Protein for improving yield of nuclear polyhedrosis virus and application thereof

Abstract

The invention relates to a protein for improving the yield of a nuclear polyhedrosis virus and application of the protein. The amino acid sequence of the protein is shown as SEQ ID No. 1 or SEQ ID No. 5. Under the condition of producing the same amount of the nuclear polyhedrosis virus, the production cost of the nuclear polyhedrosis virus as a biopesticide can be greatly reduced by using the two proteins.

Description

Technical Field

[0001] The invention relates to the field of biocides, and in particular to a protein for increasing the yield of nuclear polyhedrosis virus and an application thereof. Background Art

[0002] Nuclear polyhedrovirus (NPV) is a type of virus belonging to the Baculoviridae family, which mainly infects insects, especially Lepidoptera, Hymenoptera, and Diptera. The main species are Autographa californica NPV and Spodoptera litura NPV. Among them, Autographa californica NPV is one of the most representative viruses in the Baculoviridae family and is also the most intensively studied NPV. Autographa californica ) as its natural host, but it can also infect a variety of lepidopteran insect cell lines. This type of virus is named because it forms polyhedrons (protein inclusion bodies) in host cells. Among them, the polyhedrons contain a large number of virus particles or virus bundles.

[0003] Polyhedrosis can remain active in the environment for a long time and release virus particles to infect new hosts under appropriate conditions. Therefore, polyhedrosis can be prepared into virus products as biological pesticides to control harmful insects that it can infect. However, the production of polyhedrosis viruses has been in need of improvement in order to reduce production costs. Summary of the invention

[0004] One aspect of the present invention provides a protein, whose amino acid sequence is shown as SEQ ID No. 1 or SEQ ID No. 5.

[0005] The second aspect of the present invention provides a nucleic acid encoding the protein as described in one of the present invention.

[0006] In a specific embodiment, the base sequence of the nucleic acid is shown as SEQ ID No. 2 or SEQ ID No. 6.

[0007] The third aspect of the present invention provides the use of the protein according to the first aspect of the present invention or the nucleic acid according to the second aspect of the present invention in increasing the yield of nuclear polyhedrosis virus.

[0008] In a specific embodiment, the nuclear polyhedrosis virus is Autographa californica nuclear polyhedrosis virus and / or Spodoptera litura nuclear polyhedrosis virus.

[0009] In a specific embodiment, the cells used for preparing the nuclear polyhedrosis virus are Sf9 cells and / or High Five cells.

[0010] In a specific embodiment, the insects used for preparing the nuclear polyhedrosis virus are Autographa californica ( Autographa californica ), and / or Spodoptera litura ( Spodoptera litura ).

[0011] Advantages of the present invention: The present invention discovers that the proteins encoded by the two genes Innexin1 and Innexin2 of Microplitis bicoloratus can increase the yield of nuclear polyhedrosis virus. In the case of producing the same amount of nuclear polyhedrosis virus, this will greatly reduce the production cost of nuclear polyhedrosis virus as a biological pesticide. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shows the Western blot of the expression and nucleocytoplasmic separation of pIZT / V5-His-Mb-inx1 recombinant plasmid and pIZT / V5-His-Mb-inx2 recombinant plasmid in High Five cells.

[0013] Figure 2 Shows the plasmid map of the donor plasmid DpieG-polh-pFB.

[0014] Figure 3 Shows the ectopic expression of Mb-Inx1 in Sf9 cells infected with reBac-GFP-Polh and the detection of the production of nuclear polyhedra.

[0015] Figure 4 Shows the ectopic expression of Mb-Inx2 in Sf9 cells infected with reBac-GFP-Polh and the detection of the production of nuclear polyhedra. DETAILED DESCRIPTION OF THE INVENTION

[0016] The above content of the present invention will be further described in detail below in the form of preferred implementation cases, but it does not constitute a limitation to the present invention.

[0017] Unless otherwise specified, the strains, plasmid vectors, and reagents in the examples of the present invention can be purchased through commercial channels. Example 1: Expression and localization of Mb-inx1 and Mb-inx2 in High Five cells 1. Cloning of the full lengths of Mb-inx1 and Mb-inx2

[0018] The Innexin1 protein of Microplitis bicoloratus (Mb-inx1) consists of 364 amino acids, as shown in SEQ ID No. 1; the cDNA of Mb-inx1 is 1095 bp in total, as shown in SEQ ID No. 2. The specific primers for amplifying the full length of Mb-inx1 are Mb-inx1-F (as shown in SEQ ID No. 3) and Mb-inx1-R (as shown in SEQ ID No. 4).

[0019] The Innexin2 protein of Microplitis bicoloratus (Mb-inx2) consists of 358 amino acids, as shown in SEQ ID No. 5; the cDNA of Mb-inx2 is 1077 bp in total, as shown in SEQ ID No. 6. The specific primers for amplifying the full length of Mb-inx2 are Mb-inx2-F (as shown in SEQ ID No. 7) and Mb-inx2-R (as shown in SEQ ID No. 8).

[0020] Total RNA was extracted from 10 3-day-old larvae of Microplitis bicoloratus, and then reverse transcription was carried out using the PrimeScript™ RT reagent Kit with gDNA Eraser kit (product number RR047A) to obtain cDNA.

[0021] Using the obtained cDNA as a template and Mb-inx1-F and Mb-inx1-R as primers, PCR amplification of Mb-inx1 was performed to obtain the Mb-inx1 PCR product. After detecting the Mb-inx1 PCR product, it was ligated to the cloning vector pBM16A-T (Biomed, product number CL071-01), transformed into Escherichia coli DH5α, single colonies were picked, and after plasmid extraction, double digestion of the extracted plasmid with Sac I and Hind III, PCR amplification and correct sequencing identification, the pBM16A-Mb-inx1 recombinant plasmid containing the target fragment and the DH5α / pBM16A-Mb-inx1 recombinant strain were obtained.

[0022] Using the obtained cDNA as a template and Mb-inx2-F and Mb-inx2-R as primers, PCR amplification of Mb-inx2 was performed to obtain the Mb-inx2 PCR product. After detecting the Mb-inx2 PCR product, it was ligated to the cloning vector pBM16A-T, transformed into Escherichia coli DH5α, single colonies were picked, and after plasmid extraction, double digestion of the extracted plasmid with Sac I and Xba I, PCR amplification and correct sequencing identification, the pBM16A-Mb-inx2 recombinant plasmid containing the target fragment and the DH5α / pBM16A-Mb-inx2 recombinant strain were obtained.

[0023] Extract the pBM16A-Mb-inx1 recombinant plasmid from the DH5α / pBM16A-Mb-inx1 recombinant strain. After double-digesting pBM16A-Mb-inx1 with Sac I and Hind III, use a gel extraction kit (product number D2500-02-200) to recover the Mb-inx1 gene fragment. Double-digest the pIZT / V5-His plasmid (Thermo fisher, containing 6 histidine tags) with Sac I and HindIII in the same way, and then use a gel extraction kit (product number D2500-02-200) to recover the linear fragment of the pIZT / V5-His vector. Ligate the recovered Mb-inx1 gene fragment with the recovered linear fragment of the pIZT / V5-His vector using T4 ligase for 12 h, transform it into Escherichia coli DH5α, pick single colonies, and after extracting the plasmid, double-digesting the extracted plasmid with Sac I and Hind III, and PCR amplification and sequencing identification are correct, obtain the positive pIZT / V5-His-Mb-inx1 recombinant plasmid and the DH5α / pIZT / V5-His-Mb-inx1 recombinant strain.

[0024] Extract the pBM16A-Mb-inx2 recombinant plasmid from the DH5α / pBM16A-Mb-inx2 recombinant strain. After double-digesting pBM16A-Mb-inx2 with Sac I and Xba I, use a gel extraction kit (product number D2500-02-200) to recover the Mb-inx2 gene fragment. Double-digest the pIZT / V5-His plasmid (Thermo fisher, containing 6 histidine tags) with Sac I and Xba I in the same way, and then use a gel extraction kit (product number D2500-02-200) to recover the linear fragment of the pIZT / V5-His vector. Ligate the recovered Mb-inx2 gene fragment with the recovered linear fragment of the pIZT / V5-His vector using T4 ligase for 12 h, transform it into Escherichia coli DH5α, pick single colonies, and after extracting the plasmid, double-digesting the extracted plasmid with Sac I and Xba I, and PCR amplification and sequencing identification are correct, obtain the positive pIZT / V5-His-Mb-inx2 recombinant plasmid and the DH5α / pIZT / V5-His-Mb-inx2 recombinant strain. 2. Transfection of pIZT / V5-His-Mb-inx1 into High Five and extraction of total protein and separation of nuclear protein

[0025] (2,3-Dioleoxypropyl)-trimethylammonium chloride (DOTAP) was purchased from Sigma, product number D-6182; 1,2-Dioleayl-sn-glycero-3-phosphoethanolamine (1,2-Dioleagl-sn-gly -3-phosphoethanolamine) was purchased from Sigma, product number BBCB6052.

[0026] Preparation of transfection reagent Cell fection II: First, dissolve 0.01 g (2,3-dioleyloxypropyl)-trimethylammonium chloride in 1 mL of chloroform to obtain (2,3-dioleyloxypropyl)-trimethylammonium chloride solution; then take 60 μL of (2,3-dioleyloxypropyl)-trimethylammonium chloride solution and mix it with 40 μL of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine to obtain Cell fection II, and store it at 4 degrees Celsius away from light.

[0027] Complete medium used for culturing cells: 45.7 g of Gibco powdered medium (purchased from Thermofisher), 3.33 g of yeast extract (purchased from Thermofisher), 3.33 g of casein hydrolysate (purchased from Thermofisher) and 0.35 g of sodium bicarbonate (purchased from Thermofisher) were dissolved in 1 liter of water, filtered and sterilized with a vacuum filter (NEST), and then a final volume of 10% FBS fetal bovine serum (purchased from Rambolide) was added.

[0028] Plasmid extraction: pIZT / V5-His-Mb-inx1 recombinant plasmid was extracted from DH5α / pIZT / V5-His-Mb-inx1 recombinant strain according to the instructions of EZNA Endo-free Plasmid DNA Mini Kit I (Cat. No. D6950-01B).

[0029] Prepare the transfection reagent complex: take 100 μl of serum-free and antibiotic-free double-free culture medium and place it in a 1.5 ml centrifuge tube, then add 2 μg of the extracted pIZT / V5-His-Mb-inx1 recombinant plasmid to obtain a transfection plasmid dilution solution; take 100 μl of serum-free and antibiotic-free double-free culture medium and place it in another 1.5 ml centrifuge tube, then add 6 μl of transfection reagent Cell fection II to it, shake and mix, and obtain a transfection reagent dilution solution; add the transfection reagent dilution solution to the transfection plasmid dilution solution, mix evenly, then let it stand at room temperature for 45 minutes, and shake a few times every 15 minutes to obtain a transfection reagent complex.

[0030] Transfection of High Five cells (from Trichoplusia ni): Place complete culture medium in a 25 cm 2A culture flask is used to culture High Five cells. When the growth density of High Five cells reaches 80% to 90%, the old culture medium is removed, 5 mL of fresh complete culture medium is added, and the suspended cells are gently pipetted with a pipette tip. Subsequently, 90 μL of cell suspension is taken and 10 μL of trypan blue is added, gently mixed, left standing for 3 min, and counted using a hemocytometer; after counting, 2×10 6 cells are added to a 6-well plate and cultured adherently at 27 °C for more than 2 hours until the cells adhere and are in good condition. After the cells adhere completely, High Five cells are starved for 30 min using serum-free and antibiotic-free medium. 800 μL of serum-free and antibiotic-free medium is added to the transfection reagent complex, mixed evenly, and then added drop by drop to a 60 mm culture dish containing High Five cells that have been starved for 30 min. Gently shake to make the mixture uniform, and then continue to culture at 27 °C for 5 hours. Discard the culture solution to obtain HighFive / pIZT / V5-His-Mb-inx1 recombinant transient expression cells. Subsequently, complete culture medium is added thereto and cultured for 72 h.

[0031] Extraction of total protein: (1) After culturing High Five / pIZT / V5-His-Mb-inx1 recombinant transient expression cells for 72 hours, aspirate the culture medium, wash 3 times with 1 ml of 1×PBS buffer, and then add 1 ml of 1×PBS; (2) Blow the cells off the adherent state, transfer them to a 1.5 ml EP tube, place on ice, centrifuge at a relative centrifugal force of 5000 g at 4 °C for 5 min, and discard the supernatant; (3) Add 80 μL of RIPA mixture (RIPA:PMSF = 100:1; RIPA is purchased from Solarbio, product number R0010; PMSF is purchased from Solarbio, product number P0100), shake once every 5 min for a total of three times, and then centrifuge at a relative centrifugal force of 13000 g at 4 °C for 10 min; (4) Transfer the supernatant to a new EP tube to obtain a total protein extract, and the total protein extract contains the Mb-inx1 fusion protein fused with V5 and His tags; (5) Use a BCA protein quantification kit (DingGuo, product number BCA02) to measure the total protein concentration in the total protein extract.

[0032] Nuclear protein isolation: (1) After culturing High Five / pIZT / V5-His-Mb-inx1 recombinant transient expression cells for 72 hours, aspirate the culture medium, wash 3 times with 1 ml of 1×PBS, and then add 1 ml of 1×PBS; (2) Blow the cells off the adherent state, transfer them to a 1.5 ml EP tube, place on ice, centrifuge at 5000 g relative centrifugal force at 4 °C for 5 min, and discard the supernatant; (3) Use a nuclear protein kit (BestBio, BB-3102-50T, Shanghai, China) to extract nuclear proteins and cytoplasmic proteins; (4) Use a BCA protein quantification kit to measure the concentrations of nuclear proteins and cytoplasmic proteins. 3. Transfection of pIZT / V5-His-Mb-inx2 into High Five and extraction of total proteins and isolation of nuclear proteins

[0033] Transfect the pIZT / V5-His-Mb-inx2 recombinant plasmid into HighFive cells by the same operation as in the second subsection of this example to obtain High Five / pIZT / V5-His-Mb-inx2 recombinant transient expression cells. Then extract the total proteins to obtain a total protein extract, which contains the fusion protein of Mb-inx2 fused with V5 and His tags. Use a BCA protein quantification kit to measure the concentrations of total proteins, nuclear proteins, and cytoplasmic proteins. 4. Transfection of pIZT / V5-His into High Five and extraction of total proteins

[0034] Transfect the pIZT / V5-His empty vector into High Five cells by the same operation as in the second subsection of this example to obtain High Five / pIZT / V5-His recombinant transient expression cells. Then extract the total proteins to obtain a total protein extract. As a negative control group, the extracted total proteins are called negative control total proteins. Use a BCA protein quantification kit to measure the concentration of total proteins in the total protein extract. 5. Extraction of total proteins from High Five

[0035] Extract the total proteins by the same operation as in the second subsection of this example to obtain a total protein extract. As a blank control group, the extracted total proteins are called blank control total proteins. Use a BCA protein quantification kit to measure the concentration of total proteins in the total protein extract. 6. Western blot detection

[0036] After performing SDS-PAGE on the total proteins of High Five / pIZT / V5-His-Mb-inx1 (denoted as pIZT / V5-His-Mb-inx1), HighFive / pIZT / V5-His-Mb-inx2 (denoted as pIZT / V5-His-Mb-inx2), High Five / pIZT / V5-His (denoted as pIZT / V5-His), and High Five cells (denoted as Ctrl), PVDF membrane transfer was carried out. Incubation was performed with Anti-V5 (R960-25, purchased from Invirotrogen) as the primary antibody. After washing the membrane, incubation was carried out with HRP-labeled goat anti-mouse IgG (H+L) (A0216, purchased from Beyotime) as the secondary antibody. After washing the membrane again, chemiluminescence was performed using a horseradish peroxidase (HRP) chemiluminescent substrate, and imaging was carried out by exposure with Flour Chem EFE0511, as shown in Figure 1 。

[0037] Next, the nuclear reference protein antibody Anti-LaminA / C (GB11407, purchased from Servicebio) was used as the primary antibody, and the PVDF membrane after Anti-V5 imaging was incubated with it. After washing the membrane, incubation was carried out with HRP-labeled goat anti-rabbit IgG (H+L) (A0208, purchased from Beyotime) as the secondary antibody. After washing the membrane again, chemiluminescence was performed using a horseradish peroxidase (HRP) chemiluminescent substrate, and imaging was carried out by exposure with FlourChem EFE0511, as shown in Figure 1 。

[0038] Finally, the internal reference tubulin antibody Anti-Tubulin (M1000130, purchased from Solarbio) was diluted as the primary antibody, and the PVDF membrane after Anti-LaminA / C imaging was incubated with it. After washing the membrane, incubation was carried out with HRP-labeled goat anti-mouse IgG (H+L) (A0216, purchased from Beyotime) as the secondary antibody. After washing the membrane again, chemiluminescence was performed using a horseradish peroxidase (HRP) chemiluminescent substrate, and imaging was carried out by exposure with FlourChem EFE0511, as shown in Figure 1 。

[0039] It can be seen from Figure 1 that Mb-inx1 and Mb-inx2 were successfully expressed in High Five cells. In addition, nuclear protein separation showed that Mb-Inx1 and Mb-Inx2 were localized in the nucleus and cytoplasm. Example 2: Mb-inx1 and Mb-inx2 Promote the Formation of Polyhedra and Increase the Yield of Baculovirus 1. Preparation of Baculovirus

[0040] Autographa californica multiple nucleopolyhedrovirus exists in nature in two forms. One form is the occlusion-derived virus (ODV), which is the nucleopolyhedrovirus form. It mainly forms in the nuclei of infected hosts and is responsible for spreading between hosts. In this state, virions are present within occlusion bodies, and the occlusion bodies protect the virions, enabling them to remain stable in the environment. The other form is the budded virus (BV), which is released by budding through the plasma membrane of infected cells and is responsible for spreading between the cells of the host, that is, it helps the virus to spread within the host. These two forms play different roles in the virus's life cycle to ensure the effective spread of the virus both inside and outside the host.

[0041] The plasmid map of the donor plasmid DpieG-polh-pFB is shown in Figure 2 , which was kindly provided by Northwest A&F University. In this plasmid, IEI-GFP-polyA is the GFP expression cassette, and PP-Polh is the Polh expression cassette. Among them, IEI is the promoter of the GFP gene, and PP is the promoter of the Polh gene (i.e., the gene of polyhedrin). Two tandem GFP expression cassettes and one Polh expression cassette constitute the target fragment to be transposed in the donor plasmid.

[0042] Escherichia coli DH10Bac was purchased from Thermo Fisher Scientific. This Escherichia coli contains Bacmid (which was modified from Autographa californica multiple nucleopolyhedrovirus) and a helper plasmid (Helper). For detailed information, see the product manual. Among them, Bacmid produces budded virus after infecting cells.

[0043] The donor plasmid DpieG-polh-pFB was transformed into Escherichia coli DH10Bac. Through transposition, the target fragment in the donor plasmid was transposed into Bacmid, and the recombinant Bacmid (named reBac-GFP-Polh) was obtained. The DH10Bac containing this recombinant Bacmid reBac-GFP-Polh is the DH10Bac / reBac-GFP-Polh recombinant strain. The recombinant Bacmid reBac-GFP-Polh produces occlusion-derived virus, that is, nucleopolyhedrovirus, after infecting cells.

[0044] reBac-GFP-Polh was extracted from the DH10Bac / reBac-GFP-Polh recombinant strain according to the instructions of the E.Z.N.A. Endo-free Plasmid DNA Mini Kit I (product number D6950-01B).

[0045] Preparation of plasmid transfection reagent complex: Pipette 100 μL of serum-free and antibiotic-free medium into a 1.5 mL centrifuge tube, and then add 2 μg of reBac-GFP-Polh DNA to obtain a transfected plasmid dilution; Pipette 100 μL of serum-free and antibiotic-free medium into another 1.5 mL centrifuge tube, and then add 6 μL of transfection reagent Cell fection II, shake and mix well to obtain a transfection reagent dilution; Add the transfection reagent dilution to the transfected plasmid dilution, mix evenly, and then let it stand at room temperature for 45 min, shaking it a few times every 15 min to obtain a transfection reagent complex.

[0046] Transfection of Sf9 cells (derived from Spodoptera frugiperda): Place the complete medium in a 25 cm 2 culture flask for culturing Sf9 cells. When the growth density of Sf9 cells reaches 80% to 90%, remove the old medium, add 5 mL of new complete medium, gently pipette the suspended cells, then take 90 μL of cell suspension and add 10 μL of trypan blue, mix gently, let it stand for 3 min, and count with a hemocytometer; After counting, take 2×10 6 cells and add them to a 6-well plate, and culture them adherently at 27 °C for more than 2 h until the cells adhere and are in good condition. After the cells adhere completely, starve the Sf9 cells with serum-free and antibiotic-free medium for 30 min. Add 800 μL of serum-free and antibiotic-free medium to the transfection reagent complex, mix well, and add it drop by drop to a 60 mm culture dish containing Sf9 cells that have been starved for 30 min, shake gently to make the mixture uniform, and then continue to culture at 27 °C for 5 h, discard the culture medium to obtain Sf9 / reBac-GFP-Polh cells for the preparation of P1 generation nucleopolyhedrovirus.

[0047] Preparation of P1 generation nucleopolyhedrovirus: After discarding the culture medium as above, culture the above Sf9 / reBac-GFP-Polh cells with fresh complete medium. When cultured for 72 h, a small amount of cells lysed and released P1 generation nucleopolyhedrovirus. At this time, the fluorescent cells basically accounted for about 80%. When cultured for 96 h, a large amount of P1 generation nucleopolyhedrovirus was released. At this time, start to collect the virus, that is, gently suspend the cells with a pipette, transfer the cell suspension to a centrifuge tube, and then centrifuge at a relative centrifugal force of 500 g at room temperature for 5 min, take the supernatant, which contains P1 generation nucleopolyhedrovirus, wrap it with tin foil and store it at 4 °C in the dark.

[0048] Preparation of P2 generation nucleopolyhedrovirus: Spread 2×10 6A number of Sf9 cells were cultured in complete medium. In each well, three concentration gradients of 1%, 5%, and 10% of the P1 generation nuclear polyhedrosis virus were inoculated according to the volume of the medium. Subsequently, the cell status was observed every 24 hours. It was observed whether the number of cells doubled after 24 hours of infection, whether the cells stopped proliferating 48 hours and later after infection, and whether the cells lysed massively; after 96 hours, the wells with an inoculation concentration of 10% were selected to collect the cell suspension and centrifuged at a relative centrifugal force of 500 g for 5 min at room temperature. The supernatant was taken, which contained the P2 generation nuclear polyhedrosis virus, wrapped with tin foil and stored in the dark at 4 °C.

[0049] Preparation of P3 generation nuclear polyhedrosis virus: 2×10 6 A number of Sf9 cells were cultured in complete medium. In each well, three concentration gradients of 0.5%, 1%, and 5% of the P2 generation nuclear polyhedrosis virus were inoculated according to the volume of the medium. Subsequently, the cell status was observed every 24 hours. It was observed whether the number of cells doubled after 24 hours of infection, whether the cells stopped proliferating 48 hours and later after infection, and whether the cells lysed massively; subsequently, after 96 hours, the wells with an inoculation concentration of 5% were selected to collect the cell suspension and centrifuged at a relative centrifugal force of 500 g for 5 min at room temperature. The supernatant was taken, which contained the P3 generation nuclear polyhedrosis virus, wrapped with tin foil and stored in the dark at 4 °C. 2. Detection of the effect of Mb-Inx1 on nuclear polyhedrosis virus

[0050] Plasmid extraction: The pIZT / V5-His-Mb-inx1 recombinant plasmid was extracted from the DH5α / pIZT / V5-His-Mb-inx1 recombinant strain according to the instructions of the E.Z.N.A. Endo-free Plasmid DNA Mini Kit I (product number D6950-01B).

[0051] Preparation of plasmid transfection reagent complex: Pipette 100 μl of serum-free and antibiotic-free medium into a 1.5 ml centrifuge tube, and then add 2 μg of the pIZT / V5-His-Mb-inx1 recombinant plasmid to obtain a transfected plasmid dilution; take 100 μl of serum-free and antibiotic-free medium and place it in another 1.5 ml centrifuge tube, and then add 6 μl of the transfection reagent Cell fection II, shake and mix well to obtain a transfection reagent dilution; add the transfection reagent dilution to the transfected plasmid dilution, mix evenly, and then let it stand at room temperature for 45 min, and shake it a few times every 15 minutes to obtain the transfection reagent complex.

[0052] Transfection of Sf9 cells with pIZT / V5-His-Mb-inx1 and infection with reBac-GFP-Polh nucleopolyhedrovirus: Place complete medium in a 25 cm 2 culture flask for culturing Sf9 cells. When the growth density of Sf9 cells reaches 80%-90%, remove the old medium, add 5 mL of fresh complete medium, gently pipette to resuspend the cells, then take 90 μL of cell suspension and add 10 μL of trypan blue, mix gently, let stand for 3 min, and count using a hemocytometer; after counting, take 2×10 6 cells and add them to a 6-well plate, and culture adherently at 27 °C for more than 2 hours until the cells adhere and are in good condition. After the cells adhere completely, starve Sf9 cells with serum-free medium for 30 min. Add 800 μL of serum-free medium to the transfection reagent complex, mix well, and add it drop by drop to a 60 mm culture dish containing Sf9 cells that have been starved for 30 min, gently shake to mix evenly, then continue to culture at 27 °C for 5 hours, discard the culture medium to obtain Sf9 / pIZT / V5-His-Mb-inx1 recombinant transient expression cells. Then add fresh complete medium to it. 48 hours after transfection, remove the medium in the culture dish, add fresh complete medium again, and then inoculate 5% of the P3 generation of reBac-GFP-Polh nucleopolyhedrovirus into each well. Take this as zero time, and then take pictures every 24 hours, continuously shooting until 120 hours. Use Sf9 cells transfected with pIZT / V5-His empty plasmid and infected with reBac-GFP-Polh nucleopolyhedrovirus as a control. After taking pictures, count the percentage of the number of cells producing nucleopolyhedrovirus in each treatment group accounting for the total number of cells. Data analysis uses t-test, ns indicates no difference, p<0.01, ** indicates significant difference, p<0.001, *** indicates extremely significant difference, see Figure 3 . It can be seen from Figure 3 that transfection of pIZT / V5-His-Mb-inx1 and infection with reBac-GFP-Polh nucleopolyhedrovirus significantly increased the production of nucleopolyhedrovirus in Sf9 cells with ectopic expression of Mb-Inx1 compared with the control group. 3. Detection of the effect of Mb-Inx2 on nucleopolyhedrovirus

[0053] Plasmid extraction: Extract the pIZT / V5-His-Mb-inx2 recombinant plasmid from the DH5α / pIZT / V5-His-Mb-inx2 recombinant strain according to the instructions of E.Z.N.A. Endo-free Plasmid DNA Mini Kit I (product number D6950-01B).

[0054] Transfect the pIZT / V5-His-Mb-inx2 recombinant plasmid into Sf9 cells and infect them with reBac-GFP-Polh nucleopolyhedrovirus using the same operation as in the second subsection of this example. Take pictures every 24 hours starting from the time point when the P3 generation of reBac-GFP-Polh nucleopolyhedrovirus is added, and continuously shoot until 120 hours. Use Sf9 cells transfected with the pIZT / V5-His empty plasmid and infected with reBac-GFP-Polh nucleopolyhedrovirus as a control. After shooting, count the percentage of cells producing nucleopolyhedra in each treatment group among the total number of cells. Data analysis uses the t-test, ns indicates no difference, p < 0.01, and ** indicates significant difference. See Figure 4 . It can be seen from Figure 4 that when the cells are transfected with pIZT / V5-His-Mb-inx1 and infected with reBac-GFP-Polh nucleopolyhedrovirus, the heterologous expression of Mb-Inx2 in Sf9 cells significantly increases the yield of nucleopolyhedrovirus compared with the control group.

[0055] The above results indicate that Mb-Inx1 and Mb-Inx2 of Microplitis bicoloratus can promote the formation of nucleopolyhedra and increase the yield of nucleopolyhedrovirus.

Claims

1. A protein, the amino acid sequence of which is shown in SEQ ID No. 1 or SEQ ID No.

5.

2. A nucleic acid encoding the protein according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The base sequence of the nucleic acid is shown as SEQ ID No. 2 or SEQ ID No.

6.

4. Use of the protein according to claim 1 or the nucleic acid according to claim 2 or 3 in increasing the yield of nuclear polyhedrosis virus.

5. The use according to claim 4, characterized in that: The nuclear polyhedrosis virus is Spodoptera californica nuclear polyhedrosis virus and / or Spodoptera litura nuclear polyhedrosis virus.

6. The use according to claim 4, characterized in that: The cells used to prepare the nuclear polyhedrosis virus are Sf9 cells and / or High Five cells.

7. The use according to claim 4, characterized in that: The insect used to prepare the nuclear polyhedrosis virus is Spodoptera californica ( Autographa californica ) and / or Spodoptera litura ( Spodoptera litura ).

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