Synergistic protein ORF42 of insect baculovirus and application thereof

Abstract

The invention relates to the technical field of prevention and control of agricultural and forestry pests, and discloses a synergistic protein ORF42 of insect baculovirus and application of the synergistic protein ORF42. According to the invention, a synergistic protein (ORF42) is obtained from an insect biocontrol resource, i.e., a Pinus fumosa nuclear polyhedrosis virus, and protein function verification shows that the protein has a remarkable synergistic effect on a fall webworm nuclear polyhedrosis virus, an apocheima cinerarius nuclear polyhedrosis virus and a Pinus fumosa nuclear polyhedrosis virus; the compound can be used as a synergistic factor to be added into insect viruses, and has important significance on prevention and control of major forestry pests (fall webworms, spring inchworm and smoke wing pine bees).

Description

Technical Field

[0001] This invention relates to the field of agricultural and forestry pest control technology, specifically to an enhancing protein ORF42 of insect baculoviruses and its applications. Background Technology

[0002] *Diprion infuscalae* (Wang & Wei, 2019), belonging to the family Diprionidae and genus *Diprion* in the order Hymenoptera, is a newly discovered species first discovered in Jiangxi, China in 2019 (Wang et al., 2019; 2024; Li et al., 2023; Ruan Jiaqing et al., 2020). The larvae of *Diprion infuscalae* feed gregariously on the needles of *Pinus massoniana* Lamb., and have already caused outbreaks and infestations in Youxi County, Fujian Province, and Huichang County, Jiangxi Province, China (Ruan Jiaqing et al., 2020). In 2019, *Diprion infuscalae* severely damaged 118 hectares of *Pinus massoniana* forests in Youxi State-owned Forest Farm and surrounding villages in Fujian Province alone. 2 (Ruan Jiaqing et al., 2020). According to the survey, the pine sawfly can have 2-3 generations per year in Xiwei Township, Youxi County. When it breaks out, the insect population density can reach tens of thousands per tree. The affected forest stands are 100% infested, and the needles are completely eaten, resembling fire damage, which seriously affects the normal growth and development of Masson pine forests.

[0003] Insect viral insecticides are one of the important means of biological control of agricultural and forestry pests, and baculoviruses are playing an increasingly important role as efficient expression systems for exogenous eukaryotic genes and as microbial insecticides in the control of agricultural and forestry pests. However, the low virulence and slow insecticidal speed of baculoviruses are key limiting factors for their application. Therefore, improving the virulence of baculoviruses and accelerating their insecticidal speed has become an important research topic for baculovirus insecticides. Constructing biosynergists into engineered baculoviruses is the main strategy to solve this problem, and developing and utilizing such synergists is also an important way to expand the application of baculovirus insecticides. Summary of the Invention

[0004] In view of this, the present invention provides an enhancing protein ORF42 for insect baculoviruses and its application, in order to solve the problems of low virulence and slow insecticidal speed of existing insect baculoviruses.

[0005] In a first aspect, the present invention provides an enhancing protein ORF42 for insect baculoviruses, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] Secondly, the present invention provides a nucleotide sequence for encoding the aforementioned synergistic protein ORF42.

[0007] Thirdly, the present invention provides an expression frame comprising the above-mentioned nucleotide sequence.

[0008] Fourthly, the present invention provides a recombinant plasmid containing the above-mentioned nucleotide sequence or the above-mentioned expression frame.

[0009] Fifthly, the present invention provides a recombinant bacterial strain, which is obtained by transfecting competent host bacteria with the above-mentioned recombinant plasmid.

[0010] In a sixth aspect, the present invention provides the application of the above-mentioned synergistic protein ORF42 in the preparation of insect virus insecticides or insect virus insecticide synergistic factors.

[0011] In a seventh aspect, the present invention provides a composition for insecticidal purposes, the composition comprising an insect virus and the aforementioned synergistic protein ORF42.

[0012] Eighthly, the present invention provides an insect virus insecticide comprising the above-described composition.

[0013] It should be noted that in the composition and the insect virus insecticide, the insect virus and the synergistic protein ORF42 can be stored together or separately. In addition to the composition, the insect virus insecticide may also include commonly used excipients or other functional ingredients in insect virus insecticides.

[0014] In one alternative implementation, the insect virus includes at least one of the following: fall webworm nucleopolyhedrovirus, spring inchworm nucleopolyhedrovirus, and smoke-winged pine sawfly nucleopolyhedrovirus.

[0015] In a ninth aspect, the present invention provides the application of the above-mentioned synergistic protein ORF42 or its composition or insect virus insecticide in the control of forest pests, wherein the forest pests include at least one of the fall webworm, spring inchworm and tobacco pine sawfly.

[0016] Based on the above technical solution, the present invention has at least the following beneficial effects:

[0017] This invention yields an synergistic protein (ORF42) from the nucleopolyhedrovirus of the pine sawfly, a biological control resource for insects. Functional verification of the protein shows that it has a significant synergistic effect against nucleopolyhedroviruses of the fall webworm, spring inchworm, and pine sawfly. It can be added as a synergistic factor to insect viruses and is of great significance for the control of major forest pests (fall webworm, spring inchworm, and pine sawfly).

[0018] The synergistic protein ORF42 provided by this invention enhances the composition of baculovirus synergistic factors, providing a theoretical basis for the molecular improvement and efficient utilization of baculovirus insecticides, and providing selected proteins and genes for the development of protein pesticides and insect-resistant breeding, which is of great significance for the sustainable control of forest pests. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is an agarose gel electrophoresis result of the ORF42 protein gene fragment with double restriction sites in an embodiment of the present invention;

[0021] Figure 2 This is a flowchart illustrating the construction process of the pGEX-4T-1-ORF42 prokaryotic expression vector in this embodiment of the invention.

[0022] Figure 3 This is a 12% SDS-PAGE gel electrophoresis result of the expression product induced by the pGEX-4T-1-ORF42 prokaryotic expression vector in this embodiment of the invention.

[0023] Figure 4 This is a 12% SDS-PAGE gel electrophoresis result of the purified ORF42 recombinant protein sample in this embodiment of the invention. Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0025] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0026] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0027] I. Obtaining Enhancer Proteins

[0028] The applicant collected diseased and dead larvae of the Gammabaculovirus diprinfuscalae from a state-owned forest farm in Youxi County, Fujian Province, China, and isolated and purified a new virus from them. Ultrastructural identification confirmed it to be a nucleopolyhedrovirus, named Gammabaculovirus diprinfuscalae (DiinNPV), and deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 46470, deposited on June 17, 2025, and classified as Gammabaculovirus diprinfuscalae. Bioactivity experiments were conducted to determine the LC50 and LT50 of DiinNPV against 3rd instar Gammabaculovirus larvae, as well as its virulence against larvae of different instars. Verification has shown that the isolation and identification of this virus strain is likely to become an effective biocontrol resource for controlling the tobacco pine sawfly.

[0029] Based on the isolation of DiinNPV (nucleopolyhedrovirus of the pine sawfly), a gene for enhancing the function of the virus was discovered through high-throughput sequencing. A positive clone of this gene was obtained from the whole genome of the virus. This gene for enhancing the function of the virus has 66% homology with unknown functional proteins of other hymenopteran baculoviruses and was named ORF42.

[0030] Sequencing revealed the amino acid sequence of the enhancing protein ORF42 as shown in SEQ ID NO.1, and the nucleotide sequence encoding ORF42 as shown in SEQ ID NO.2.

[0031] MSRLCMNEISQLYSITRSESKALLKTDVYSNGDIFIVTLVYKNTITRGTA KRCREARNIAARNMLRILNHTDDESKNYHVLQKYVIRRHQPCVTFAYFNDK WCCEVEYNEWCAHGFGVSKIDAKTHACKHMLKIINENKNGCDYIN(SEQ ID NO.1);

[0032] atgtcgcgcttatgtatgaacgagatatcgcaattgtattcgataacacgatcggaatccaaagcattattaaaaactgacgtatattccaacggtgatatatttatagta actttagtttacaaaaacacaattactcgtggaactgccaagcgatgtcgagaagccagaaatattgcggcaagaaatatgttacgtatactcaaccatacagatgatgaa tcaaaaaattatcatgtattacagaagtacgttatacgcagacaccaaccgtgtgttacatttgcatattttaatgataaatggtgttgtgaggtagagtataatgaatgg tgtgctcatggatttggcgtttctaagatagatgcaaaaacacatgcgtgtaaacatatgttaaaaataataaacgaaaataaaaatggctgcgattacatcaattga (SEQ IDNO.2).

[0033] II. Prokaryotic expression of the synergist protein ORF42

[0034] 1. Double digestion of the enhancing protein ORF42 gene and the pGEX-4T-1 vector

[0035] Using the genomic DNA of DiinNPV virus as a template, primers (SEQ ID NO.3 and SEQ ID NO.4) with EcoRI and XhoI restriction sites at both ends were designed based on the nucleotide sequence shown in SEQ ID NO.2. The primer sequences are shown in Table 1.

[0036] Table 1 Primer sequences

[0037] Primer name Sequence (5'-3') Enzyme cleavage sites serial number ORF42-F accagaattcatgtcgcgcttatgtatgaa EcoR I SEQ ID NO.3 ORF42-R aaacctcgagattgatgtaatcgcagcca Xho I SEQ ID NO.4

[0038] The specific steps are as follows:

[0039] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 5 min, followed by denaturation at 95℃ for 30 s, annealing at 65℃ for 45 s, extension at 72℃ for 1 min, for 33 cycles, and a final extension at 72℃ for 10 min. The PCR reaction system is shown in Table 2.

[0040] The products obtained from the PCR amplification reaction were detected by 1.0% agarose gel electrophoresis. After successful detection, the gel was excised and purified. The purified products were then subjected to double digestion with restriction endonucleases EcoRI and XhoI at 37℃ for 3 h. The double digestion system is shown in Table 3. After digestion, the digestion products were detected by 1.0% agarose gel electrophoresis. After successful detection, the target gene fragment was excised and recovered using a kit method. Simultaneously, the pGEX-4T-1 vector plasmid was transformed into E. coli DH5α competent cells using a heat shock method and plated on LB solid medium containing ampicillin (Amp, 20 mg / mL) resistance. The cells were incubated overnight at 37℃, then the plasmids were picked, shaken, and extracted using a plasmid miniprep kit. The plasmid was then double-digested as above. The digestion products were detected by 1.0% agarose gel electrophoresis. After successful detection, the vector fragment was excised and recovered using a kit method.

[0041] Table 2 PCR reaction system

[0042]

[0043] Table 3. Double enzyme digestion reaction system

[0044]

[0045] 2. Construction of recombinant plasmid expression vectors

[0046] The target gene fragment (generally 2-5 times the concentration of the vector fragment) and the vector fragment, purified by the above enzyme digestion, were ligated overnight at 4°C using T4 DNA ligase. The ligation reaction system is shown in Table 4. After the ligation reaction, the ligation solution was transferred into E. coli DH5α competent cells and cultured overnight at 37°C. Colonies were then selected for PCR identification, and those with the correct band positions were sequenced.

[0047] Table 4. T4 Connection Reaction System

[0048]

[0049]

[0050] 3. Induced expression of pGEX-4T-1-ORF42 prokaryotic expression vector

[0051] (1) Activation of the strain

[0052] Take 1 μL of the recombinant expression vector pGEX-4T-1-ORF42 constructed above and transform it into BL21 and Rosetta competent cells, respectively. After culturing overnight at 37°C, select colonies and inoculate them into LB liquid medium containing Amp (20 mg / mL). Culturing overnight at 37°C and 200 r / min with shaking, the activated bacterial solution is obtained.

[0053] (2) Induction of strains

[0054] The activated bacterial culture was inoculated into LB liquid medium containing Amp at a ratio of 1.0% and cultured at 37℃ and 200rpm / min for 3h. Then IPTG was added and the culture was induced under specific temperature conditions. Samples were then taken to obtain induced bacterial samples.

[0055] (3) Sample processing

[0056] Take 1 mL of the induced bacterial sample, centrifuge at 12000 rpm for 1 min, discard the supernatant, add 1 mL of 0.1 M phosphate buffer (1×PBS) to resuspend the bacterial pellet, centrifuge at 12000 rpm for 1 min to wash, discard the supernatant, add 50 μL of 1×PBS to resuspend the bacterial pellet again, add an equal volume of 2×SDS loading buffer, boil at 100℃ for 5 min to denature the protein, centrifuge at 12000 rpm for 3 min, take the supernatant, and load it onto 12% SDS-PAGE for detection.

[0057] 4. Screening of conditions for inducing expression of the synergist protein ORF42

[0058] (1) Screening of protein expression strains

[0059] Following the induction expression method described in section 3 above, under the same IPTG induction concentration (1.0 mM), induction time (8 h), and induction temperature (25 °C), the recombinant plasmid pGEX-4T-1-ORF42 was expressed in BL21 and Rosetta expression strains, respectively. The induced protein samples were analyzed by SDS-PAGE, and the optimal expression strain was selected based on the expression level. Uninduced BL21 and Rosetta expression strains served as negative controls.

[0060] (2) Screening of protein expression induction temperature

[0061] Following the induction expression method in item 3 above, the optimal expression strain selected in item (1) above was chosen, and then the protein was induced to express at an induction concentration of 1.0 mM IPTG at induction temperatures of 16℃, 25℃, 30℃ and 37℃ respectively. After 8 h, samples were taken for SDS-PAGE analysis, with the uninduced expression strain as the negative control.

[0062] (3) Screening of protein expression induction concentration

[0063] Following the induction expression method described in section 3 above, the optimal expression strain and optimal induction temperature were selected. Samples expressing the strain at different induction concentrations (0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM IPTG) under the same conditions were subjected to SDS-PAGE analysis, with the uninduced expression strain serving as a negative control.

[0064] (4) Screening of protein expression induction time

[0065] Following the induction expression method described in section 3 above, and after screening under the above conditions, samples were taken for SDS-PAGE analysis at 4h, 6h, 8h, and 12h after induction, respectively, under the optimal expression strain, optimal induction temperature, and optimal induction concentration. Samples that were not induced and cultured for 4h, 6h, 8h, and 12h served as negative controls.

[0066] 5. Purification of ORF42 recombinant protein

[0067] In the optimal expression strain, the recombinant ORF42 protein was induced to express in large quantities under optimal protein induction conditions. 50–100 mL of the induced sample was collected, and the bacteria were sonicated to break down the protein. The collected supernatant was the soluble protein. Purification was performed using an NGC Quest10 protein purification system in buffer A (for washing the column before sample loading) and buffer B (for eluting and purifying the protein after sample loading). The protein was filtered through a GST-tagged affinity chromatography column to remove impurities, thus separating and purifying the ORF42-containing fusion protein while maintaining its biological activity.

[0068] Buffer A: 50 mmol / L Tris, 0.2 mol / L sodium chloride, 2 mmol / L dithiothreitol (DTT), pH 8.0. Buffer B: 50 mmol / L Tris, 0.2 mol / L sodium chloride, 2 mmol / L DTT, 20 mmol / L reduced glutathione, pH 8.0.

[0069] 6. Quantitative analysis of recombinant ORF42 protein

[0070] The protein concentration in the purified protein solution was quantified using a BCA protein concentration assay kit via microplate reader. The specific procedure is as follows:

[0071] (1) Take an appropriate amount of 25 mg / mL protein standard and dilute it with PBS solution to a final concentration of 0.5 mg / mL;

[0072] (2) Based on the number of samples, add 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) to prepare an appropriate amount of BCA working solution and mix thoroughly.

[0073] (3) Add the standard to the wells of the 96-well plate in amounts of 0, 1, 2, 4, 8, 12, 16, and 20 μL, and add standard diluent to make up to 20 μL. This is equivalent to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively.

[0074] (4) Add an appropriate volume of sample to the well of the 96-well plate. If the sample is less than 20 μL, add standard diluent to make up to 20 μL. Please record the sample volume.

[0075] (5) Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 minutes;

[0076] (6) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at A562 or other wavelengths between 540-595 nm, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0077] III. Synergistic Effect of the Synergistic Protein ORF42

[0078] 1. Determination of the synergistic effect of ORF42 recombinant protein on DiinNPV

[0079] DiinNPV is a diprion infuscalaenucleopolyhedrovirus (DiinNPV) found on the pine sawfly. Healthy third-instar larvae of the pine sawfly were selected and placed in cylindrical rearing containers (Φ20cm×25cm). After 24 hours of starvation, they were fed fresh pine needles coated with a virus solution. The virus-laden pine needles were evenly brushed with 1 mL of virus solution at varying concentrations using a brush, dried, and then fed to the larvae. A blank control consisted of fresh pine needles brushed with an equal volume of sterile water. The experiment included one blank control and five concentration gradients of pine sawfly virus: 5.0 × 10⁻⁶. 6 1.0×10 6 5.0×10 5 1.0×10 5 and 4.0×10 4 OBs·mL -1Each group was treated with 30 larvae, repeated 3 times. After 2 days, the larvae had largely consumed the pine needles, and were then replaced with fresh, non-toxic pine needles. Rearing conditions remained the same. Starting from the 3rd day after infection, larval mortality was recorded until the larvae died or pupated (larvae exhibiting obvious symptoms of nucleopolyhedrovirus infection were recorded as virus-induced deaths, such as swollen bodies, easily broken by touch, or hanging upside down on pine branches; larvae killed by other pathogens, such as bacteria or fungi, were not recorded and discarded). The same treatment was applied, with ORF42 recombinant protein added to the five different concentrations of virus solution mentioned above, resulting in protein concentrations of 50 ppm and 100 ppm. Starting from the 3rd day after inoculation, larvae were observed continuously for 11 days, with mortality counts recorded every 24 hours. The cumulative mortality rate was calculated, and the LC50 was calculated. 50 .

[0080] 2. Determination of the synergistic effect of recombinant ORF42 protein on HycuNPV

[0081] HycuNPV is the nucleopolyhedrovirus of the fall webworm (Hyphantria cuneanucleopolyhedrovirus). Healthy third-instar larvae of the fall webworm were selected and placed in rearing cups (Φ6.3cm×5.2cm) for 24 hours of starvation. The purified HycuNPV suspension was then serially diluted to a concentration of 3.0×10⁻⁶. 7 9.0×10 6 3.0×10 6 9.0×10 5 and 3.0×10 5 OBs·mL -1 The virus fluid was used. ORF42 recombinant protein was added at doses of 50 ppm and 100 ppm. Ten healthy larvae were selected from each group, with three replicates. Starting from day 3 after inoculation, the mortality symptoms and number of American white moth larvae were recorded daily until all larvae died or pupated. This observation was repeated for 11 days, and the cumulative mortality rate was calculated. The LC50 was then calculated. 50 .

[0082] 3. Determination of the synergistic effect of recombinant ORF42 protein on ApciNPV

[0083] ApciNPV is a nucleopolyhedrovirus of the spring inchworm (Apocheima cinerarius nucleopolyhedrovirus, ApciNPV). Healthy early third-instar larvae of the spring inchworm were selected and placed in rearing cups (Φ6.3cm×5.2cm). After starvation for 24 hours, they were fed once with artificial feed containing viral fluid / or viral fluid + purified protein and continued to be reared. The indoor rearing conditions were (25±1)℃ and a photoperiod of 16L:8D. The purified ApciNPV virus suspension was diluted sequentially to 1.0×10⁻⁶. 7, 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 OBs·mL -1 Five concentration gradients of virus solution were used to infect test insects. 200 μL of virus suspension was inoculated into each insect rearing cup. The number of dead larvae was recorded starting from the 3rd day after virus inoculation until the insects died or pupated (dead larvae with obvious symptoms of cytoplasmic polyhedrosis virus infection were recorded as virus-induced death, such as the body becoming rigid and shortened, pink liquid or feces discharged from the head or tail. Deaths caused by other pathogens were not recorded, such as those caused by bacteria or fungal infections, and they were discarded). Another group with sterile water as the control was set up, with 30 insects in each treatment group and repeated 3 times. In the same treatment, the ORF42 recombinant protein was added to the above 5 different concentrations of virus solution respectively, so that the protein concentrations were 50 ppm and 100 ppm. Starting from the 3rd day after virus inoculation and rearing, it was continuously observed for 11 days. The number of dead insects was checked and counted every 24 hours, and the cumulative mortality was calculated, and LC 50 was calculated.

[0084] 4. Data Statistics and Analysis

[0085] The Probit regression analysis in SPSS 22.0 software was used to calculate the virulence regression equations, LC 50 etc. of different viruses and virus + recombinant protein. The synergistic effect was measured by the synergistic ratio (SR) and co-toxicity coefficient (CTC), and the calculation formulas are as follows:

[0086] SR = LC 50 (virus NPV) / LC 50 (virus NPV + recombinant protein) - 1;

[0087] CTC = LC 50 (virus NPV) / LC 50 (virus NPV + recombinant protein) × 100.

[0088] Among them, when the synergistic ratio (SR) > 0, it indicates a synergistic effect; when (SR) < 0, it indicates an antagonistic effect; when (SR) = 0, it indicates no synergistic effect. When the co-toxicity coefficient (CTC) ≥ 120, the compounding of the agents indicates a synergistic effect; if CTC ≤ 80, it indicates an antagonistic effect; if 80 < CTC < 120, it indicates an additive effect.

[0089] IV. Results

[0090] 1. Cloning of the ORF42 protein gene with double restriction enzyme sites

[0091] Using DiinNPV genomic DNA as a template, the ORF42 protein gene fragment with double restriction enzyme sites was amplified using the primers in Table 1. The fragment was 441 bp in size. The results of agarose gel electrophoresis are shown below. Figure 1 As shown. After ligation and transformation, the sample was sent to Kingwise Biotechnology Co., Ltd. for sequencing. The sample size was consistent with the provided gene fragment and contained double restriction enzyme sites. Figure 1 In the diagram, M stands for DNA marker, and the lengths from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0092] 2. Construction of the pGEX-4T-1-ORF42 recombinant plasmid expression vector

[0093] The ORF42 protein gene and the pGEX-4T-1 vector plasmid were double-digested using EcoRI and XhoI restriction endonucleases, respectively. The digested target gene fragment was ligated into the vector plasmid and transformed. Colony PCR was performed to identify colony clusters. Successfully identified bacterial cultures were sent to the company for sequencing, confirming the successful construction of the pGEX-4T-1-ORF42 prokaryotic expression vector. The construction process is as follows: Figure 2 As shown.

[0094] 3. Induced expression of pGEX-4T-1-ORF42 prokaryotic expression vector

[0095] The optimal induction conditions for ORF42 protein are: induction in Rosetta strain with 1.0 mM IPTG at 16°C for 20 h. The results of 12% SDS-PAGE gel electrophoresis are as follows: Figure 3 As shown. (Molecular weight of protein + molecular weight of GST fusion protein) Based on bioinformatics analysis, the molecular weight of ORF42 protein is estimated to be 17.065 kDa, while the size of the GST tag protein on the pGEX-4T-1 vector is 26 kDa. Therefore, the size of the induced ORF42 fusion protein is estimated to be 43.06 kDa.

[0096] Figure 3 In the middle, lane 5 is the sample obtained by inducing the ORF42 recombinant protein in Rosetta strain with 0.8mM IPTG at 16℃ for 20h; lanes 2 and 4 are the negative control of Rosetta strain; lane 3 is the protein marker (from top to bottom: 66kDa, 45kDa, 35kDa, 27kDa, 20kDa).

[0097] 4. Purification of ORF42 recombinant protein

[0098] (1) Identification of ORF42 protein expression

[0099] To further determine whether ORF42 protein is a soluble protein or an inclusion body protein, ORF42 protein was induced in large quantities in the optimal expression strain under optimal induction conditions. The bacterial culture was collected, centrifuged, and the resulting bacterial pellet was resuspended in lysis buffer (20 mmol / L Tris + 0.5 mol / L NaCl, pH 8.0). After sonication and centrifugation, the supernatant and pellet were collected separately. The bacterial cells were resuspended in 50 μL of 1×PBS, and an equal volume of 2×SDS loading buffer was added. The mixture was boiled at 100°C for 5 min to denature the protein, centrifuged at 12000 rpm for 3 min, and the supernatant was used for 12% SDS-PAGE analysis. The results showed that ORF42 protein was soluble in the supernatant.

[0100] (2) Purification of recombinant ORF42 protein

[0101] The sample was analyzed using a GST column (5 mL column volume) on an NGC Quest 10 protein purification system. The sample from the peak was collected and analyzed by 12% SDS-PAGE electrophoresis. Results are shown below. Figure 4 .

[0102] Figure 4 In the diagram, lane M represents the protein markers (from top to bottom: 66kDa, 45kDa, 35kDa, 27kDa, 20kDa); lanes B1-B2 represent the patterns of the ORF42 recombinant protein-induced expression strain in the precipitate and supernatant; and lane B3 represents the purified ORF42 recombinant protein.

[0103] 5. Synergistic effect of soluble proteins

[0104] (1) The toxicity of ORF42 protein mixed with DiinNPV to the pine sawfly is shown in Table 5.

[0105] Table 5. Toxicity test results of 3rd instar larvae of the smoke-winged pine sawfly (11 days)

[0106]

[0107]

[0108] (2) The toxicity of ORF42 protein mixed with HycuNPV to the American white moth is shown in Table 6.

[0109] Table 6. Toxicity test results of third instar larvae of the fall webworm (11 days)

[0110] Treatment concentration, ppm <![CDATA[LC 50 (OBs / mL)]]> 95% confidence limit (OBs / mL) Enhancement factor Cotoxicity coefficient HycuNPV - <![CDATA[9.02×10 5 ]]> <![CDATA[4.23×10 5 ~2.26×10 6 ]]> - - HycuNPV 50 <![CDATA[5.74×10 5 ]]> <![CDATA[9.31×10 4 ~8.19×10 5 ]]> 0.57 157 HycuNPV 100 <![CDATA[3.06×10 4 ]]> <![CDATA[1.56×10 4 ~7.12×10 4 ]]> 1.95 294

[0111] (3) The toxicity of ORF42 protein and ApciNPV mixture to spring inchworm is shown in Table 7.

[0112] Table 7. Toxicity test results of 3rd instar larvae of the spring inchworm (11 days)

[0113] Treatment concentration, ppm <![CDATA[LC 50 (OBs / mL)]]> 95% confidence limit (OBs / mL) Enhancement factor Cotoxicity coefficient ApciNPV - <![CDATA[6.89×10 4 ]]> <![CDATA[2.43×10 4 ~1.19×10 5 ]]> - - ApciNPV 50 <![CDATA[3.21×10 4 ]]> <![CDATA[1.17×10 4 ~8.77×10 4 ]]> 1.14 215 ApciNPV 100 <![CDATA[1.17×10 4 ]]> <![CDATA[6.56×10 3 ~6.63×10 4 ]]> 4.89 589

[0114] As shown in Tables 5-7, the synergistic protein ORF42 provided in this application has a significant synergistic effect on the nucleopolyhedrovirus of the fall webworm, the nucleopolyhedrovirus of the spring inchworm, and the nucleopolyhedrovirus of the tobacco pine sawfly. It can be added as a synergistic factor to the application of insect viruses and is of great significance for the control of major forest pests (fall webworm, spring inchworm, and tobacco pine sawfly).

[0115] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An enhancing protein ORF42 for insect baculoviruses, characterized in that, The amino acid sequence of the enhancing protein ORF42 is shown in SEQ ID NO.

1.

2. A nucleotide sequence for encoding the synergistic protein ORF42 of claim 1.

3. An expression box, characterized in that, The expression frame includes the nucleotide sequence of claim 2.

4. A recombinant plasmid, characterized in that, The recombinant plasmid contains the nucleotide sequence of claim 2, or the expression cassette of claim 3.

5. A recombinant bacterial strain, characterized in that, The recombinant strain was obtained by transfecting competent host bacteria with the recombinant plasmid as described in claim 4.

6. The use of the synergistic protein ORF42 as described in claim 1 in the preparation of insect virus insecticides or insect virus insecticide synergistic factors.

7. A composition for insecticidal purposes, characterized in that, The composition comprises an insect virus and the enhancing protein ORF42 as described in claim 1.

8. An insect virus insecticide, characterized in that, The insect virus insecticide includes the composition of claim 7.

9. The application according to claim 6, the composition according to claim 7, or the insect virus insecticide according to claim 8, characterized in that, Insect viruses include at least one of the following: fall webworm nucleopolyhedrovirus, spring inchworm nucleopolyhedrovirus, and smoke-winged pine sawfly nucleopolyhedrovirus.

10. The application of the synergistic protein ORF42 of claim 1, the composition of claim 7, or the insect virus insecticide of claim 8 in the control of forest pests, wherein the forest pests include at least one of the fall webworm, spring inchworm, and tobacco pine sawfly.

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