRISSEVDKNVVSLEDNSANKK。

[0057]

[0058] Using the synthesized codon-optimized Bombyx mori cytokine-like gene sequence as a template, PCR amplification was performed with the primers in Table 2 to obtain the Bombyx mori cytokine-like fragment. The amplified product was purified and recovered. The amplified product and the pET-28a plasmid were respectively double-digested with BamHI and SacI enzymes. After purifying the digested products, they were ligated with T4 ligase, transformed into Escherichia coli DH5α competent cells, positive clones were screened, plasmids were extracted, and digestion verification was carried out. The recombinant vector verified by digestion was sent to BGI Genomics Guangzhou Branch for sequencing confirmation, and the recombinant plasmid pET-28a-BmLOC101736094 Bombyx mori cytokine-like was obtained.

[0059] Table 2

[0060]

[0061] Example 4

[0062] Expression and purification of BmLOC101736094 Bombyx mori cytokine-like

[0063] The successfully constructed recombinant plasmid pET-28a-BmLOC101736094 of Bombyx mori cytokine-like was transferred into Escherichia coli Rosetta(DE3) competent cells by heat shock method. Positive clones were screened on kanamycin and chloramphenicol resistant plates, and double digestion identification was carried out to further verify the prokaryotic expression recombinant positive plasmid.

[0064] The re-identified Bombyx mori cytokine-like Rosetta(DE3) E.coli / pET-28a-BmLOC101736094 was inoculated into LB liquid medium containing kanamycin and cultured with shaking at 37°C. When the OD 600 value reached 0.6, IPTG with a final concentration of 0.1 mmol / L was added, and induction continued at 32°C for 8 h. Then, the cells were collected by centrifugation at 8000 g / min for 5 min, resuspended in PBS at a volume ratio of 1:10, sonicated, and the supernatant / precipitate was collected for SDS-PAGE detection.

[0065] From Figure 6 it can be seen that compared with the empty vector group, protein contents were detected in the whole bacteria, precipitate, and supernatant of three different positive strains; and the protein expression levels in lanes 5, 9, and 13 were significantly higher than those in lanes 6, 10, and 14, that is, the protein content in the strain precipitate was higher than that in the supernatant.

[0066] Example 5

[0067] Extraction of expression product and protein renaturation and purification

[0068] (1) Extraction and treatment of inclusion bodies:

[0069] Centrifuge 500 ml of the recombinant bacteria E. coli / pET-28a-BmLOC101736094 induced for expression at 8000 g / min for 10 min to collect the bacterial cells, resuspend the bacterial cells in ultrasonic buffer at a ratio of 1:10, and ultrasonically disrupt them sufficiently on ice. The ultrasonic buffer is 100 mM NaH2PO4, 300 mM NaCl, 100 mM Tris, pH 7.2; then centrifuge at 4°C and 12000 g / min for 20 min to collect the precipitate, and this precipitate is the crude inclusion body.

[0070] Wash the crude inclusion body collected by centrifugation with the washing buffer by magnetic stirring at room temperature for 1 h. The washing buffer is 100 mM NaH2PO4, 300 mM NaCl, 100 mM Tris, 2% NP-40, pH 7.2, and then centrifuge at 4°C and 12000 g / min for 20 min.

[0071] Then wash the inclusion body with washing solution 2 and washing solution 3 in sequence; the washing solution 2 is 100 mM NaH2PO4, 300 mM NaCl, 100 mM Tris, 2 M Urea, pH 7.2; the washing solution 3 is 100 mM NaH2PO4, 300 mM NaCl, 100 mM Tris, 4 M Urea, pH 7.2.

[0072] (2) Denaturation of inclusion body

[0073] Add the inclusion body denaturation solution to the washed inclusion body at a ratio of 1:10. The denaturation solution is 100 mM NaH2PO4, 300 mM NaCl, 100 mM Tris, 8 M Urea, pH 7.2, stir magnetically at room temperature for 2 h, centrifuge at 4°C and 12000 g / min for 30 min to collect the supernatant; filter the supernatant with a 0.45 mm filter membrane and store it at 4°C.

[0074] (3) Purification of inclusion body

[0075] Purify the supernatant with His-tag according to the nickel column purification instruction manual of Solarbio company, and then perform SDS-PAGE analysis on the purified product.

[0076] The results are as Figure 7 、 Figure 8 shown, where Figure 7The results of SDS-PAGE purified protein are shown as follows. In lane 1, the expression of the target protein was detected in the stock solution. In lane 2, the flow-through solution after the stock solution containing the target protein was bound to the nickel column. Lanes FT1, FT2, and FT3 were elution solutions containing 20 mM, 50 mM, and 100 mM imidazole respectively, which could remove some non-target proteins. Then, the target protein eluted from the nickel column was detected in the elution solutions of lanes E1, E2, E3, E4, and E5. Figure 8 The results of Western Blot verification of the purified protein showed that the electrophoretic band of the SDS-PAGE purified protein was single.

[0077] Example 6

[0078] (1) Cytotoxicity experiment

[0079] According to the instructions of the Cell Counting Kit-8 kit (purchased from Beyotime Biotechnology Co., Ltd.), BmN cells were inoculated into a 96-well plate with a cell density of 1×10 4 cells / mL. BmN cells were treated with silkworm cytokine-like protein at concentrations of 0.6 μg / mL, 1.8 μg / mL, and 3 μg / mL for 24 h. Then, the medium in each well was replaced with a medium containing CCK-8 (10 μL per well) and cultured for another 4 h. The OD 450nm value was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0080] It can be seen from Figure 9 that with the increase of the protein dosage, there was no obvious effect on cell growth activity. According to the principle of the minimum protein dosage, 0.6 μg / ml was determined as the optimal concentration of the protein.

[0081] (2) Cell antiviral experiment

[0082] After infecting the STAT KO cell line with 1 MOI of BmNPV-eGFP, the supernatant was replaced with Grace medium (10% FBS) 1 h later. Cell samples were collected at 48 h and 72 h after virus infection. Total RNA was extracted according to the instructions of the Feijie Total RNA Rapid Extraction Kit, and the expression changes of the viral vp39 gene were detected using real-time fluorescence quantitative PCR.

[0083] Figure 10 The results showed that after knocking out the key gene STAT in the Jak-STAT signaling pathway in the STAT KO cell line, the expression level of the BmNPV-eGFP virus vp39 gene in the STAT KO cell line was significantly up-regulated. It can be seen from this that the Jak-STAT signaling pathway plays an important role in inhibiting the replication of the BmNPV-eGFP virus.

[0084] (3) The antiviral function of the silkworm cytokine-like protein is exerted through the Jak-STAT signaling pathway

[0085] Subsequently, BmN cells were treated with 0.6 μg / mL of the silkworm cytokine-like protein for 24 h and then infected with 1 MOI of BmNPV-eGFP. PBS was used as the control group. After 1 h, the supernatant was replaced with Grace medium (10% FBS). Cell samples were collected at 24 h, 48 h, and 72 h after virus infection. Total RNA was extracted according to the instructions of the Feijie Total RNA Rapid Extraction Kit, and the expression changes of the viral vp39 gene were detected using real-time fluorescence quantitative PCR.

[0086] Figure 11 The results showed that after adding the silkworm cytokine-like protein to BmN cells, it was found by real-time fluorescence quantitative PCR that compared with the control group, in BmN cells transfected with the silkworm cytokine-like protein, within 24 hpi, 48 hpi, and 72 hpi after infection with BmNPV-eGFP virus, the expression levels of the vp39 gene in BmNPV-eGFP virus all showed a significant decrease, and further observation Figure 11 showed that the inhibitory effect of the silkworm cytokine protein on BmNPV-eGFP virus was stronger at 24 hpi after BmNPV-eGFP virus infection, decreasing from 1.0 to about 0.8, a decrease of 0.2, and only decreasing to 0.7 and 0.6 at 48 hpi and 72 hpi respectively, a decrease of 0.1.

[0087] In summary, the silkworm cytokine-like protein can effectively inhibit the replication of BmNPV-eGFP virus in BmN cells.

[0088] Meanwhile, KO-STAT cell lines were treated with 0.6 μg / mL of the silkworm cytokine-like protein for 24 h and then infected with 1 MOI of BmNPV-eGFP. PBS was used as the control group. After 1 h, the supernatant was replaced with Grace medium (10% FBS). Cell samples were collected at 48 h and 72 h after virus infection. Total RNA was extracted according to the instructions of the Feijie Total RNA Rapid Extraction Kit, and the expression changes of the viral vp39 gene were detected using real-time fluorescence quantitative PCR.

[0089] Figure 12 The results showed that in STAT KOAfter adding the silkworm cytokine-like protein to the cell line, real-time fluorescence quantitative PCR detection showed that the expression level of the vp39 gene in the BmNPV-eGFP virus was significantly increased at 48 hpi. At the same time, after observing for another 72 hpi and comparing with the control group, it was also found that the expression level of the vp39 gene in the BmNPV-eGFP virus was still significantly increased.

[0090] And further observation Figure 11 、 Figure 12 showed that in normal BmN cells, the BmLOC101736094 protein could inhibit the replication of the BmNPV-eGFP virus, while in the STAT KO cells with the Jak-STAT signaling pathway knocked out, the BmLOC101736094 protein could not inhibit the proliferation of the virus. From this, it can be seen that the silkworm cytokine protein BmLOC101736094 inhibits the infection of BmNPV-eGFP through the Jak-STAT signaling pathway.

[0091] 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a cytokine-like protein of Bombyx mori in the preparation of a drug for inhibiting Bombyx mori nucleopolyhedrovirus; The cytokine-like protein of Bombyx mori as an active ingredient of a drug for inhibiting Bombyx mori nucleopolyhedrovirus; The amino acid sequence of the cytokine-like protein of Bombyx mori is as shown in SEQ ID NO: 1; The coding nucleotide sequence of the cytokine-like protein of Bombyx mori is as shown in SEQ ID NO:

2.

2. The use according to claim 1, wherein The cytokine-like protein of Bombyx mori inhibits the replication of Bombyx mori nucleopolyhedrovirus through the Jak-STAT signaling pathway.

Images