Scale moth blood lymphocyte line, expanded production method of nuclear polyhedrosis virus and application of red moth blood lymphocyte line and nuclear polyhedrosis virus

Abstract

The invention discloses a yellow thorn moth blood lymphocyte line, an expanded production method of a nuclear polyhedrosis virus and application of the yellow thorn moth blood lymphocyte line and the nuclear polyhedrosis virus. The name of the dalbergia mandshurica blood lymphocyte line is dalbergia mandshurica blood lymphocyte CFW-CHY, the dalbergia mandshurica blood lymphocyte line is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: C2025104, and the preservation date is March 27, 2025; the yellow thorn moth blood lymphocyte line is used for expanded production of the nuclear polyhedrosis virus, so that industrial production of the nuclear polyhedrosis virus is realized, and the yellow thorn moth blood lymphocyte line can be used in the fields of preparation of insecticides, insecticidal pesticides and the like.

Description

Technical Field

[0001] This invention relates to the field of insect cell technology, specifically to a method for the large-scale production of a yellow-spotted moth hemolymphocyte line, a nucleopolyhedrovirus, and their applications. Background Technology

[0002] Insect cells, as important tools in biotechnology and scientific research, have demonstrated broad application prospects in multiple fields due to their unique advantages. Insect cell lines play a vital role in baculovirus production and recombinant protein expression. The insect cell-baculovirus expression system has become a classic platform for efficient recombinant protein expression. Utilizing ovarian cells from lepidopteran insects such as Sf9, Sf21, and High Five, and baculoviruses such as AcMNPV (Autographa Californica Multiple Nucleopolyhedrovirus), this system has shown outstanding performance in vaccine, antibody, and enzyme production. It not only achieves high-level protein expression but also possesses post-translational modification capabilities, making the expressed proteins closer to their natural state. Furthermore, insect cells are widely used in gene function research, drug screening, biopesticide development, and cell biology research, providing strong support for scientists to explore gene regulation mechanisms, screen potential drug targets, develop environmentally friendly pesticides, and elucidate the laws governing cellular life activities.

[0003] With continuous technological advancements, insect cells have demonstrated unique technological advantages and ecological value in the biological control of pests. Insect cell culture technology enables the large-scale production of insect viruses (such as baculoviruses). These viruses exhibit high specificity for specific pests, effectively controlling agricultural pests while minimizing negative impacts on non-target organisms and the environment. For example, nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) produced using insect cells have been widely used to control pests in Lepidoptera and Coleoptera. Compared to traditional chemical pesticides, insect cell-based biological control methods are environmentally friendly, less prone to developing resistance, and have high ecological compatibility, meeting the needs of sustainable agriculture. Furthermore, insect cell technology can be used to study the interaction mechanisms between pests and pathogens, providing theoretical support for developing novel biological control strategies. With advancements in genetic engineering, insect cells have shown potential in improving viral virulence, expanding host range, and enhancing field stability, injecting new research directions into the field of biological pest control. Therefore, insect cells, with their high efficiency, safety, and multifunctionality, have become an indispensable tool in modern biotechnology and life science research.

[0004] Yellow-spotted moth ( Cnidocampa flavescensThe yellow-spotted tussock moth, also known as the hairy caterpillar, is an important pest in the family Limacodidae of the order Lepidoptera, widely distributed in East Asia. Among tussock moth pests in my country, the yellow-spotted tussock moth is particularly prominent in terms of occurrence frequency and damage, especially in orchards, gardens, and forest ecosystems, where it often becomes one of the major pests. Its larvae are omnivorous, feeding on more than 120 kinds of plants, including fruit trees and forest trees (Yang Hongzhen et al., Understanding my country's Medicinal Insects—The Yellow-Spotted Tussock Moth. Encyclopedia Knowledge, 2023). Furthermore, it has strong reproductive capacity and high adaptability, making it prone to outbreaks and disasters under suitable environmental conditions. Because its larvae have poisonous stingers on their bodies, they can cause itching and pain when they come into contact with the skin, and in severe cases, allergic reactions. They pose a dual threat to humans, animals, agricultural production, and the ecological environment. The yellow-spotted moth is considered one of the most harmful species among tussock moths (Li Shilong et al., Investigation of Tussock Moth Species in Zhaoyuan, Liaoning, Northern Sericulture, 2020; Hong Yong et al., Investigation of Common Tussock Moth Species in Prunus cerasifera and Platanus orientalis in Wuhu City, Chinese Journal of Schistosomiasis Control, 2016; Gao Yong et al., Control of Tussock Moths in Blueberry Cultivation, Horticulture and Seedlings, 2016).

[0005] Existing technology has identified and isolated the nucleopolyhedrovirus (NPV) of the yellow-spotted moth. The budding virus particles (BV) of this virus can specifically infect the cells of the yellow-spotted moth and spread within the population, achieving long-term control and reducing the frequency and cost of pest control. This makes it a potential biopesticide for controlling the yellow-spotted moth. Furthermore, primary cells of the yellow-spotted moth can be used to enrich research on the mechanism of action of NPV in infecting lepidopteran insects, which is of great significance for preventing damage to lepidopteran insects. However, the production of NPV using primary cells of the yellow-spotted moth results in low yields, hindering large-scale industrial application and limiting the promotion and application of the NPV industrialization.

[0006] Based on this, the present invention provides a method for the scale-up production of *Spodoptera litura* nucleopolyhedrovirus using a *Spodoptera litura* hemolymphocyte line, thereby realizing the industrial application of *Spodoptera litura* nucleopolyhedrovirus. Summary of the Invention

[0007] The main objective of this invention is to provide a method for the large-scale production of the yellow-spotted moth hemolymphocyte line and nucleopolyhedrovirus, as well as their applications, to overcome the shortcomings of the prior art.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution.

[0009] As the first aspect of the invention, this invention discloses a yellow-spotted moth hemolymph cell line, named: yellow-spotted moth hemolymph cell CFW-CHY, which has been deposited at the China Center for Type Culture Collection, accession number CCTCC NO:C2025104, and the deposit date is March 27, 2025.

[0010] As a second aspect of the invention, the present invention also discloses the use of the above-mentioned yellow tussock moth hemolymphocyte line in the large-scale production of nucleopolyhedrovirus.

[0011] As a third aspect of the invention, the present invention also discloses a method for scaling up the production of the above-mentioned nucleopolyhedrovirus, which includes inoculating the yellow-spotted moth nucleopolyhedrovirus into the above-mentioned yellow-spotted moth hemolymphocyte line, incubating and purifying it, thereby scaling up the production of nucleopolyhedrovirus.

[0012] Preferably, the concentration of nucleopolyhedrovirus obtained from scale-up production is 1.0 × 10⁻⁶. 7 ~1.0×10 9 PIB / mL.

[0013] Preferably, the concentration of nucleopolyhedrovirus inoculated with *Spodoptera litura* is 4-6 v / v.

[0014] Preferably, the incubation conditions include a culture temperature of 25~37℃, a culture time of 48~120h, and a shaking speed of 100~200 rpm.

[0015] As a fourth aspect of the invention, this invention provides a suspension of *Spodoptera litura* nucleopolyhedrovirus, comprising at least 20% by mass of 300 million PIB / mL *Spodoptera litura* nucleopolyhedrovirus. The suspension also includes: Additives: nonionic carboxylic acid hydrochloride DS733 2.5%, ethoxylated mixed sulfonate TT-1 2.5%, thickener magnesium aluminum silicate 0.08%, thickener xanthan gum 0.02%, preservative potassium sorbate 0.3%, photoprotectant fluorescent whitening agent 0.1%, balance is pure water.

[0016] As a fifth aspect of the invention, the present invention also discloses an insecticide comprising at least a nucleopolyhedrovirus prepared using the scale-up production method of nucleopolyhedrovirus as described above.

[0017] Preferably, the insecticide has a kill rate of 85-95% against the larvae of the yellow-spotted moth.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The technical solution of this invention uses the hemolymph cell line of the yellow-spotted moth to scale up the production of nucleopolyhedrovirus of the yellow-spotted moth, which has the advantages of high yield and the virulence of the obtained nucleopolyhedrovirus of the yellow-spotted moth is comparable to that of the nucleopolyhedrovirus of the yellow-spotted moth extracted directly from the insect body. This solves the problems of long production cycle, low yield and inability to be applied on a large scale in the existing technology of nucleopolyhedrovirus of the yellow-spotted moth.

[0019] 2. By adopting the technical solution of this invention, the production time of the nucleopolyhedrovirus of the yellow tussock moth is greatly shortened, and the entire production process is simple and low in cost.

[0020] 3. The nucleopolyhedrovirus of the yellow-spotted moth provided by this invention can also be used for the prevention and control of the yellow-spotted moth pest. Attached Figure Description

[0021] Figure 1 This is a 100x microscope image of the adherent hemolymphocytes of the yellow-spotted moth in Example 1 of the present invention.

[0022] Figure 2 This is a 100x microscope image of the primary generation hemolymphocytes of the yellow-spotted moth cultured for 28 days in Example 1 of this invention.

[0023] Figure 3 This is a 400x microscope image of the hemolymphocytes of the yellow-spotted moth cultured to 90% capacity at the bottom of the flask in Example 1 of this invention.

[0024] Figure 4 This is a 200x microscope image of the hemolymphocytes of the yellow-spotted moth revived in Example 1 of the present invention.

[0025] Figure 5 This is a 400x microscope image of the hemolymphocytes of the yellow-spotted moth passaged to the 40th generation in Example 1 of this invention.

[0026] Figure 6 This is a 400x microscope image of the proliferation of the nucleopolyhedrovirus of the yellow-spotted moth in the hemolymphocytes of the yellow-spotted moth in Example 2 of the present invention. The "particles" in the cells are the nucleopolyhedrovirus of the yellow-spotted moth.

[0027] Figure 7 The image shows a typical "liquefaction" symptom of a fourth-instar larva of the yellow-spotted moth that died after ingesting the yellow-spotted moth nucleopolyhedrovirus in the virulence experiment of Example 2 of this invention.

[0028] Figure 8 The left, middle, and right figures are experimental diagrams of the storage stability, persistent foaming, and suspension dispersibility tests of the yellow tussock moth nucleopolyhedrosis suspension agent in Example 3 of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0030] This invention discloses a hemolymph cell line of the yellow-spotted moth, named Cnidocampa flavescens walker CFW-CHY, which has been deposited at the China Center for Type Culture Collection, accession number CCTCC NO:C2025104, on March 27, 2025.

[0031] In a specific embodiment of the present invention, a method for extracting and culturing the above-mentioned yellow-spotted moth hemolymphocyte line is provided, specifically including: First, the hemolymphocytes of the yellow-spotted moth are isolated and cultured. This method can successfully establish the primary hemolymphocyte cell line of the yellow-spotted moth. The consumables and steps are simple, and no antibiotics are used. It has the advantages of low contamination rate and high success rate. This method has also been tested and is applicable to the isolation and culture of hemolymphocytes of other tussock moths.

[0032] Specifically, the method for isolating and culturing hemolymphocytes of the yellow-spotted moth includes the following steps: (1) Preparation of insect disinfectant, cleaning solution and culture medium; (2) Cleaning of yellow tussock moth insects and acquisition of sterile hemolymphocytes; (3) Adhesion of yellow tussock moth hemolymphocytes and treatment with deoxidase; (4) Culture and passage of yellow tussock moth hemolymphocytes; (5) Cryopreservation and revival of passaged yellow tussock moth hemolymphocytes.

[0033] The technical solution of this invention can, on the one hand, shorten the collection time of hemolymph, avoid prolonged exposure of hemolymphocytes to contamination, and prevent oxidative enzymes in hemolymphocytes from coming into contact with air, thus preventing cells from being easily oxidized and blackened, resulting in cell damage and death; on the other hand... In particular, it helps to study the yellow-spotted moth under in vitro conditions, especially the interaction between yellow-spotted moth cells and yellow-spotted moth nucleopolyhedrovirus, providing a new approach to the prevention and control of yellow-spotted moth damage.

[0034] As a preferred embodiment, the method for isolating, culturing, and preserving hemolymphocytes of the yellow-spotted moth specifically includes the following steps: (1) Prepare the required solutions, including: 75% (v / v) alcohol, 2wt% sodium hypochlorite solution, and sterile PBS (phosphate buffered saline) solution.

[0035] (2) Disinfect the body surface of the yellow tussock moth larvae by immersing them in 75% alcohol and 2% sodium hypochlorite for 10 minutes each. Preferably, mature larvae of the yellow tussock moth are selected, as they are larger and have abundant hemolymph, making it easier to collect hemolymphocytes.

[0036] (3) Wash the yellow tussock moth with sterile PBS solution and then air dry the surface of the insect; (4) Place the clean worm body in a sterile culture dish, with the head of the worm body facing down, so that the hemolymph can be fully gathered in the head and thorax of the worm body; (5) Cut open the side of the thorax of the yellow tussock moth, and the hemolymph will flow out slowly and naturally. Collect the hemolymph that flows out.

[0037] Preferably, the size of the incision is 2-3 mm.

[0038] Preferably, T-12.5 cm is selected. 2 Collection in culture bottles; each bottle is suitable for collecting hemolymph from 4-5 yellow-spotted moths.

[0039] Preferably, the time to collect the hemolymph of each yellow-spotted moth is about 20 to 30 seconds, and the time to collect the hemolymph of each bottle of 4 to 5 yellow-spotted moths is about 1.5 to 3 minutes.

[0040] However, existing technologies use capillary methods to collect hemolymph, relying on the principle of capillary action. Therefore, the collection speed primarily depends on the inner diameter of the capillary and the viscosity of the fluid. For example, using a capillary with an inner diameter of 0.5 × 100 mm to collect hemolymph from each *Spodoptera litura* moth requires at least one minute, and isolation and culture experiments require collecting hemolymph from 4-5 moths each time, totaling more than five minutes. Therefore, the collection method of this invention significantly reduces the collection time, overcomes the limitations of capillary methods, and significantly improves collection efficiency.

[0041] In some specific embodiments, the hemolymph obtained is naturally squeezed out by the pressure inside the insect body, which can naturally separate the fat bodies in the hemolymph. By controlling the size of the chest wound, the hemolymph is ensured to seep out from the chest wound, ensuring the collection time while greatly avoiding contamination problems.

[0042] (6) Add 2 mL of fetal bovine serum and SF-SFM medium mixture to the collected yellow tussock moth hemolymph at a volume ratio of 1:9, let stand, and wait for the hemolymph cells to settle and adhere to the wall.

[0043] (7) After the yellow tussock moth hemolymph cells adhere to the wall, discard the upper culture medium and add fresh cell culture medium. Repeat 2-3 times to reduce the effect of oxidase in the hemolymph oxidizing the cells and producing toxic cells.

[0044] (8) Continue to incubate at 27°C with minimal observation and movement. Change the medium for the first time after 10 days, replacing only 50% of the old cell culture medium and adding 50% of fresh cell culture medium. Repeat the medium change every 7 days thereafter. Observe cell growth and division during medium changes. Once the cells have proliferated to fill more than 90% of the bottom area of ​​the culture flask, they can be passaged.

[0045] This yields primary hemolymphocytes of the yellow-spotted moth. Further, these primary hemolymphocytes are passaged to obtain a hemolymphocyte cell line, specifically comprising: (1) When passage, tap the primary cell culture flask 3-5 times to dislodge the adhered cells. Transfer 50% of the cell culture medium in the primary cell flask to a new culture flask and then add 50% of the fresh cell culture medium. (2) When the hemolymphocytes of the yellow-spotted moth are stably passaged and cultured to a volume of 50 mL, the cells can be cryopreserved. After counting the number of viable cells, the cultured cells are placed in sterile centrifuge tubes and centrifuged at 1400 rpm for 4 minutes. The supernatant is discarded, and the cell pellet is resuspended in a certain volume of cryopreservation buffer to achieve a final cell concentration of 6 × 10⁻⁶. 7 Cells were divided into 1 mL cryovials and aliquoted into cryovials. The cells were placed in a cryovial box for programmed gradient cooling and transferred to liquid nitrogen for cryopreservation after 24 hours.

[0046] (3) Thawing the cryopreserved cells. Remove the cryopreservation tubes containing the cells from liquid nitrogen and immediately place them in a 37°C water bath to thaw. When 90% of the cells have thawed, transfer them to new sterile centrifuge tubes and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in a small amount of fresh culture medium. Finally, transfer the cells to a culture flask and add a certain amount of fresh culture medium to achieve a cell concentration of 1×10⁻⁶. 6 Cells / mL, incubated statically at 27℃.

[0047] The method for scaling up the production of nucleopolyhedrovirus (NPV) using the hemolymphocyte line of *Spodoptera litura* prepared by the above method includes inoculating the NPV into the hemolymphocyte line, incubating and purifying it, thereby scaling up the production of NPV.

[0048] Preferably, the concentration of nucleopolyhedrovirus obtained from scale-up production is 1.0 × 10⁻⁶. 7 ~1.0×10 9 PIB / mL.

[0049] Preferably, the concentration of nucleopolyhedrovirus inoculated with *Spodoptera litura* is 4-6 v / v.

[0050] Preferably, the incubation conditions include a culture temperature of 25~37℃, a culture time of 48~120h, and a shaking speed of 100~200 rpm.

[0051] As a fourth aspect of the invention, the present invention also discloses an insecticide comprising at least a nucleopolyhedrovirus prepared using the scale-up production method of nucleopolyhedrovirus as described above.

[0052] Preferably, the insecticidal rate against the yellow-spotted moth is 85-95%.

[0053] The technical solution of the present invention will be described in detail below through specific embodiments.

[0054] Example 1 This embodiment provides a method for separating and culturing hemolymphocytes by directly collecting hemolymph from the thorax of the yellow-spotted moth. The specific steps include: I. Preparation of insect disinfectant, cleaning solution, cell culture medium, cryopreservation solution and cell staining solution (1) Preparation of 75% alcohol disinfectant: To prepare 100 mL of 75% alcohol using 95% alcohol, measure 78.95 mL of 95% alcohol and add it to 21.05 mL of distilled water. Stir well to obtain 100 mL of 75% alcohol. Store at room temperature.

[0055] (2) Preparation of 2% sodium hypochlorite solution: To prepare 100 mL of 2% sodium hypochlorite solution using 10% sodium hypochlorite stock solution, take 20 mL of 10% sodium hypochlorite stock solution and add it to 80 mL of distilled water. Stir well to obtain 100 mL of 2% sodium hypochlorite solution. Store at room temperature.

[0056] (3) Preparation of sterile PBS solution for washing: Prepare 1 L of PBS with pH 7.4 as follows: 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and 1 L distilled water. Add the weighed reagents to about 800 mL of distilled water and stir until completely dissolved; measure the pH of the solution with a pH meter and adjust it to 7.4 with hydrochloric acid or sodium hydroxide; transfer the solution to a 1 L volumetric flask, add distilled water to the mark, and mix well; sterilize by autoclaving at 121℃ for 15-20 minutes or by filtration through a 0.22 μm filter membrane to obtain sterile PBS solution. Incubate at 27℃ for 1 week, and after checking for contamination, store at 4℃.

[0057] (4) Preparation of cell culture medium: In a sterile laminar flow hood, 500 mL of cell culture medium was prepared using SF-SFM medium (Suzhou Womei Biotechnology Co., Ltd.) and fetal bovine serum. 50 mL of fetal bovine serum was added to 450 mL of SF-SFM medium to make the content of fetal bovine serum 10%. After mixing, 500 mL of cell culture medium was obtained. The medium was placed in an incubator at 27℃ for 1 week. After checking for contamination, it was stored at 4℃.

[0058] (5) Preparation of cryopreservation solution: In a sterile laminar flow hood, prepare 50 mL of cell culture medium using SF-SFM medium, fetal bovine serum and dimethyl sulfoxide (DMSO). The volume ratio of each component of the cryopreservation solution is SF-SFM medium: fetal bovine serum: DMSO = 5:4:1, that is, take 25 mL of SF-SFM medium, 20 mL of fetal bovine serum and 5 mL of DMSO, mix well and set aside. The cryopreservation solution should be prepared and used immediately.

[0059] (6) Preparation of 0.4% trypan blue staining solution: To prepare 100 mL of 0.4% trypan blue, weigh 0.4 g of trypan blue powder and add it to about 80 mL of distilled water. Stir until completely dissolved, bring the volume up to 100 mL, filter with a 0.22 μm filter membrane and store in the dark.

[0060] II. Isolation and Culture of Lymphocytes from Yellow-spotted Moth (1) Disinfect the body surface of the yellow tussock moth larvae by soaking them in 75% alcohol and 2% sodium hypochlorite for 10 minutes respectively; (2) In a sterile laminar flow hood, rinse the surface of the yellow tussock moth larvae three times with sterile PBS solution, and then use sterile gauze to dry the surface of the larvae. (3) Place the worm in a disposable sterile culture dish with a diameter of 3 cm. Hold the posterior end of the worm with pointed forceps, lift the worm, and make a 3 mm incision on the side of the thorax with ophthalmic surgical scissors. Wait for the hemolymph to drain naturally. Use a sterile T-12.5 cm... 2 Collect culture flasks, each flask T-12.5 cm. 2 Five yellow-spotted moths were collected from the culture flask. It took about 30 seconds to collect the hemolymph from each yellow-spotted moth, and it took about 2 to 3 minutes to complete the separation of primary cells from the hemolymph of one flask of yellow-spotted moths. (4) Immediately apply the yellow-spotted moth hemolymph to a T-12.5 cm area. 2 Add 2 mL of cell culture medium to the culture flask, let it stand for 20 minutes, and examine the cell adhesion under a microscope. (See attached image.) Figure 1 ; (5) After standing, discard the upper layer of culture medium, add 2 mL of new culture medium, and repeat 3 times; (6) Tighten T-12.5 cm2 Place the cap on the bottle and incubate statically at 27°C in a cell culture medium without moving it. After 10 days, change the medium, discarding 50% of the old culture medium and adding 50% fresh culture medium. Repeat this medium change every 7 days. After 28 days, all cells will show a stringy growth pattern and spread outwards. (See [reference needed]) Figure 2 This is a microscope image of cells cultured for 28 days.

[0061] III. Establishment of the Yellow-spotted Moth Hemolymphocyte Line The cells were cultured for proliferation following the above steps. When the cells filled to a T-12.5 cm⁻¹ scale... 2 Subculture should be performed when the bottom area of ​​the culture flask is more than 90% full. (See [reference needed]) Figure 3 A microscope photograph showing cells covering the bottom of the flask; During passage, tap the side of the cell culture flask five times with your palm to dislodge the adherent cells. Transfer 50% of the cell culture volume from the primary flask to a fresh T-12.5 cm cell culture medium. 2 The culture flask was then replenished with 50% fresh culture medium; this was used for subculturing, and after two stable subculturings, the hemolymphocyte line of the yellow-spotted moth was initially established.

[0062] By the sixth generation, the hemolymphocytes of the yellow-spotted moth gradually adapted to the in vitro culture system, exhibiting a significantly accelerated growth and reproduction rate, with cells evenly spreading across the bottom of the culture flask. (See reference...) Figure 3 At this time, the cells grow at a rate of 1×10⁻⁶. 6 The initial density for passage was cells / mL, which increased to 2×10⁻⁶ after 24 hours. 7 The number of cells / mL increased to 1×10⁻⁶ after 48 hours. 8 The cell concentration was too high (number of cells / mL), causing the culture medium to become cloudy. The passage frequency could then be adjusted to 48 hours. Yellow-spotted moth hemolymphocytes were passaged at a frequency of 48 hours and remained stably cultured continuously even after 40 passages. (See [reference needed]). Figure 5 This indicates that the hemolymphocyte line of the yellow-spotted moth was successfully established.

[0063] III. Cryopreservation and Resuscitation of Lymphocytes from the Yellow-spotted Moth (1) Cryopreservation: In a sterile laminar flow hood, after counting the number of viable cells, adjust the cell culture concentration to 6 × 10⁻⁶ using cell culture medium. 7 Cells were collected at a density of 1 / mL and placed in sterile 50 mL centrifuge tubes. The tubes were centrifuged at 1400 rpm for 4 minutes. After discarding the supernatant, the cell pellet was resuspended in 50 mL of cryopreservation solution. The pellet was then aliquoted into 2 mL cryopreservation tubes, with 1 mL of cell solution in each tube. The tubes were placed in a cryopreservation box for programmed gradient cooling. After 24 hours, the cells were transferred to liquid nitrogen for cryopreservation.

[0064] (2) Thawing: Remove the cryovial containing cells from liquid nitrogen and immediately place it in a 37°C water bath to thaw. When 90% of the cells have thawed, transfer them to a new sterile 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 1 mL of cell culture medium. After resuspending, transfer the cells to a culture flask containing 59 mL of cell culture medium to achieve a cell concentration of 1×10⁻⁶ cells / mL. 6 Cells / mL were cultured in a static cell culture incubator at 27°C.

[0065] (3) Cell counting: 48 hours after resuscitation, 100 μL of well-mixed cell solution was taken from a sterile laminar flow hood and mixed with 100 μL of 0.4% trypan blue solution at a 1:1 ratio. 10 μL of the mixture was then dropped into the counting chamber of a hemocytometer. Under a 400x microscope, cells stained blue (dead cells) and unstained cells (live cells) were observed. Cell counts were performed, and cell viability was calculated. (See [reference needed]). Figure 4 Cells with a survival rate of over 80% can be successfully revived and cultured.

[0066] In the prior art, High Five cells derived from the white-striped armyworm have been used to propagate the tussah silkworm nucleopolyhedrovirus (Fan Qi et al., Application of Insect Cell High Five in the Culture of Tussah Silkworm Nucleopolyhedrovirus, 201310562776.8[P], 2016-06-22), and SF9 cells derived from the fall armyworm can not only be used for large-scale in vitro culture of the alfalfa silver-striped armyworm nucleopolyhedrovirus, but also for subsequent domestication to enhance viral virulence (Teng Xiaonuo et al., Domestication Method and Application of Nucleopolyhedrovirus Adapted to Fall Armyworm, 202010059629.9[P]. 2020-05-08). Specifically, the yellow-striped moth hemolymphocyte cell line provided by this invention can be used to propagate the yellow-striped moth nucleopolyhedrovirus. Furthermore, the hemolymph cell line of the yellow-spotted moth provided by this invention belongs to the insect cell line of the Limacodidae family of Lepidoptera. It is the first cell line of the slug moth, filling the gap in the cell line of the Limacodidae family. Therefore, the establishment of this cell line can be applied to in vitro experimental research on slug moths, such as the study of insect gene function and signaling pathways, the replication, assembly and interaction of baculoviruses or insect-specific viruses with their hosts, etc.

[0067] Because the larvae of Limacodidae insects have similar morphology and physiological structure, the method for isolating and culturing hemolymphocytes of the yellow-spotted moth provided in this invention is also applicable to the isolation and culture of primary hemolymphocytes of other Limacodidae insects, and has a certain degree of versatility.

[0068] Example 2 As a second aspect of the invention's objective, this invention discloses a method for producing *Tadpa spp.* nucleopolyhedrovirus using the *Tadpa spp.* hemolymphocyte cell line obtained in Example 1, the specific steps of which include: I. Cell Counting and Adjustment of Cell Concentration (1) Cell count Mix 100 μL of cell fluid with 0.4% trypan blue solution at a 1:1 ratio, add the mixture to the counting chamber of a hemocytometer, count the cells, and determine the number of viable cells.

[0069] (2) Adjusting cell concentration Adjust the viable cell concentration to 2.5 × 10⁻⁶ using cell culture medium. 6 The cell culture medium was a mixture of fetal bovine serum and SF-SFM medium at a volume ratio of 1:9, and the culture vessel used was a 250 mL shake flask.

[0070] II. Acquisition and purification of nucleopolyhedrovirus of the yellow-spotted moth (1) Acquisition of the virus The liquefied carcasses of yellow-spotted moth larvae infected with the virus were collected from the wild. The carcasses were ground with a mortar and pestle, and an appropriate amount of sterile water was added during the grinding process. The ground material was then filtered through three layers of sterile gauze to remove impurities. The resulting ground material was a crude extract of yellow-spotted moth nucleopolyhedrovirus.

[0071] (2) Virus purification The crude virus extract was transferred to a 50 mL centrifuge tube and centrifuged at 6000 rpm for 25 minutes. The supernatant was discarded, and the precipitate was resuspended in 50 mL of sterile water. This centrifugation process was repeated three times. The resulting suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant was retained, and the precipitate was discarded. This process was repeated three times, and the supernatants from the three low-speed centrifugations were combined. The mixture was then centrifuged at 10000 rpm for 30 minutes. After centrifugation, the supernatant was discarded, and the resulting precipitate was the purified *Spodoptera litura* nucleopolyhedrovirus. The purified *Spodoptera litura* nucleopolyhedrovirus solution was filtered through a 0.22 μm syringe filter in a sterile laminar flow hood. The resulting product was the purified *Spodoptera litura* nucleopolyhedrovirus.

[0072] III. Cell inoculation and virus harvesting (1) Cell inoculation The purified yellow-spotted moth nucleopolyhedrovirus was inoculated into the adjusted concentration of cell culture at 5% of the total culture volume, that is, 2.5 mL of yellow-spotted moth nucleopolyhedrovirus was inoculated into 50 mL of yellow-spotted moth hemolymphocytes. The cells were then placed in a shaker and cultured at 27°C and 110 rpm.

[0073] (2) Harvesting the virus After inoculation, the culture should be sampled every 24 hours to test cell viability and viral load. (See attached document.) Figure 6 .

[0074] Referring to Table 1, after approximately 120 hours of culture, cell viability drops below 30%, at which point the viral particle yield in the culture is at its maximum. The culture is then terminated, and the culture flask is collected. Following the virus purification steps described above, the *Spodoptera litura* nucleopolyhedrovirus produced from its hemolymph can be harvested. Cellular toxin production data are shown in Table 1 below. The highest toxin yield (2.47 × 10⁻⁶) was observed between 96 and 120 hours of culture. 9 PIB / mL.

[0075] Table 1. Data on nucleopolyhedrovirus produced by hemolymphocytes of the yellow-spotted moth. 24 h 48 h 72 h 96 h 120 h Virus concentration (PIB / mL) <![CDATA[7.3×10 6 ]]> <![CDATA[1.2×10 7 ]]> <![CDATA[2.18×10 8 ]]> <![CDATA[1.76×10 9 ]]> <![CDATA[2.47×10 9 ]]> IV. Toxicity Testing The nucleopolyhedrovirus produced by the hemolymphocytes of the yellow-spotted moth was concentrated under a microscope. The experimental procedures and methods followed NY / T 1154.14-2008, "Guidelines for Indoor Bioassay Testing of Pesticides (Insecticides Part 14: Leaf Dipping Method)". Fourth-instar larvae of the yellow-spotted moth were used as the test insects. Water (CK) and the virus extracted from the yellow-spotted moth were used as controls for virulence determination. Tukey multiple comparison data analysis was performed after correcting for cumulative mortality. The results are shown in Table 2 below, indicating that the nucleopolyhedrovirus produced by the hemolymphocytes of the yellow-spotted moth has a relatively ideal insecticidal effect on fourth-instar larvae. Compared with the nucleopolyhedrovirus extracted from the yellow-spotted moth, its virulence is weaker, but it still achieves a relatively ideal insecticidal effect. (See also...) Figure 7 The image on the left shows the corpse of an insect that died after consuming nucleopolyhedrovirus produced by the hemolymphocytes of the yellow-spotted moth, while the image on the right shows the corpse of an insect that died after consuming virus extracted from the diseased yellow-spotted moth. Both insects exhibited a typical "liquefaction" appearance when they became ill.

[0076] Table 2. Virulence determination of nucleopolyhedrovirus produced by hemolymphocytes of the yellow-spotted moth.

[0077] Note: The data processing above used the Tukey HSD multiple comparison method. Lowercase letters indicate the comparison results between different groups. The presence of the same letter indicates that the difference between the data at the significance level α = 0.05 is not significant.

[0078] The isolation and culture method provided by this invention can efficiently isolate and culture hemolymphocytes of the yellow-spotted moth. The hemolymphocytes of the yellow-spotted moth can be used to proliferate the nucleopolyhedrovirus of the yellow-spotted moth, and the produced nucleopolyhedrovirus has an ideal insecticidal effect on the fourth instar larvae of the yellow-spotted moth. This cell line has a good application prospect in the control of the yellow-spotted moth.

[0079] The nucleopolyhedrovirus of the yellow-spotted moth obtained by the above method can be used for the preparation of insecticides. In particular, it has a significant effect on the control of the yellow-spotted moth, and its virulence is comparable to that of the virus extracted directly from the yellow-spotted moth. After scale-up production through the yellow-spotted moth lymphocyte line, the nucleopolyhedrovirus of the yellow-spotted moth can be produced in large quantities, thereby meeting the needs of industrial application.

[0080] Example 3 This embodiment tests the virulence of the *Tabarella tigrinosa* nucleopolyhedrovirus produced from the aforementioned *Tabarella tigrinosa* hemolymphocyte line, specifically including: The virulence of *Tadpa spp.* nucleopolyhedrovirus (NPV) prepared by scaling up production using the *Tadpa spp.* hemolymphocyte cell line from Example 2 was tested. This included the preparation of a NPV suspension, the composition and mass percentages of which were as follows: 20% *Tadpa spp.* nucleopolyhedrovirus (300 million PIB / mL), 2.5% nonionic carboxylic acid hydrochloride DS733 (Guangzhou Fangzhong Chemical Co., Ltd.), 2.5% ethoxylated mixed sulfonate TT-1 (Guangzhou Fangzhong Chemical Co., Ltd.), 0.08% magnesium aluminum silicate (Shanghai Maclean Biochemical Technology Co., Ltd.), 0.02% xanthan gum (Shanghai Maclean Biochemical Technology Co., Ltd.), 0.3% potassium sorbate (Shanghai Maclean Biochemical Technology Co., Ltd.), 0.1% photoprotectant and fluorescent whitening agent VBL (Dinghai Plastics Chemical Co., Ltd.), and the remainder being pure water. The *Tadpa spp.* nucleopolyhedrovirus served as the parent drug in this formulation.

[0081] I. Preparation of Yellow-spotted Moth Nucleopolyhedrovirus Suspension The specific preparation steps include: (1) Weigh nonionic carboxylic acid hydrochloric acid, ethoxylated mixed sulfonate, fluorescent whitening agent, potassium sorbate and some pure water into beakers according to the weight ratio, and premix them in a high-speed homogenizer at 4000 rpm / min for 20 min. (2) Add yellow tussock moth nucleopolyhedrovirus, thickener magnesium aluminum silicate and xanthan gum to step (1), and at the same time replenish water, and reduce the speed of the high-speed homogenizer to 2000 rpm / min, and the hydration time to 30 min, so that the materials are fully mixed and uniform, and yellow tussock moth nucleopolyhedrovirus suspension is obtained.

[0082] II. Performance Index Testing of Yellow-spotted Moth Nuclear Polyhedrome Suspension The suspension prepared from the above-mentioned nucleopolyhedrovirus produced by the yellow-spotted moth hemolymph cell line was subjected to suspension performance tests. The experimental procedures and methods are as follows: GB / T 1601 Method for Determination of pH Value of Pesticides GB / T 1605 Sampling Methods for Commercial Pesticides GB / T 14825—2023 Method for Determination of Pesticide Suspension Rate GB / T 16150—1995 Methods for Determination of Pesticide Powders and Wettable Powders GB / T 28137 Determination of persistent foaming properties of pesticides GB / T 31737 Determination of Pesticide Dumping Properties GB / T 19136—2021 Determination of thermal storage stability of pesticides GB / T 19137-2003 Method for Determination of Low-Temperature Stability of Pesticides The results are shown in Table 3 below. The results indicate that all performance indicators of the *Tabarella asiatica* nucleopolyhedrovirus suspension meet industry and national standards. For details on the suspension system and key indicator determination, please refer to [reference needed]. Figure 8 .

[0083] Table 3 Technical indicators and test results of the yellow-spotted moth nucleopolyhedrovirus suspension

[0084] III. Toxicity Testing The toxicity of the suspension prepared from the nucleopolyhedrovirus produced by the *Tadpa spp.* hemolymph cell line was determined. The experimental procedures and methods followed NY / T 1154.14-2008, Guidelines for Indoor Bioassays of Pesticides (Insecticides Part 14: Leaf Dipping Method). Fourth-instar larvae of *Tadpa spp.* were used as the test insects, and water (CK) was selected as the control. Toxicity tests were conducted on the 2 billion spores / *Bacillus thuringiensis* suspension (Lu Kang Bio) and the *Tadpa spp.* nucleopolyhedrovirus suspension. The experimental results were calculated using DPS data processing software, and the toxicity regression equations and LC-1 values ​​for the control and test pesticides were derived. 50 LC 90 The results were calculated using a 95% confidence interval. Table 4 shows that the nucleopolyhedrovirus suspension of the yellow-spotted moth has a relatively ideal insecticidal effect on the fourth instar larvae of the yellow-spotted moth. Compared with the commercially available 2 billion spores / Bacillus thuringiensis suspension, its toxicity is similar, achieving a relatively ideal insecticidal effect.

[0085] Therefore, the nucleopolyhedrovirus suspension of the yellow tussock moth provided in this embodiment has an insecticidal rate of 85-95% against the fourth instar larvae of the yellow tussock moth.

[0086] Table 4. Results of indoor toxicity experiments of the nucleopolyhedrovirus suspension of the yellow-spotted moth against 4th instar larvae of the yellow-spotted moth. Test sample (experimental reagent) virulence regression equation <![CDATA[LC50(95%CL)(×10 7 PIB / mL)]]> <![CDATA[LC90(95%CL)(×10 7 PIB / mL)]]> Yellow-spotted moth nucleopolyhedrovirus suspension Y = 4.9825 + 1.9517X 1.0209（0.85~1.22） 4.6305（3.42~7.31） Table 5. Results of indoor toxicity tests of Beauveria bassiana suspension against 4th instar larvae of the yellow-spotted moth. Test sample (control reagent) virulence regression equation <![CDATA[LC50(95%CL)(×10 8 (spores / mL) <![CDATA[LC90(95%CL)(×10 8 (spores / mL) 2 billion spores / Bacillus thuringiensis suspension Y = 3.4760 + 2.2231X 4.8477（4.12~5.69） 18.2818（14.18~25.96） The experimental results in Table 4 show that the tested sample, the nucleopolyhedrovirus suspension of the yellow-spotted moth, has good insecticidal activity against fourth-instar larvae of the yellow-spotted moth. (LC50) 50 The value is 1.0209 × 10 7 PIB / mL, LC 90 The value is 4.6305 × 10 7 PIB / mL; As shown in Table 5, the commercially available Bacillus thuringiensis suspension, the control agent, also exhibits good toxicity, LC50 / mL. 50 The value is 4.8477 × 10 8 spores / mL, LC 90 The value was 18.2818 spores / mL. The yellow tussock moth nucleopolyhedrovirus suspension system prepared in Example 3 was stable, and all indicators met industry and national standards. It also showed good insecticidal ability against the fourth instar larvae of the target pest, the yellow tussock moth. Obviously, it can be used as a microbial pesticide insecticide and can be industrially produced and applied.

[0087] The successful isolation and culture of the *Tadpa fulvidraco* hemolymphocyte line provides crucial technical support for overcoming the core bottleneck in the industrial production of *Tadpa fulvidraco* nucleopolyhedrovirus (NPV) insecticides. Compared to traditional methods of producing NPV using live insects, conventional methods suffer from low virus production efficiency, high production costs, and, most importantly, unstable batch quality, severely hindering the widespread application of viral insecticides. This invention, utilizing the *Tadpa fulvidraco* lymphocyte line, achieves highly efficient NPV amplification in an in vitro, controlled environment. This signifies a shift in production methods from traditional, extensive "live-batch farming" to modern, precise "cell factories," significantly increasing virus yield, reducing production costs, and, most importantly, ensuring batch stability, thus providing technical support for its industrial-scale production.

[0088] In summary, the technical solution of this invention, which utilizes the hemolymph cell line of the yellow-spotted moth for virus amplification, not only greatly improves virus yield and production stability and significantly reduces production costs, but also lays a solid foundation for the standardization, large-scale and industrial production of NPV products. This has milestone significance for promoting the industrial upgrading of biological pesticides and reducing environmental pollution from chemical pesticides.

[0089] It should be noted that the above description is only a preferred embodiment of this application and is intended to provide a detailed description of this application. It is merely an example and is not intended to limit this application. Although those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A hemolymph cell line of the yellow-spotted moth, named: Yellow-spotted moth hemolymph CFW-CHY, has been deposited at the China Center for Type Culture Collection, accession number CCTCC NO:C2025104, and the deposit date is March 27, 2025.

2. The use of the *Tabarella tigrinosa* hemolymphocyte line as described in claim 1 in the large-scale production of nucleopolyhedrovirus.

3. A method for large-scale production of nucleopolyhedrovirus, characterized in that, The method involves inoculating the yellow-spotted moth nucleopolyhedrovirus into the yellow-spotted moth hemolymphocyte line as described in claim 1, incubating and purifying it, thereby enabling the large-scale production of the nucleopolyhedrovirus.

4. The method for large-scale production of nucleopolyhedrovirus according to claim 3, characterized in that, The concentration of nucleopolyhedrovirus obtained from scale-up production was 1.0 × 10⁻⁶. 7 ~1.0×10 9 PIB / mL.

5. The method for large-scale production of nucleopolyhedrovirus according to claim 3, characterized in that, The concentration of nucleopolyhedrovirus (NPV) used for inoculation of the yellow-spotted moth was 4–6 v / v.

6. The method for large-scale production of nucleopolyhedrovirus according to claim 3, characterized in that, The incubation conditions include a culture temperature of 25-37℃, a culture time of 48-120 h, and a shaking speed of 100-200 rpm.

7. The use of a method for scaling up the production of a nucleopolyhedrovirus as described in any one of claims 3-6 in the preparation of an insecticide for the yellow-spotted moth.

8. A suspension of nucleopolyhedrovirus from the yellow-spotted moth, characterized in that, It includes at least 300 million PIB / mL of yellow tussock moth nucleopolyhedrovirus at a mass percentage of 20%.

9. An insecticide comprising at least a nucleopolyhedrovirus prepared by the method for large-scale production of nucleopolyhedrovirus as described in any one of claims 3-6, or at least a suspension of the yellow-spotted moth nucleopolyhedrovirus as described in claim 8.

10. The insecticide according to claim 7, characterized in that, The insecticide has a kill rate of 85-95% against the larvae of the yellow-spotted moth.

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