Rhabdovirus-negative spodoptera frugiperda insect cell line, and screening, identification and application thereof

Abstract

The invention pertains to the technical fields of genetic engineering and cell engineering, and in particular relates to a rhabdovirus-negative spodoptera frugiperda insect cell line, and screening, identification and application thereof. According to the invention, the rhabdovirus-negative spodoptera frugiperda insect cell line WSK-Sf9, with a CCTCC accession number C202246, is obtained through screening and identification. The cell line is verified through various high-sensitivity test methods such as nested PCR, transcriptome next-generation sequencing , realtime fluorescence quantitative PCR and Taqman probe-based real-time PCR, and finally obtained the Sf-rhabdovirus-negative spodoptera frugiperda insect cell line WSK-Sf9. The cell is tested for sterility, mycoplasma, exogenous virus and tumorigenicity according to pharmacopoeial requirements, and the results show that all indicators satisfy the requirements, and the cell can be used in or for the production of recombinant proteins and recombinant protein vaccines based on the baculovirus expression system. (FIG 1)

Description

Rhabdovirus-negative Spodoptera frugiperda insect cell line and its screening, identification and application Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and cell engineering, and relates to a new fall armyworm insect cell strain, in particular to a rhabdovirus-negative fall armyworm insect cell strain and its screening, identification and application. Background Art

[0002] Insect cell expression systems have been widely used in the production of recombinant proteins. Compared with other expression systems, they have many advantages, such as: insect baculovirus specifically parasitizes invertebrates and is highly safe; high levels of recombinant protein expression; correct folding and post-translational modification of recombinant proteins to obtain biologically active proteins; adaptability to complex designs of multi-gene expression such as virus-like particles; and suitability for large-scale serum-free culture.

[0003] At present, many recombinant protein vaccines produced by insect cell expression systems have been approved for marketing around the world and have demonstrated good efficacy and safety. Among them, the Cervarix cervical cancer vaccine produced by GSK, the Provenge prostate cancer vaccine produced by Dendreon, and the FluBlok influenza vaccine produced by ProteinSciences are representative. In addition, many recombinant protein vaccines in the preclinical trial stage also show good application prospects.

[0004] Sf9 cells (Spodoptera frugiperda cells) are the most commonly used insect cell line in insect baculovirus expression systems for the expression and production of exogenous proteins, including antibodies, vaccines, and recombinant proteins. Sf9 cells are derived from the IPLBSF-21 cell line (also known as Sf21), which originated from the ovarian tissue of the fall armyworm (Spodoptera frugiperda) pupae isolated and cultured in 1977. Spodoptera frugiperda rhabdovirus (Sf-rhabdovirus) is a new negative-strand RNA virus discovered by US FDA researchers in 2014 in the Spodoptera frugiperda insect cell line Sf9 and its parental cell line Sf21. It contains genes encoding five structural proteins: N, P, M, G, and L. An additional gene sequence, X, of unknown function, was also detected between G and L. Although the Sf-rhabdovirus has not been integrated into the host cell genome, it has a complete genome and may be packaged into complete viral particles, which brings potential risks to the production and use of recombinant proteins such as vaccines based on the Sf9 cell baculovirus expression system.

[0005] Summary of the Invention

[0006] The present invention obtains the rhabdovirus-negative Spodoptera frugiperda Sf9 cell derivative line WSK-Sf9, referred to as WSK-Sf9, through screening and identification. This cell line can produce recombinant proteins based on the baculovirus expression system and is used for the production of recombinant protein vaccines.

[0007] The present invention provides a rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9, with a deposit number of CCTCC NO: C202246. The cell line is designated as the Spodoptera frugiperda Sf9 cell-derived line WSK-Sf9 and dated February 16, 2022. The cell line is deposited with the China Center for Type Culture Collection (CCTCC), located at Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, 430072.

[0008] The rhabdovirus-negative fall armyworm insect cell line WSK-Sf9 of the present invention is screened for a single clone using the limiting dilution method and verified by a variety of different high-sensitivity detection methods, including nested PCR, second-generation transcriptome sequencing, fluorescence quantitative PCR, and probe-based quantitative PCR, to obtain the rhabdovirus-negative fall armyworm insect cell line WSK-Sf9. At the same time, according to the requirements of the pharmacopoeia, the cell line was tested for sterility, mycoplasma, exogenous viruses, and tumorigenicity. The results showed that all indicators met the requirements and the cell line could be used as a cell matrix for production.

[0009] Another object of the present invention is to provide the use of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 in the production of recombinant proteins based on a baculovirus expression system.

[0010] Among them, the application includes the application of producing recombinant protein drugs or vaccines based on the baculovirus expression system.

[0011] Wherein, the recombinant protein drugs include cytokines, hormones, recombinant enzymes or antibodies.

[0012] The cytokines include recombinant human interleukin, recombinant human epidermal growth factor, recombinant human interferon, recombinant human fibroblast growth factor, recombinant human erythropoietin or recombinant human granulocyte macrophage stimulating factor.

[0013] Wherein, the hormone includes recombinant human growth hormone, recombinant human insulin, insulin analogs or recombinant human follicle-stimulating hormone.

[0014] Wherein, the recombinant enzymes include recombinant human α-glucosidase or recombinant human urokinase.

[0015] Wherein, the antibody includes a monoclonal antibody, a Fab antibody, a scFv antibody or a nanobody.

[0016] Wherein, the vaccine includes a recombinant protein vaccine or a virus-like particle vaccine.

[0017] Among them, the recombinant protein vaccine includes a new coronavirus protein vaccine, a hepatitis B virus protein vaccine or a rabies virus protein vaccine; preferably, it is a new coronavirus protein vaccine.

[0018] The virus-like particle vaccine includes a novel coronavirus virus-like particle vaccine (SARS-CoV-2-VLP), a human papillomavirus-like particle vaccine (HPV-VLP), an influenza virus-like particle vaccine (HA-VLP), a polio virus-like particle vaccine (PV-VLP), a respiratory syncytial virus-like particle vaccine (RSV-VLP), or a hand, foot and mouth disease virus-like particle vaccine (EV71-VLP). Preferably, it is a novel coronavirus virus-like particle vaccine.

[0019] The technical solution for realizing the application of WSK-Sf9 in the production of recombinant proteins based on a baculovirus expression system or in the vaccine production process of the present invention is: utilizing the Bac-to-Bac insect baculovirus expression system to package and produce baculovirus, infecting WSK-Sf9 cells to express the target protein, and obtaining the target recombinant protein by affinity purification technology.

[0020] Beneficial effects: The present invention uses the limiting dilution method to screen single clones, and the screening is verified by a variety of different high-sensitivity detection methods such as nested PCR, second-generation transcriptome sequencing, fluorescent quantitative PCR, and probe-based quantitative PCR to obtain the rhabdovirus-negative fall armyworm insect cell line WSK-Sf9; at the same time, according to the requirements of the pharmacopoeia, its sterility, mycoplasma, exogenous viruses, and tumorigenicity are tested. The results show that all indicators meet the requirements and can be used to produce recombinant protein products such as protein vaccines for clinical use. In addition, the present invention uses the Bac-to-Bac insect baculovirus expression system to package and produce baculovirus, infect WSK-Sf9 cells to express the target protein, and obtain the target recombinant protein through technical means such as affinity purification.

[0021] The rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening and identification in the present invention has a deposit number of CCTCC NO: C202246. The deposit date is February 16, 2022; the deposit center is the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072. The name and identification characteristics of the deposited culture are "Spodoptera frugiperda Sf9 cell derivative line WSK-Sf9." BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 is a nested PCR agarose gel electrophoresis result of the present invention for identifying WSK-Sf9 cells;

[0023] FIG2 shows the morphological characteristics of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 screened by the present invention;

[0024] FIG3 is a karyotype analysis of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 screened by the present invention;

[0025] FIG4 is a growth curve of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the present invention;

[0026] FIG5 is a continuous passage culture curve of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the present invention;

[0027] FIG6 is a time gradient detection of the expression of exogenous recombinant protein by the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the present invention. DETAILED DESCRIPTION

[0028] Below with reference to specific embodiment, the scheme of the present invention will be explained. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Reagents or instruments used that do not indicate the manufacturer are conventional products that can be obtained commercially.

[0029] The following examples will further illustrate the screening, identification and application of the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9. The present invention will be further described below with reference to the accompanying drawings.

[0030] Example 1: Screening and identification of the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9

[0031] Currently, the only commercially available Sf-rhabdovirus-negative Sf-RVN cells (Sf-rhabdovirus-negative Sf9) were screened from Sf9 by Professor Jarvis' team through limiting dilution combined with antiviral drug treatment (Maghodia AB, et al. Protein expression and purification. 2016; 122: 45-55.). Nested PCR targeting the L gene confirmed their Sf-rhabdovirus-negative status. These cells are owned by GlycoBac (http: / / www.glycobac.com / sf-rvn-cells), which has entered into a partnership with Millipore / Sigma to sell these cells.

[0032] According to the non-patent reference: Ma H, Nandakumar S, Bae EH, Chin PJ, Khan AS. The Spodoptera frugiperda Sf9 cell line is a heterogeneous population of rhabdovirus-infected and virus-negative cells: Isolation and characterization of cell clones containing rhabdovirus X-gene variants and virus-negative cell clones. Virology 2019; 536: 125-33., the fall armyworm Sf9 cell line is a heterogeneous cell population, which includes two types of cell populations, Sf- rhabdovirus-positive and Sf- rhabdovirus-negative, and a single Sf- rhabdovirus-negative cell population can be obtained by limiting dilution. The present invention uses the limiting dilution method to pick a single cell clone, and then screens and identifies Sf- rhabdovirus-negative cells by nested PCR, transcriptome sequencing, fluorescence quantitative PCR (Q-PCR), and probe-based quantitative PCR detection.

[0033] 1) The Sf9 parental cell line was purchased from ThermoFisher (Lot No: 2043331). The repurchased cells were defined as passage P0. The passage used in this screening experiment was P4. The cell culture medium was SIM SF serum-free medium (Beijing Sino Biological Science and Technology Co., Ltd., MSF1). The suspension-cultured Sf9 cells were diluted in a 10-fold limiting dilution series into well plates. The cells were observed every few days. When a single cell cluster formed, the cells were transferred to a new well plate for expansion. An appropriate number of cells were then collected, and total RNA was extracted. Candidate cells were initially identified using nested PCR primers targeting the Sf-Rhabdovirus-specific M gene (Table 1).

[0034] Table 1. Nested PCR primer sequences targeting the Sf-rhabdosome-specific M gene

[0035]

[0036] 2) The candidate Sf-Rhabdovirus-negative cells were serially subcultured, and cell samples were collected and frozen at -80°C for later use. After 45 consecutive passages, nested PCR analysis revealed that one of the cells, designated WSK-Sf9, was negative for the Sf-Rhabdovirus M gene from passages P1 to P45 (Figure 1).

[0037] 3) Transcriptome sequencing: 5×10 6 Transcriptome sequencing was performed on the parental Sf9 cells and WSK-Sf9 cells. The results showed that the RNA gene information of Sf-Rhabdovirus (GenBank: KF947078.1) was detected in the transcriptome of the parental Sf9 cells, but not in WSK-Sf9.

[0038] 4) Fluorescence quantitative PCR: Two pairs of quantitative PCR primers targeting the Sf-Rhabdovirus M gene were designed (Table 2). Fluorescence quantitative PCR detection was performed using the Bio-Rad SsoFastEvaGreensupermix kit. The results showed that no fluorescent signal was detected in WSK-Sf9 P3 and P28, indicating that the WSK-Sf9 cells were negative for Sf-Rhabdovirus (Table 3).

[0039] Table 2. Specific Q-PCR primers for the Sf-Rhabdovirus M gene

[0040]

[0041] Table 3. Q-PCR experimental data

[0042]

[0043] 5) Probe-based quantitative PCR: A taqMan probe targeting the Sf-Rhabdovirus M gene was designed (Table 4). Quantitative PCR detection revealed that no fluorescent signal was detected in either the WSK-Sf9 master cell bank (MCB) or the working cell bank (WCB), further demonstrating that the WSK-Sf9 cells were negative for Sf-Rhabdovirus (Table 5).

[0044] Table 4. TaqMan probe primers specific for the Sf-Rhabdovirus M gene

[0045]

[0046] Table 5. Q-PCR experimental data

[0047]

[0048] Example 2: Growth Characteristics of the Sf-Rhabdovirus-Negative Spodoptera frugiperda Insect Cell Line WSK-Sf9

[0049] 1) WSK-Sf9 culture characteristics: The cells can grow adherently or in suspension in serum-free culture medium at 27°C. The average diameter of the cells when growing in suspension is 16.28±0.34 μm, as shown in Figure 2.

[0050] 2) Karyotype analysis of WSK-Sf9 cells: Karyotype analysis of the parental Sf9 cells revealed that the chromosome number in 60 metaphases was mainly distributed between 180 and 250, with an average of 215 chromosomes per metaphase. In contrast, karyotype analysis of the WSK-Sf9 cells revealed that the chromosome number in 60 metaphases was mainly distributed between 191 and 538, with an average of 299 chromosomes per metaphase (Figure 3). This indicates that the Sf-Rhabdovirus-negative WSK-Sf9 cell line is different from its parental Sf9 cells.

[0051] 3) WSK-Sf9 cell growth curve: WSK-Sf9 cells in the logarithmic growth phase were diluted to 1×10 6 / ml, and then subcultured into 250ml breathable cap shake flasks with a culture volume of 100ml. The cells were continuously cultured in a 27℃ constant temperature shaker. The cell density and viability were counted every 24 hours. After three different batches of culture, the growth curves and viability are shown in Figure 4. The cells grew to 1×10 after 96 hours. 7 / ml and remained at this level for 6 days, with the highest cell density approaching 1.2×10 7 / ml, and starting from the 11th day, the cell viability gradually decreased, and the average cell doubling time was about 23 hours.

[0052] 4) Continuous subculture curve of WSK-Sf9 cells: WSK-Sf9 cells in the logarithmic growth phase were diluted to 1×10 6 / ml, subcultured into 250ml breathable cover shake flasks with a culture volume of 100ml, placed in a 27℃ constant temperature shaker for continuous culture, counted the cell concentration and viability every 3 days, and subcultured. Generally, after 3 days of culture, the cell concentration can reach 6-8×10 6 / ml, the viability was greater than 98%, and the continuous subculture curve was shown in Figure 5. After 100 generations of culture, the cell growth characteristics were stable.

[0053] Example 3: Safety Testing of the Sf-Rhabdovirus-Negative Spodoptera frugiperda Insect Cell Line WSK-Sf9

[0054] According to the 2020 edition of the Chinese Pharmacopoeia, Part III, the following series of tests were performed on WSK-Sf9 cells.

[0055] 1) Species identification: DNA barcoding assay revealed that the WSK-Sf9 cells were derived from Spodoptera frugiperda.

[0056] 2) Sterility test: using membrane filtration method, the result shows sterile growth;

[0057] 3) Mycobacterium examination: The culture method was used for testing, and the result showed negative for mycobacteria;

[0058] 4) Mycoplasma testing: using culture, indicator cell culture, and touchdown PCR, the results showed negative for mycoplasma;

[0059] 5) Spiroplasma test: using fluorescent PCR method, the result showed negative for Spiroplasma;

[0060] 6) Exogenous virus inspection:

[0061] (1) Using in vitro cell culture observation, hematocrit test and hematocrit test, different cell cultures including monkey Vero cells, human MRC-5 cells, Sf9 cells, BHK-21 cells, mosquito cells Aedes and Drosophila cells D.Mel were tested. All cells had normal morphology and the test results were negative.

[0062] (2) The in vivo method was used to inoculate suckling mice, adult mice and chicken embryos (5-6 day old chicken embryos, 9-11 year old chicken embryos), and the results showed that they all met the requirements.

[0063] 7) Retrovirus testing: No virus-like particles were observed using transmission electron microscopy, HEK293 cell inoculation and passage infectivity assays, and chemical reagent-induced virus testing, and the results met the requirements;

[0064] 8) Both bovine and porcine virus tests are negative;

[0065] 9) Other specific virus tests, including Baculoviruses, T.ni Furry Stall Virus Variant (FHVvar), Rhabdoviruses, Arboviruses Bluetongue Viruses (Reoviridae), Togaviruses, Flaviviruses, Bunyaviruses, Asfaviruses, Invertebrate Viruses Vesicular Viruses (Ascoviridae), Iridoviridae, Poxviridae The virus strains of the Chinese medicine laboratory were tested for Poxviridae, Baculoviridae, Poldnaviridae, Parvoviridae, Birnaviridae, Reoviridae, Picornaviralses, Dicistrovitidae, Nodaviridae and Tetraviridae, and the results were all negative, which was in compliance with the regulations.

[0066] 10) Tumorigenicity test: WSK-Sf9 cells were used to inoculate experimental mice, and the results showed that the cells were not tumorigenic, which met the regulations.

[0067] In summary, all safety tests of the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 were in compliance with regulations and met all requirements for cell matrices for the production of biological products.

[0068] Example 4: Expression of exogenous recombinant protein in Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9

[0069] A baculovirus expression vector containing the RBD domain of the novel coronavirus SARS-CoV-2 was constructed, and WSK-Sf9 was used to package the baculovirus for recombinant protein expression. Sf9 and WSK-Sf9 cells were cultured separately until the concentration reached 2.5×10 6 / ml, the virus was inoculated at a ratio of MOI = 0.5 to infect Sf9 and WSK-Sf9 cells respectively, and the supernatants were collected before infection (0 hours) and 24 hours, 48 ​​hours, 72 hours and 96 hours after infection. The supernatants were detected by western-blot using antibodies against the His tag. The results showed that the expression level of the recombinant protein in WSK-Sf9 cells was upregulated compared with that in Sf9 cells. Produced and purified in a GMP workshop, and after compounding with an adjuvant, the recombinant protein can be used to prevent infection with the new coronavirus. This shows that the Sf-rhabdovirus-negative fall armyworm insect cell line WSK-Sf9 can be used to express exogenous recombinant proteins such as protein vaccines, and its expression level is higher than that of Sf9 cells.

Claims

1. The rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 is deposited with CCTCC NO: C202246.

2. Use of the cell line WSK-Sf9 according to claim 1 in the production of recombinant proteins based on a baculovirus expression system.

3. The use according to claim 2, characterized in that: Including the application of producing recombinant protein drugs or vaccines based on baculovirus expression system.

4. The use according to claim 3, characterized in that: The recombinant protein drugs include cytokines, hormones, recombinant enzymes or antibodies.

5. The use according to claim 4, characterized in that: The cytokines include recombinant human interleukin, recombinant human epidermal growth factor, recombinant human interferon, recombinant human fibroblast growth factor, recombinant human erythropoietin or recombinant human granulocyte macrophage stimulating factor.

6. The use according to claim 4, characterized in that: The hormones include recombinant human growth hormone, recombinant human insulin, insulin analogs or recombinant human follicle-stimulating hormone.

7. The use according to claim 4, characterized in that: The recombinant enzymes include recombinant human α-glucosidase or recombinant human prourokinase.

8. The use according to claim 4, characterized in that: The antibodies include monoclonal antibodies, Fab antibodies, scFv antibodies or nanobodies.

9. The use according to claim 3, characterized in that: The vaccine includes a recombinant protein vaccine or a virus-like particle vaccine.

10. The use according to claim 9, characterized in that: The recombinant protein vaccine includes a novel coronavirus protein vaccine, a hepatitis B virus protein vaccine or a rabies virus protein vaccine; preferably, it is a novel coronavirus protein vaccine.

11. The use according to claim 9, characterized in that: The virus-like particle vaccine includes a new coronavirus virus-like particle vaccine, a human papillomavirus-like particle vaccine, an influenza virus-like particle vaccine, a polio virus-like particle vaccine, a respiratory syncytial virus-like particle vaccine or a hand, foot and mouth disease virus-like particle vaccine; preferably, it is a new coronavirus virus-like particle vaccine.