Use of a Bombyx mori lysozyme, a lysozyme gene, an overexpression system and a cell

Abstract

The present invention belongs to the biological field and discloses a bombyx mori lysozyme, whose nucleotide coding sequence is shown in SEQ ID NO.1 or SEQ ID NO.2. This bombyx mori lysozyme has not been found in previous studies, and no one has studied and analyzed the properties of these two bombyx mori lysozymes. By overexpressing the coding nucleotide of this lysozyme, the function of inhibiting the replication of BmNPV can be achieved very well. At the same time, the present invention also discloses the uses of the lysozyme gene, as well as the overexpression system and cells.

Description

Technical Field

[0001] The present invention belongs to the field of biology, and specifically relates to the uses of a silkworm lysozyme and a lysozyme gene, as well as an overexpression system and cells. Background Art

[0002] Insect innate immune pathways, such as Imd (Immune deficiency), JAK / STAT (Janus kinase / signal transducer and activator of transcription), STING (Stimulator of interferon genes), and autophagy, play important roles in antiviral immune responses. These innate immune signaling pathways can regulate the expression of effector molecules, including antimicrobial peptides (AMPs) and lysozyme (LYZ), to effectively combat bacterial and viral infections.

[0003] Lysozyme is a hydrolase that can dissolve bacterial cell walls. It is mainly expressed in the skin, mucosa, and immune cells, showing strong bactericidal activity. As an antimicrobial peptide, lysozyme has also been found to have antiviral properties. Different from antibiotics, lysozyme has different antibacterial mechanisms. Therefore, it is less likely to induce antibiotic resistance in pathogenic bacteria, indicating that lysozyme may have great potential in resisting pathogen invasion.

[0004] Silkworms are important economic insects with a long history, significantly increasing farmers' income. Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical baculovirus that can rapidly cause the death of silkworms and shows strong infectivity. Silkworms have several lysozyme genes, all belonging to the c-type lysozyme category. Among them, BmC-LZM has been determined to have the ability to inhibit the replication of BmNPV in silkworm larvae. However, it is currently unclear whether other lysozymes in silkworms have the same antiviral function.

[0005] The technical problem to be solved by the present invention is: how to discover new lysozyme genes to enhance or enrich related preparations and applications for inhibiting baculoviruses. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a silkworm lysozyme that has not been found in previous studies, and no one has studied and analyzed the performance of these two silkworm lysozymes. By overexpressing the coding nucleotides of this lysozyme, the function of inhibiting the replication of BmNPV can be very good.

[0007] Meanwhile, the present invention also discloses the uses of the lysozyme gene, as well as an overexpression system and cells.

[0008] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0009] A silkworm lysozyme, whose nucleotide coding sequence is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0010] Meanwhile, the present invention also discloses the use of the nucleotide coding sequences shown in SEQ ID NO.1 or SEQ ID NO.2 to prepare silkworm cells overexpressing the lysozyme gene or a virus expression system overexpressing the lysozyme gene.

[0011] In the above use, the silkworm cells are silkworm BmN cells;

[0012] The virus expression system is a baculovirus expression system.

[0013] Meanwhile, the present invention also discloses the use of the above-mentioned silkworm lysozyme to prepare a preparation for inhibiting the replication of baculovirus or a drug for preventing and treating silkworms infected with baculovirus.

[0014] Finally, the present invention also discloses a silkworm BmN cell overexpressing the lysozyme gene, which is obtained by transfecting silkworm BmN cells with an overexpression plasmid; the silkworm lysozyme gene carried by the overexpression plasmid is as shown in SEQ ID NO.1 and / or SEQ ID NO.2.

[0015] In addition, a baculovirus expression system overexpressing the lysozyme gene, in which the silkworm lysozyme gene shown in SEQ ID NO.1 and / or SEQ ID NO.2 is inserted and can be expressed.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention provides two exogenous overexpression vectors, baculovirus expression vectors and products corresponding to two silkworm lysozyme genes through genetic engineering related technologies, and proves that these two genes have the function of improving the resistance of silkworm cells and larvae to BmNPV infection. Constructing BmC-LZM-like2 and BmC-LZM-like4 of the present invention into an insect expression vector and transferring them into silkworm BmN cells for overexpression has the function of inhibiting the replication of BmNPV; constructing BmC-LZM-like2 and BmC-LZM-like4 of the present invention into a silkworm baculovirus expression system and infecting silkworm larvae has the function of inhibiting the replication of BmNPV.

[0018] 2. No one has discovered and analyzed the functions of the above-mentioned BmC-LZM-like2 and BmC-LZM-like4 genes, and the research of the present invention has strong foresight. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] BmC-LZM1 (nucleotide sequence SEQ ID NO.5, amino acid sequence SEQ ID NO.6), BmC-LZM2 (nucleotide sequence SEQ ID NO.7, amino acid sequence SEQ ID NO.8), BmC-LZM3 (nucleotide sequence SEQ ID NO.9, amino acid sequence SEQ ID NO.10), BmC-LZM-like1 (nucleotide sequence SEQ ID NO.11, amino acid sequence SEQ ID NO.12), BmC-LZM-like2 (nucleotide sequence SEQ ID NO.1, amino acid sequence SEQ ID NO.3), BmC-LZM-like3 (nucleotide sequence SEQ ID NO.13, amino acid sequence SEQ ID NO.14), and BmC-LZM-like4 (nucleotide sequence SEQ ID NO.2, amino acid sequence SEQ ID NO.4)

[0020] Figure 1 is the transcription level of BmC-LZM1 in silkworm cells;

[0021] Figure 2 is the transcription level of BmC-LZM2 in silkworm cells;

[0022] Figure 3 is the transcription level of BmC-LZM3 in silkworm cells;

[0023] Figure 4 is the transcription level of BmC-LZM-like1 in silkworm cells;

[0024] Figure 5 is the transcription level of BmC-LZM-like2 in silkworm cells;

[0025] Figure 6 is the transcription level of BmC-LZM-like3 in silkworm cells;

[0026] Figure 7 is the transcription level of BmC-LZM-like4 in silkworm cells;

[0027] Figure 8 is the transcription level of BmC-LZM-like2 in larval tissues;

[0028] Figure 9 is the transcription level of BmC-LZM-like4 in larval tissues;

[0029] Figure 10 is the plasmid map of pIEX-BmC-LZM-like4-v5;

[0030] Figure 11 It is the plasmid map of pIEX - BmC - LZM - like2 - v5;

[0031] Figure 12A It is the over - expression result of BmC - LZM - like2 - v5 in Bombyx mori BmN cells

[0032] Figure 12B It is the over - expression result of BmC - LZM - like4 - v5 in Bombyx mori BmN cells;

[0033] Figure 12C It is the over - expression result of BeGFP in Bombyx mori BmN cells;

[0034] Figure 12D It is the result figure of detecting that over - expression of BmC - LZM - like2 - v5 inhibits the replication of BmNPV gene vp39 by real - time fluorescence quantitative PCR;

[0035] Figure 12E It is the result figure of detecting that over - expression of BmC - LZM - like4 - v5 inhibits the replication of BmNPV gene vp39 by real - time fluorescence quantitative PCR;

[0036] Figure 12F It is the result figure of over - expression of BmC - LZM - like2 - v5 inhibiting the replication of BmNPV virus particles;

[0037] Figure 12G It is the result figure of over - expression of BmC - LZM - like4 - v5 inhibiting the replication of BmNPV virus particles;

[0038] Figure 13A It is the schematic diagram of the construction of the Bombyx mori baculovirus expression system;

[0039] Figure 13B It is the schematic diagram of using the recombinant strain to infect BmN cells to produce recombinant baculoviruses BmNPV - eGFP - BmC - LZM - like2 - v5 and BmNPV - eGFP - BmC - LZM - like4 - v5 (scale bar: 200 microns);

[0040] Figure 13C It is the result figure of using the recombinant baculovirus to infect Bombyx mori larvae to express BmC - LZM - like2 and BmC - LZM - like4.

[0041] Figure 13DInject the recombinant viruses BmNPV-eGFP-BmC-LZM-like2-v5 and BmNPV-eGFP-BmC-LZM-like4-v5 into the larvae of Bombyx mori, and use the BmNPV-eGFP recombinant virus as a control. Extract the total DNA 96 hours after inoculation, and the result graph of measuring the BmNPV DNA level by real-time fluorescence quantitative PCR;

[0042] Figure 14A Results of overexpression of BmC-LZM-like2-v5 and BmC-LZM-like4-v5 detected by real-time fluorescence quantitative PCR after virus inoculation;

[0043] Figure 14B Statistical results between overexpression of BmC-LZM-like2-v5 and BmC-LZM-like4-v5 and survival rate after virus inoculation. Specific implementation mode

[0044] The technical solution of the present invention will be further described below through specific implementation modes. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0045] First part: Identification and identification of Bombyx mori BmCLZM gene

[0046] Lysozyme genes are widely distributed in various species including insects. In this embodiment, by analyzing the silkworm of the "Dazao" variety, 7 lysozyme genes were screened and named BmC-LZM1 (nucleotide sequence SEQ ID NO.5, amino acid sequence SEQ ID NO.6), BmC-LZM2 (nucleotide sequence SEQ ID NO.7, amino acid sequence SEQ ID NO.8), BmC-LZM3 (nucleotide sequence SEQ ID NO.9, amino acid sequence SEQ ID NO.10), BmC-LZM-like1 (nucleotide sequence SEQ ID NO.11, amino acid sequence SEQ ID NO.12), BmC-LZM-like2 (nucleotide sequence SEQ ID NO.1, amino acid sequence SEQ ID NO.3), BmC-LZM-like3 (nucleotide sequence SEQ ID NO.13, amino acid sequence SEQ ID NO.14) and BmC-LZM-like4 (nucleotide sequence SEQ ID NO.2, amino acid sequence SEQ ID NO.4);

[0047] The amino acid sequence comparison of the above 7 lysozymes with that of silkworm showed a similarity of 31.88%. Domain analysis revealed that BmC-LZM1, BmC-LZM-like1, BmC-LZM-like2, and BmC-LZM-like4 of silkworm have typical LYZ1 domains, which are similar to those of rapeseed lysozyme. On the other hand, BmC-LZM2, BmC-LZM3, and BmC-LZM-like3 exhibited the pam-unstable enzyme domains typical of the lysozyme family. Most silkworm lysozyme genes consist of 3 - 5 exons; unexpectedly, the BmC-LZM-like4 gene has no intron region.

[0048] The relevant properties of the above 7 lysozymes can be referred to Figures 1 to 7 , in which there are obvious performance differences between the five of BmC-LZM1, BmC-LZM2, BmC-LZM3, BmC-LZM-like1, and BmC-LZM-like3 and the two of BmC-LZM-like4 and BmC-LZM-like2;

[0049] Figures 1 to 7 In, the expression levels of BmC-LZM-like4 and BmC-LZM-like2 were significantly increased compared with the NC blank group as the infection time increased.

[0050] Based on the above analysis, BmC-LZM-like2 gene and BmC-LZM-like4 were used for subsequent overexpression studies.

[0051] The second part: Changes in the transcriptional levels of BmC-LZM-like2 and BmC-LZM-like4 after BmNPV infection

[0052] 1.1 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 hour, replace it with fresh Grace medium containing 10% FBS (fetal bovine serum, purchased from Gibco, USA). Collect cell samples at 24, 48, and 72 hours after infection respectively.

[0053] 1.2 Inject 5 μL of the BmNPV-eGFP recombinant fluorescent reporter virus into the bodies of the 5th instar larvae of the "Dazao" variety of silkworm by tail-foot injection. Collect the fat body tissue samples of silkworm at 24, 48, and 72 hours after infection respectively.

[0054] 1.3 Add 100 μL of Trizol lysis buffer (RNA extraction reagent, purchased from Invitrogen, USA) to the harvested cells, and add 1 mL of Trizol solution to the silkworm tissues for homogenization. Total RNA was extracted from both cells and tissues using Kit RNA fast 2000 (RNA extraction kit, purchased from Feijie Co., China) according to the manufacturer's instructions, and the RNA was reverse-transcribed into cDNA using the gDNA Eraser (reverse transcription kit, purchased from TaKaRa, Japan).

[0055] 1.4 Using TIF4A as an internal reference, the transcriptional levels of BmC-LZM-like2 and BmC-LZM-like4 after infection with BmNPV were detected by real-time fluorescence quantitative PCR. Configure the PCR reaction system according to the instructions of the iTaqTM Universal GreenSupermix Kit reagents from Bio-Rad, USA. Use a fluorescence quantitative PCR instrument (CFX TM Optics Module from Bio-Rad, USA) for qPCR.

[0056] The transcriptional level of BmC-LZM-like2 in silkworm cells can be seen Figure 5 and Figure 8 ; The transcriptional level of BmC-LZM-like4 in silkworm cells can be seen Figure 7 and Figure 9 ; The results showed that after infection with BmNPV, the transcriptional levels of BmC-LZM-like2 and BmC-LZM-like4 in silkworm cells and larval tissues were significantly up-regulated.

[0057] Part III Overexpression of BmC-LZM-like2 and BmC-LZM-like4 in silkworm BmN cells can inhibit the replication of BmNPV

[0058] 2.1 Using the cDNA of BmN cells as a template, the gene sequences encoding BmC-LZM-like2 and BmC-LZM-like4 were amplified by PCR, as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0059] The BmC-LZM-like2 gene and the BmC-LZM-like4 gene were respectively cloned into the pIEX insect overexpression plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) using two restriction enzyme sites, SacⅠ and SalⅠ, to construct BmC-LZM-like2 and BmC-LZM-like4 overexpression plasmids with a V5-tag (tag sequence), namely pIEX-BmC-LZM-like2-v5 (the plasmid structure diagram can be seen in Figure 11 ) and pIEX-BmC-LZM-like4-v5 plasmid (the plasmid structure diagram can be seen in Figure 10 ).

[0060] The nucleotide sequence of the BmC-LZM-like2 gene is shown in SEQ ID NO.1;

[0061] The nucleotide sequence of the BmC-LZM-like4 gene is shown in SEQ ID NO.2;

[0062] The amino acid sequence corresponding to the BmC-LZM-like2 gene is shown in SEQ ID NO.3;

[0063] The amino acid sequence corresponding to the BmC-LZM-like4 gene is shown in SEQ ID NO.4;

[0064] 2.2 Transfect BmN cells with pIEX-BmC-LZM-like2-v5 and pIEX-BmC-LZM-like4-v5 plasmids (1 μg / well), and pIEX-eGFP as a control. Collect cells at 24, 48, 72, and 96 hours after transfection and prepare protein samples. Detect the expression of BmC-LZM-like2-v5, BmC-LZM-like4-v5, and eGFP proteins by Western Blot (WB) using a mouse V5 antibody (purchased from Thermo, USA) and a mouse anti-GFP (purchased from Beyotime, China), and α-tubulin was used as an internal reference.

[0065] 2.3 After 24 hours of transfection, infect with BmNPV (multiplicity of infection is 5). Collect total cell RNA and supernatant at 24, 48, and 72 hours after infection to detect the infection of BmNPV using real-time fluorescence quantitative PCR and virus titer (TCID 50 ).

[0066] The results are as shown in Figures 12A to 12G .

[0067] The results showed that after transfection of pIEX-BmC-LZM-like2-v5, pIEX-BmC-LZM-like4-v5 and pIEX1-eGFP plasmids into BmN cells, the expression of BmC-LZM-like2-v5 ( Figure 12A ), BmC-LZM-like4-v5 ( Figure 12B ) and eGFP ( Figure 12C ) was successfully achieved in BmN cells. Through real-time fluorescence quantitative PCR and virus titer detection, it was found that overexpression of BmC-LZM-like2-v5 ( Figure 12D , Figure 12F ) and BmC-LZM-like4-v5 ( Figure 12E , Figure 12G ) could significantly inhibit the replication of vp39 protein and BmNPV in BmN cells.

[0068] Part 4 Overexpression of BmC-LZM-like2 and BmC-LZM-like4 in silkworm larvae using the baculovirus expression system can inhibit the replication of BmNPV

[0069] 3.1 First, to achieve the expression of BmC-LZM-like2 and BmC-LZM-like4 proteins, BmC-LZM-like2 and BmC-LZM-like4 together with the v5 tag were cloned into the pFBDM vector (model: LM-1183) through two restriction enzyme sites, SalⅠ and SacⅠ ( Figure 13A ).

[0070] Secondly, to observe the replication of baculovirus, the eGFP fluorescent reporter gene was also cloned into the pFBDM vector, and the dual-expression transfer vectors pFBDM-eGFP-BmC-LZM-like2-v5 and pFBDM-eGFP-BmC-LZM-like4-v5 were successfully constructed ( Figure 13A ).

[0071] Use the DAP-deficient rBmSw106-inv strain unique to our laboratory (full name: DAP auxotrophic E. coli BmMultiBac / △asd Sw106 / PGB2ΩInv strain, preserved by our laboratory, and relevant information can be found in "Study on the Effects of ie1, gp64 and ie0 Genes on the Proliferation and Replication of BmNPV by RNAi" by Yan Donghua, Master's Thesis, 2017) and the method of using the infection protein-mediated production of recombinant baculoviruses as an example. Transfer the dual-expression transfer vectors pFBDM-eGFP-BmC-LZM-like2-v5 and pFBDM-eGFP-BmC-LZM-like4-v5 into the rBmSw106-inv strain through the Tn7 transposition site to obtain the recombinant strains rBmSw106-eGFP-BmC-LZM-like2-v5 and rBmSw106-eGFP-BmC-LZM-like4-v5( Figure 13B ).

[0072] Use the recombinant strains to directly infect BmN cells, and finally obtain the recombinant baculoviruses BmNPV-eGFP-BmC-LZM-like2-v5 and BmNPV-eGFP-BmC-LZM-like4-v5 expressing BmC-LZM-like2, BmC-LZM-like4 and eGFP proteins( Figure 13C ).

[0073] Detect the expression of exogenous BmC-LZM-like2 and BmC-LZM-like4 proteins in the fat body tissue of silkworms by Western blot experiment( Figure 13D ).

[0074] By the method of caudal foot injection, infect silkworm larvae (fifth instar, day 1) with 5 μL of BmNPV-eGFP-BmC-LZM-like2-v5 and BmNPV-eGFP-BmC-LZM-like4-v5 (1×10 7.71 TCID 50 / mL), using the BmNPV-eGFP reporter virus as a control.

[0075] 3.2 After 96 hours of infection, dissect the silkworm bodies and collect the fat bodies. Extract the fat body DNA and detect the replication of viral DNA by real-time fluorescence quantitative PCR. At the same time, count the survival rate of silkworms.

[0076] The results are as Figure 14A and Figure 14BAs shown, the overexpression of BmC-LZM-like2 and BmC-LZM-like4 was successfully achieved in the fat body of silkworms using the baculovirus expression system of silkworms. Through real-time fluorescence quantitative PCR detection and survival rate statistics, it was found that the overexpression of BmC-LZM-like2-v5 and BmC-LZM-like4-v5 could significantly inhibit the replication of BmNPV in the fat body of silkworm larvae.

[0077] Result analysis:

[0078] 1. Two groups of lysozyme genes with significant effects were screened from several genes in the present invention; among them, the expression level of the BmC-LZM-like2 gene was significantly higher than that of BmC-LZM-like4 in both silkworm cells and larval tissues.

[0079] 2. In subsequent experiments, it was verified that the antiviral performance after the overexpression of BmC-LZM-like2 was significantly better than that of BmC-LZM-like4. Especially at 48 h of infection, its inhibition rate of vp39 protein and control of virus titer were significantly advantageous.

[0080] 3. In subsequent experiments, the overexpression of BmC-LZM-like2 and BmC-LZM-like4 was carried out in the fat body of silkworms using the baculovirus expression system of silkworms, further proving that the overexpression of BmC-LZM-like2-v5 was significantly superior to BmC-LZM-like4-v5 in inhibiting the replication of BmNPV.

[0081] 4. In summary, the greatest advantage of this case is that effective lysozyme genes were screened, and it was found that the BmC-LZM-like2 gene has the most powerful function.

[0082] The applicant declares that the present invention uses the above embodiments to illustrate the process method of the present invention, but the present invention is not limited to the above process steps, nor does it mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of the raw materials selected for the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of silkworm lysozyme in the preparation of a drug for treating silkworms infected with baculovirus; the baculovirus is Bombyx mori nucleopolyhedrovirus; the drug is a baculovirus expression system overexpressing the lysozyme gene; the silkworm lysozyme gene shown in SEQ ID NO. 1 is inserted into the baculovirus expression system and can be expressed.

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