Spodoptera exigua ovarian cell line with high yield of baculovirus and construction and application thereof

Abstract

The invention provides a beet armyworm ovary cell line with high yield of baculovirus, the cell line is named as IOZCAS-Spex18, and the preservation number of the cell line is CGMCC (China General Microbiological Culture Collection Center) No.46039. The invention also discloses a preparation method of the cell line and application of the cell line in large-scale growth of baculovirus. The cell line can be used for replicating baculovirus, large-scale production of baculovirus insecticides, construction of a baculovirus expression vector system and expression of protein with commercial or scientific value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. The present invention relates to an insect cell line, in particular to a cell line derived from insect ovarian tissue and highly sensitive to baculovirus, and also relates to a method for constructing the cell line and the application of the cell line in the large-scale growth of baculovirus. Background Art

[0002] It is reported that more than 700 insect cell lines have been established in the past 60 years since the first insect cell line was successfully established in 1965. They are derived from more than 170 insect species such as Lepidoptera, Diptera, Homoptera, Hymenoptera, Orthoptera and Coleoptera, most of which are from Lepidoptera and Diptera. As research materials, insect cell lines have always been an important tool for scientific research in physiology, developmental biology, cell biology, molecular biology and biochemistry. In addition, insect cells, as an important part of the baculovirus expression vector system, express a large number of exogenous proteins with great economic or scientific significance. At the same time, as a bioreactor, insect cells also play an important role in amplifying insect baculoviruses for use as biological insecticides, especially amplifying recombinant baculovirus insecticides containing exogenous genes.

[0003] Lepidoptera cell lines are derived from many tissues, including ovaries, embryos, blood cells, fat bodies, etc. Cell lines from different species of insects or cell lines from different tissues of the same insect have different abilities to amplify viruses or express recombinant proteins. Insect ovaries are usually paired and are the site of egg formation and development. Since Grace established the first insect cell line using ovarian tissue, the ovary has been an important tissue source for cell line establishment. Therefore, establishing and screening new and more baculovirus-sensitive cell lines (strains) is a very necessary and theoretically and practically significant task.

[0004] So far, there are 29 cell lines originally established from Spodoptera exigua recorded in the Cellosaurus database (https: / / web.expasy.org / cellosaurus), of which 8 are from (Li et al., 2012; Li et al., 2013; Zhang et al., 2012; Chaeychomsri et al., 2018; Wu et al., 2012); the virus sensitivity is summarized in Table 1. Although most of the cell lines derived from the ovaries of Spodoptera exigua listed in the above table showed a high infection rate to SeNPV and AcMNPV, there is no report on the cell lines derived from the ovaries of Spodoptera exigua that are highly sensitive to MbNPV.

[0005] Table 1 Sensitivity of the established Helicoverpa armigera pupal ovary cell line to viruses

[0006]

[0007] References:

[0008] 1. Li X, Qin Q, Zhang N, Zhu W, Zhang J, Wang H, Miao L, Zhang H. A new insect cell line from pupal ovary of Spodoptera exigua established by stimulation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In Vitro Cell Dev Biol Anim. 2012 May; 48(5):271-5.

[0009] 2. Li X., Zhang H., Qin Q.-L., Wang Y.-L., Wang H.-T., Zhang J.-H., Miao L., Meng Q., Zhang N., Zhu W., Zhou G.-L., Yang Q. Transgenic insect cell line for high-yield baculovirus, and preparation method and application thereof. Patent number CN102807969B, 25-Sep-2013 Patent=CN102807969B

[0010] 3. Zhang A.-J., Li X., Zhang H., Wang H., Miao L., Zhang J., Qin Q.-L. A new cell line from Spodoptera exigua (Lepidoptera: Noctuidae) and its differentially expressed genes. J. Appl. Entomol. 136:632-637 (2012)

[0011] 4.Chaeychomsri S.,Chaeychomsri W.,Siruntawineti J.,Ikeda M.,KobayashiM.Establishment of new cell lines from pupal ovaries of Spodoptera exiguawith differential susceptibility to homologous nucleopolyhedrovirus.J.Adv.Agric.Technol.5:281-288(2018)

[0012] 5.Wu C.-Y.,Chen Y.-W.,Lin C.-C.,Hsu C.-L.,Wang C.-H.,Lo C.-FA newcell line(NTU-SE) from pupal tissues of the beet armyworm,Spodoptera exigua(Lepidoptera:Noctuidae), is highly susceptible to S.exigua multiplenucleopolyhedrovirus(SeMNPV)and Autographa californica MNPV (AcMNPV). J. Invertebr. Pathol. 111:143-151 (2012). Summary of the invention

[0013] In view of the problem that insect cell lines sensitive to baculovirus still need to be further developed, the present invention provides a beet armyworm ovarian cell line with high baculovirus production. The present invention also provides the use of the beet armyworm ovarian cell line with high baculovirus production in the large-scale growth of baculovirus.

[0014] Specifically, on one hand, the present application relates to a Spodoptera exigua ovarian cell line with high baculovirus production, the name of the cell line is IOZCAS-Spex18, and its deposit number is CGMCC No.46039.

[0015] On the one hand, the present application relates to a method for preparing a beet armyworm ovarian cell line with high baculovirus production, which comprises the following steps: 1) culturing beet armyworm ovarian cells with cell culture medium; and 2) obtaining a beet armyworm ovarian cell line with high baculovirus production.

[0016] In some embodiments, the cell culture fluid is a mixture of insect cell culture fluid and antibiotics. The antibiotics are penicillin and streptomycin. The cell culture fluid is also supplemented with fetal bovine serum. The penicillin content is 100U / mL; the streptomycin content is 100μg / mL. The fetal bovine serum content is 10% of the volume ratio of the cell culture fluid. The cell culture fluid has a pH of 5.8-6.8. The insect cell culture fluid is selected from: Insect-XPRESSTM, TNM-FH, Grace's, Sf-900, TC-100, IPL-41 or Ex-Cell 400.

[0017] In one aspect, the present application relates to the use of a Spodoptera exigua ovarian cell line with high baculovirus production in baculovirus production.

[0018] In some embodiments, the baculovirus is Autographa californica nuclear polyhedrosis virus (AcMNPV).

[0019] In some embodiments, the baculovirus is Spodoptera brassicae nuclear polyhedrosis virus (MbNPV).

[0020] In one aspect, the present application relates to the use of a Spodoptera exigua ovarian cell line with high baculovirus production in the preparation of baculovirus insecticides.

[0021] In one aspect, the present application relates to the use of a Spodoptera exigua ovarian cell line with high baculovirus production in the preparation of a baculovirus expression vector system.

[0022] Beneficial effects:

[0023] The high-baculovirus-producing Spodoptera exigua ovarian cell line provided by the present invention can be used to replicate baculovirus, for large-scale growth of baculovirus and large-scale production of baculovirus insecticides; the cell line can also be used to construct a baculovirus expression vector system, which can express recombinant proteins with extremely high commercial and scientific research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the primary culture process of the ovarian tissue of Spodoptera exigua adherent to the wall. Cells can be seen to be free from the tissue, proving that the cells are derived from the ovarian tissue of the pupae of Spodoptera exigua. The scale bar is 100μm.

[0025] Figure 2 The cell morphology of the beet armyworm ovarian cell line IOZCAS-Spex18 of the present invention, scale bar 50 μm;

[0026] Figure 3A , Figure 3B , Figure 3CIt is the result of comparing the COI gene sequence of the beet armyworm ovarian cell line IOZCAS-Spex18 of the present invention with the sequence of the COI gene of the beet armyworm pupa and NCBI;

[0027] Figure 4 is the growth curve of the beet armyworm ovarian cell line IOZCAS-Spex18 of the present invention;

[0028] Figure 5 After IOZCAS-Spex18 was infected with Spodoptera exigua nuclear polyhedrosis virus (SeNPV), a large amount of virus polyhedrosis was obtained. The scale bar is 100 μm.

[0029] Figure 6 This is the result of IOZCAS-Spex18 infection with Autographa californica nuclear polyhedrosis virus (AcMNPV), yielding a large number of viral polyhedroses. Scale bar: 100 μm.

[0030] Figure 7 This is the result of IOZCAS-Spex18 infection with cabbage armyworm nuclear polyhedrosis virus (MbNPV), yielding a large number of viral polyhedrosis. Scale bar: 100 μm.

[0031] Figure 8 IOZCAS-Spex18 was inoculated with cabbage looper nuclear polyhedrosis virus (MbNPV), and the cells were not infected. The scale bar in the figure is 100 μm.

[0032] Deposit of biological materials

[0033] The high-yield baculovirus-producing beet armyworm ovarian cell line IOZCAS-Spex18, classified and named as the beet armyworm ovarian cell line, was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) on August 16, 2024. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No. 46039. DETAILED DESCRIPTION

[0034] The present application is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only used to illustrate the present application and are not used to limit the scope of the present application.

[0035] On the one hand, the present application provides a high-yield baculovirus beet armyworm ovarian cell line, the name of the cell line is IOZCAS-Spex18, and its deposit number is CGMCC No. 46039. The cell line is classified and named as the beet armyworm ovarian cell line, and has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on August 16, 2024.

[0036] On the other hand, the present application provides a method for preparing a beet armyworm ovarian cell line with high baculovirus production, which comprises the following steps: 1) culturing beet armyworm ovarian cells with cell culture medium; and 2) obtaining a beet armyworm ovarian cell line with high baculovirus production.

[0037] In a specific embodiment, a method for preparing a Spodoptera exigua ovarian cell line with high baculovirus production comprises the following steps:

[0038] (1) Immerse female pupae of Spodoptera exigua in an ethanol solution for 10 to 20 minutes to disinfect the surface of the insects, then wash the insects with sterile distilled water and dry the insect surface;

[0039] (2) dissecting the insects and removing the complete ovarian tissue of Spodoptera exigua;

[0040] (3) Wash the tissue obtained in (2) 2 to 3 times with physiological saline, and then wash it with cell culture medium. After washing, put the tissue into a cell culture bottle containing 1 mL of cell culture medium, cover the bottle cap tightly, and culture it in a cell culture incubator at 27° C. for 24 hours;

[0041] (4) adding an appropriate amount of cell culture medium so that the tissue block is completely or mostly immersed in the culture medium, and culturing under the same conditions as step (3). After about 21 days of culture, a large number of primary cells can be obtained;

[0042] (5) nitrogen was introduced into the three-gas incubator instead of oxygen, and the oxygen concentration in the incubator was detected by an oxygen sensor to control and reduce the oxygen content in the incubator to 5%; at the same time, the airtight bottle cap of the cell culture bottle was replaced with a breathable bottle cap so that the gas in the cell culture bottle and the gas in the incubator could circulate to achieve a hypoxic effect, and the insect primary cells were cultured at 27° C. for 9 days in hypoxia;

[0043] (6) replacing the cell culture medium and the cap of the culture flask, and culturing at 27° C. under normoxia for 10-30 days, for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days or 30 days; (7) continuing to culture the reoxygenated cells, allowing the living cells to continue to proliferate until they fill the bottom of the cell culture flask, and starting to pass the cells, thereby successfully establishing a beet armyworm cell line and obtaining the beet armyworm ovarian cell line with high baculovirus production of the present invention; the entire process is carried out under sterile conditions.

[0044] In a specific embodiment, the cell culture medium used in the above method of the present invention is a mixture of a conventional insect cell culture medium and penicillin, streptomycin and fetal bovine serum, and the pH of the prepared cell culture medium is 5.8-6.8, for example, 5.8-5.9, 5.9-6.0, 6.0-6.1, 6.0-6.2, 6.0-6.3, 6.0-6.4, 6.0-6.5, 6.0-6.6, 6.0-6.7, 6.0-6.8. The insect cell culture medium is a commercial insect cell culture medium Insect-XPRESSTM, TNM-FH, Grace's, Sf-900, TC-100, IPL-41, or Ex-Cell 400. Typically, the penicillin content in the cell culture medium is 10U / mL-500U / mL, preferably 100U / mL; the streptomycin content is 10μg / mL-500μg / mL, preferably 100μg / mL; the animal serum content is 5%-20% by volume of the cell culture medium, preferably 10% by volume of the cell culture medium.

[0045] On the other hand, the present application provides a use of a Spodoptera exigua ovarian cell line with high baculovirus production in baculovirus production.

[0046] In a specific embodiment, the baculovirus is Spodoptera exigua nuclear polyhedrosis virus (SeNPV), Autographa californica nuclear polyhedrosis virus (AcMNPV), and Spodoptera brassicae nuclear polyhedrosis virus (MbNPV).

[0047] On the other hand, the present application provides a use of a beet armyworm ovarian cell line with high baculovirus production in the preparation of a baculovirus insecticide.

[0048] On the other hand, the present application provides a use of a Spodoptera exigua ovarian cell line with high baculovirus production in preparing a baculovirus expression vector system.

[0049] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0050] Example 1. Establishment of Spodoptera exigua ovarian cell line

[0051] Take the mid-stage beet armyworm pupa (raised by the Insect Virology Research Group of the State Key Laboratory of Integrated Agricultural Pest Management, Institute of Zoology, Chinese Academy of Sciences) and immerse it in a 75% ethanol solution for 10 minutes for surface disinfection. Dissect the insect and remove the ovarian tissue, keeping it intact as much as possible during the operation. The tissue was washed 2-3 times with physiological saline, and then washed 1-2 times with cell culture medium (Insect-XPRESSTM (BioWhittaker, Lonza, Walkersville, MD, USA, 12-730Q), containing 100U / mL penicillin, 100μg / mL streptomycin and 10% (v / v) fetal bovine serum (BI, Beit Haemek, Israel, 04-001-1), pH = 6.2), and placed in a 25cm2 cell culture bottle rinsed with 1mL of the above cell culture medium, and cultured in a cell culture incubator at 27°C without light for 24 hours. Then 3mL of the above cell culture medium was added and cultured under the same conditions. After the 2-3rd day of this operation, a large number of single cells were observed to be freed from the tissue and gradually expanded to the periphery ( Figure 1 ). After about 21 days of primary culture, the cells gradually proliferated and filled the entire culture bottle. The sealed bottle cap was replaced with a breathable bottle cap, placed in a three-gas incubator, nitrogen was introduced and the oxygen concentration was controlled to 5%. After 9 days of culture under this low oxygen condition, fresh culture medium and sealed bottle cap were replaced. After culture for about 30 days under reoxygenated conditions, the newly proliferated cells, together with all the cell culture medium, were aspirated and placed in a new culture bottle, and 2 mL of the above-mentioned new cell culture medium was added. The establishment of the cell line was initially successful. The second sub-bottle subculture began 7 days after the cells began to be subcultured. The subculture time gradually shortened thereafter. When it was subcultured to the 8th generation, the cell growth began to stabilize, and the cell line was finally named IOZCAS-Spex18. IOZCAS-Spex18 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 25, 2020, with the deposit number CGMCC No.46039.

[0052] Example 2. Observation and determination of biological characteristics of IOZCAS-Spex18

[0053] (1) Morphological characteristics: Microscopic observation showed that the IOZCAS-Spex18 cell line included cells suspended in the culture medium and cells attached to the wall. The cell shapes were of two types: round and spindle-shaped. Round cells accounted for 52.17% of the IOZCAS-Spex18 cells, with an average diameter of 18.32±3.04μm (mean±SD); spindle-shaped cells accounted for 43.75%, with a length of 54.58±21.16μm and a width of 11.53±3.76μm (mean±SD). Figure 2 ).

[0054] (2) Cell growth: IOZCAS-Spex18 was inoculated into a 24-well plate at a concentration of 1×106 cells / mL and cultured at 27°C. The cell growth curve and population doubling time of the 35th generation were determined according to the method of McIntosh and Ignoffo, 1989 (McIntosh, A Hand and CM Ignoffo J. Invertebr. Pathol. 1989, 54: 97-102). That is, the cell concentration was measured every 24 hours, and the growth curve was drawn ( Figure 4 ), and the cell population doubling time was calculated according to the formula T = tlg2 / [lg(N / N0)] (where T = the time required for the average growth to double in the logarithmic phase; t = the time from inoculation to the determination of the number of cells; N0 = the number of cells at the time of inoculation; N = the total number of cells measured at time t). The IOZCAS-Spex18 cell line at the 35th generation was cultured in the Insect-XPRESSTM medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin. The cell population doubling time at the 35th generation was 69.24 hours. Currently, the cell line has been subcultured for more than 50 generations.

[0055] (3) The method of sequencing the ITS region of ribosomal RNA was used to identify that the cell line IOZCAS-Spex18 was indeed derived from Spodoptera exigua, and was not contaminated by other cell lines or Sf9 cells. DNA extracted from IOZCAS-Spex18 was used for PCR amplification with DNA from blood of Spodoptera exigua larvae. The primers used for amplification of the ITS region of ribosomal RNA were (ITS1-1: 5'-CCC CAT AAACGA GGA ATT CC-3' and ITS4: 5'-TCC TCC GCT TAT TGA TAT GC-3' ( Figure 3A , Figure 3B and Figure 3C ). The ITS domain similarity between Spex18 and beet armyworm larvae is 99.69%, while the ITS domain similarity between Spex18 and Sf9 is 53.79%, and the ITS domain similarity between Spex18 and cotton bollworm larvae is 48.13%.

[0056] (4) Cryopreservation and recovery: Use traditional cell cryopreservation methods to freeze cells, preserve cell germs, and successfully recover them.

[0057] Example 3. Determination of the sensitivity of IOZCAS-Spex18 to Spodoptera exigua nuclear polyhedrosis virus (SeNPV)

[0058] SeNPV budding virus particles BV (3 days after beet armyworm larvae feed on SeNPV polyhedrons, larval hemolymph is collected, and the supernatant solution after removing blood cells is preserved by the Institute of Zoology, Chinese Academy of Sciences) are inoculated with IOZCAS-Spex18 at a concentration of MOI (multiplicity of infection) = 1. After 7 days of culture, cells and virus polyhedrons can be harvested, and the infection rate is about 90.87%. Under an inverted phase contrast microscope, typical cytopathological characteristics can be observed, that is, enlarged cell nuclei containing a large number of virus polyhedron particles ( Figure 5 , Figure 6 ).

[0059] Example 4. Determination of the sensitivity of IOZCAS-Spex18 to Autographa californica nuclear polyhedrosis virus (AcMNPV)

[0060] AcMNPV budding virus particles BV (3 days after beet armyworm larvae feed on AcMNPV polyhedrons, the larval hemolymph is taken, and the supernatant solution after removing blood cells is preserved by the Institute of Zoology, Chinese Academy of Sciences) are inoculated with IOZCAS-Spex18 at a concentration of MOI (multiplicity of infection) = 1. After 7 days of culture, cells and virus polyhedrons can be harvested, and the infection rate is about 92.56%. Under an inverted phase contrast microscope, typical cytopathological characteristics can be observed, that is, enlarged cell nuclei containing a large number of virus polyhedron particles ( Figure 7 ).

[0061] Example 5. Determination of the sensitivity of IOZCAS-Spex18 to Spodoptera brassicae nuclear polyhedrosis virus (MbNPV)

[0062] IOZCAS-Spex18 is sensitive to cabbage armyworm nuclear polyhedrosis virus (MbNPV): MbNPV budding virus particles BV (3 days after beet armyworm larvae feed on MbNPV polyhedrons, larval hemolymph is taken, and the supernatant solution after removing blood cells is preserved by the Institute of Zoology, Chinese Academy of Sciences) are inoculated with IOZCAS-Spex18 at a concentration of MOI (multiplicity of infection) = 1. After 7 days of culture, cells and virus polyhedrons can be harvested, and the infection rate is about 86.4%. Under an inverted phase contrast microscope, typical cytopathological characteristics can be observed, that is, enlarged cell nuclei containing a large number of virus polyhedral particles ( Figure 8 ).

Claims

1. A beet armyworm ovarian cell line with high baculovirus production, wherein: The cell line is named IOZCAS-Spex18, and its deposit number is CGMCC No.46039.

2. The method for preparing an ovarian cell line according to claim 1, wherein: The method comprises the following steps: (1) culturing beet armyworm ovarian cells using cell culture medium; (2) Obtain a Spodoptera exigua ovarian cell line that produces high baculovirus.

3. The method of claim 2, wherein: The cell culture fluid is a mixture of insect cell culture fluid and antibiotics, and the pH of the cell culture fluid is 5.8-6.8; the antibiotics are penicillin and streptomycin.

4. The method of claim 2, wherein: The cell culture medium is also supplemented with fetal bovine serum, and the content of the fetal bovine serum is 5%-20% by volume of the cell culture medium.

5. The method of claim 3, wherein: The penicillin content is 10U / mL-500U / mL; the streptomycin content is 10μg / mL-500μg / mL.

6. The method of claim 2, wherein: The insect cell culture medium is selected from: Insect-XPRESSTM, TNM-FH, Grace's, Sf-900, TC-100, IPL-41 or Ex-Cell 400.

7. Use of the ovarian cell line according to claim 1 in baculovirus production.

8. The use according to claim 7, wherein The baculoviruses are Autographa californica nuclear polyhedrosis virus (AcMNPV), Cabbage looper nuclear polyhedrosis virus (MbNPV) and Beet armyworm nuclear polyhedrosis virus (SeNPV).

9. Use of the ovarian cell line according to claim 1 in the preparation of baculovirus insecticides.

10. Use of the ovarian cell line according to claim 1 in preparing a baculovirus expression vector system.

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