Rhabdovirus-negative Spodoptera frugiperda insect cell lines, their screening, their identification and their uses

Abstract

The present invention belongs to the technical field of genetic engineering and cell engineering, and particularly relates to a rhabdovirus-negative Spodoptera frugiperda insect cell line, its screening, identification and use. According to the present invention, a rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 is obtained by screening and identification, and deposited under CCTCC NO:C 202246. The cell line is verified by various different highly sensitive assay methods such as nested PCR, transcriptome next generation sequencing, fluorescence-based quantitative PCR and probe-based quantitative PCR, and finally obtained by screening. The cells are tested for sterility, mycoplasma, exogenous viruses, tumorigenicity, etc. according to the requirements of the pharmacopoeia, and the results show that the cells meet the requirements in all tests. The cells can produce recombinant proteins based on the baculovirus expression system, and can be used for the production of recombinant protein vaccines.

Description

[Technical Field]

[0001] The present invention relates to the technical fields of genetic engineering and cell engineering, and relates to a novel Spodoptera frugiperda insect cell line, in particular to a rhabdovirus-negative Spodoptera frugiperda insect cell line and its screening, identification and application. [Background technology]

[0002] Insect cell expression systems are widely used for recombinant protein production and offer many advantages over other expression systems. For example, insect baculoviruses are only parasitic on invertebrates, making them highly safe, enabling high levels of recombinant protein expression, and allowing for the correct folding and modification of post-translational recombinant proteins to yield biologically active proteins. Insect cell expression systems are compatible with complex multi-gene expression designs, such as virus-like particles, and are suitable for large-scale serum-free culture.

[0003] Currently, several recombinant protein vaccines produced using insect cell expression systems have demonstrated good efficacy and safety and have been approved worldwide, including the Cervarix cervical cancer vaccine produced by GSK, the Provenge prostate cancer vaccine produced by Dendreon, and the FluBlok influenza vaccine produced by ProteinSciences. Furthermore, many recombinant protein vaccines in the preclinical experimental stage also show good prospects for application.

[0004] Sf9 cells (Spodoptera frugiperda cells) are the most commonly used insect cells in insect baculovirus expression systems to express and produce foreign proteins, including antibodies, vaccines, and recombinant proteins. Sf9 cells are derived from the IPLBSF-21 cell line (also known as Sf21), which was isolated and cultured in 1977 from the ovarian tissue of autumn flyworm (Spodoptera frugiperda) pupae. Sf-rhabdovirus is a new negative-strand RNA virus discovered by FDA researchers in 2014 in the Spodoptera frugiperda cell line Sf9 and its parent cell line Sf21. It contains genetic information for the N, P, M, G, and L structural proteins. Furthermore, an extra gene sequence, X, with unknown function, between G and L was also tested. Sf-rhabdovirus does not integrate into the host cell genome but possesses a complete genome and can be packaged into complete viral particles. This poses a potential risk to the production and use of recombinant proteins, such as vaccines, derived from the Sf9 cell baculovirus expression system. Summary of the Invention [Problem to be solved by the invention]

[0005] According to the present invention, a rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 (abbreviated as WSK-Sf9) was obtained through screening and identification, and this cell line can be used for the production of recombinant proteins and recombinant protein vaccines based on the baculovirus expression system. [Means for solving the problem]

[0006] The present invention provides a rhabdovirus-negative Spodoptera frugiperda insect cell line, WSK-Sf9, with CTCCC accession number C202246. The cell line, designated Spodoptera frugiperda Sf9-derived cell line WSK-Sf9, was collected on February 16, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, postal code 430072.

[0007] The present invention features a rhabdovirus-negative Spodoptera frugiperda insect cell line, WSK-Sf9, which was obtained by screening monoclonal cells using the limiting dilution method and verifying them with various highly sensitive testing methods, including nested PCR, transcriptome next-generation sequencing, real-time fluorescent quantitative PCR, and TaqMan probe-based real-time PCR. The cells were tested for sterility, mycoplasma, exogenous viruses, and tumorigenicity according to Pharmacopoeia requirements. The results indicated that all indicators met the requirements and the cells could be used as a cell matrix for production.

[0008] Another object of the present invention is to provide the application of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 in the production of recombinant proteins based on the baculovirus expression system.

[0009] Such applications include those for the production of recombinant protein pharmaceuticals or vaccines based on baculovirus expression systems.

[0010] Recombinant protein pharmaceuticals include cytokines, hormones, recombinant enzymes or antibodies.

[0011] Cytokines include recombinant human interleukins, recombinant human epidermal growth factor, recombinant human interferons, recombinant human fibroblast growth factor, recombinant human erythropoietin, or recombinant human granulocyte macrophage stimulating factor.

[0012] The hormones include recombinant human growth hormone, recombinant human insulin, insulin analogs or recombinant human follicle-maturing hormone.

[0013] Recombinant enzymes include recombinant human α-glucosidase or recombinant human pro-urokinase.

[0014] Antibodies include monoclonal antibodies, Fab antibodies, scFv antibodies or nanobodies.

[0015] Vaccines include recombinant protein vaccines or virus-like particle vaccines.

[0016] The recombinant protein vaccine includes a SARS-CoV-2 protein vaccine, an influenza virus protein vaccine, a syncytial virus recombinant protein vaccine, a hepatitis B virus protein vaccine or a rabies virus protein vaccine, preferably a SARS-CoV-2 protein vaccine.

[0017] Virus-like particle vaccines include SARS-CoV-2 virus-like particle vaccines (SARS-CoV-2-VLPs), human papillomavirus-like particle vaccines (HPV-VLPs), influenza virus-like particle vaccines (HA-VLPs), poliovirus-like particle vaccines (PV-VLPs), respiratory syncytial virus-like particle vaccines (RSV-VLPs), or hand, foot, and mouth disease virus-like particle vaccines (EV71-VLPs). Preferably, the virus-like particle vaccine is a SARS-CoV-2 virus-like particle vaccine.

[0018] The technical solution for applying WSK-Sf9 to the production of recombinant proteins or vaccines based on the baculovirus expression system is as follows: Using the Bac-to-Bac insect baculovirus expression system, baculovirus is packaged and produced, and then infected into WSK-Sf9 cells to express the target protein, and the target recombinant protein is obtained by affinity purification. [Effects of the Invention]

[0019] The beneficial effects of the present invention are as follows: After screening monoclonal cells using the limiting dilution method and verifying them with various highly sensitive testing methods, including nested PCR, transcriptome next-generation sequencing, real-time fluorescent quantitative PCR, and TaqMan probe-based real-time PCR, a rhabdovirus-negative Spodoptera frugiperda insect cell line, WSK-Sf9, was obtained. The cells were tested for sterility, mycoplasma, exogenous viruses, and tumorigenicity according to Pharmacopoeia requirements. The results showed that all indicators met the requirements, indicating that the cells can be used to produce recombinant protein products, such as protein vaccines, for clinical use. Additionally, according to the present invention, a Bac-to-Bac insect baculovirus expression system is used to package and produce baculovirus, infect WSK-Sf9 cells, and express the target protein. The target recombinant protein can then be obtained by affinity purification or other technical methods.

[0020] According to the present invention, the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained through screening and identification has the CTCCC accession number C202246. The cell line was collected on February 16, 2022. The collection center is the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, zip code 430072. The name and identifying characteristics of the collected culture are Spodoptera frugiperda Sf9-derived cell line WSK-Sf9. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the results of nested PCR agarose gel electrophoresis for the identification of WSK-Sf9 cells according to the present invention. [Figure 2] FIG. 1 shows the morphological characteristics of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the present invention. [Figure 3] FIG. 1 shows the karyotype analysis of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the present invention. [Figure 4] FIG. 1 shows the growth curve of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the present invention. [Figure 5] 1 shows the serial passage curve of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the present invention. [Figure 6] FIG. 1 shows a time gradient test of exogenous recombinant proteins expressed by the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] The solutions of the present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that the following embodiments are intended to illustrate the present invention only and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the technical literature or product specifications shall prevail. If the manufacturers of the reagents or equipment used are not indicated, they are all conventional products that can be purchased from commercial reagent companies.

[0023] The screening, identification and application of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 are further illustrated in the following embodiments, and the present invention will be further described with reference to the accompanying drawings. [Example]

[0024] Example 1: Screening and identification of the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9

[0025] The only commercially available Sf-rhabdovirus-negative Sf-RVN cells (Sf-rhabdovirus-negative Sf9) were screened from Sf9 by Professor Jarvis's team using limiting dilution in combination with antiviral drug treatment (Maghodia AB et al., Protein expression and purification. 2016;122:45-55.) and confirmed to be Sf-rhabdovirus-negative by nested PCR of the L gene. The cells are attributed to GlycoBac (http: / / www.glycobac.com / sf-rvn-cells), which has partnered with Millipore / Sigma Inc. and is distributed by Millipore / Sigma.

[0026] According to the non-patent document "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 Spodoptera frugiperda Sf9 cell line is a heterogeneous cell population containing two types of cell populations: Sf-rhabdovirus-infected cells and Sf-rhabdovirus-negative cells. A single Sf-rhabdovirus-negative cell population can be obtained by limiting dilution. According to the present invention, monoclonal cells are selected by limiting dilution, and then Sf-rhabdovirus-negative cells are screened and identified by methods such as nested PCR, transcriptome next-generation sequencing, real-time fluorescent quantitative PCR, and TaqMan probe-based real-time PCR.

[0027] 1) The parental Sf9 cell line was purchased from ThermoFisher Company (Lot No.: 2043331), and the purchased cells were defined as passage 0 (P0). The passage used in this screening experiment was passage 4 (P4), and the cell culture medium was SIM SF serum-free medium (Sino Biological, Inc., MSF1). Suspended Sf9 cells were diluted 10-fold and seeded into well plates. The cells were observed every few days. When distinct monoclonal cells became apparent, they were transferred to new well plates for expansion. Subsequently, appropriate cells were collected, total RNA was extracted, and candidate cells were preliminarily identified using nested PCR primers against the Sf-rhabdovirus-specific M gene (Table 1).

[0028] [Table 1]

[0029] 2) Candidate Sf-rhabdovirus-negative cells were serially subcultured, and cell samples were collected and frozen at -80°C for subsequent use. After 45 passages of serial culture, nested PCR was used to detect cell samples. The results showed that the Sf-rhabdovirus M gene in WSK-Sf9 P1-P45 was consistently negative (Figure 1).

[0030] 3) Transcriptome Next-Generation Sequencing: 5 x 10 for next-generation sequencing 6 5 x 10 parental Sf9 cells and 5 x 10 6 WSK-Sf9 cells were collected separately, and the results showed that the RNA gene information of Sf-rhabdovirus was detected in the transcriptome of parental Sf9 cells (GenBank: KF 947078.1) but not in WSK-Sf9 cells.

[0031] 4) Real-time fluorescent quantitative PCR: Two pairs of quantitative PCR primers for the Sf-rhabdovirus M gene were designed (Table 2), and the Bio-Rad SsoFastEvaGreensupermix kit was used for fluorescent quantitative PCR testing. The results showed that no fluorescent signal was detected in WSK-Sf9 P3 and P28 generations, demonstrating that WSK-Sf9 cells were Sf-rhabdovirus negative (Table 3).

[0032] [Table 2]

[0033] [Table 3]

[0034] 5) Quantitative PCR by probe method: A TaqMan probe for the Sf-rhabdovirus M gene was designed (Table 4). The quantitative PCR results showed that no fluorescent signal was detected in the WSK-Sf9 main cell bank (MCB) and working cell bank (WCB), proving that WSK-Sf9 cells were Sf-rhabdovirus negative (Table 5).

[0035] [Table 4]

[0036] [Table 5]

[0037] Example 2: Growth characteristics of the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9

[0038] 1) Culture characteristics of WSK-Sf9: Cells can grow in either an adherent or suspension manner in serum-free medium at 27°C. The average diameter of the suspension cells is 16.28±0.34 μm. The morphological characteristics of WSK-Sf9 are shown in Figure 2.

[0039] 2) Karyotype analysis of WSK-Sf9: Karyotype analysis was performed on the parent Sf9 cells. The results showed that the chromosome numbers of 60 metaphase cells were mainly distributed between 180 and 250, with an average of 215 chromosomes per cell. Karyotype analysis was also performed on WSK-Sf9 cells. The results showed that the chromosome numbers of 60 metaphase cells were mainly distributed between 191 and 538, with an average of 299 chromosomes per cell (Figure 3). This also indicates that the Sf-rhabdovirus-negative cell line WSK-Sf9 is distinct from its parent Sf9 cells.

[0040] 3) WSK-Sf9 cell growth curve: Approximately 1 x 10 WSK-Sf9 cells in the logarithmic growth phase 6The cells were diluted to 100 cells / ml and subcultured in a 250 ml vented shake flask. The culture volume was 100 ml, and the flask was placed in a 27°C incubator for continuous culture. Cell density and viability were counted every 24 hours. The growth curves and viability of three different batches after cultivation are shown in Figure 4. The cell density was approximately 1 x 10 after 96 hours. 7 The highest cell density reached 1.2 x 10 cells / ml and remained at this level for 6 days. 7 The cell viability gradually decreased from day 11, approaching cells / ml, with the average doubling time being around 23 hours.

[0041] 4) Serial passage curve of WSK-Sf9 cells: Approximately 1 x 10 WSK-Sf9 cells in the logarithmic growth phase 6 The cells were diluted to 100 cells / ml and subcultured in a 250 ml vented shake flask. The culture volume was 100 ml, and the flask was placed in a 27°C incubator for continuous culture. Cell density and viability were counted and subcultured every 3 days. Generally, after 3 days of culture, the cell density was 6-8 × 10 6 The cell density could reach 100 cells / ml, and the viability was over 98%. The serial passage growth curve and viability are shown in Figure 5. After 100 serial passages, the cell growth characteristics remained stable.

[0042] Embodiment 3: Safety testing of the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9

[0043] The following tests were performed on WSK-Sf9 cells according to Part III of the Chinese Pharmacopoeia (2020 edition).

[0044] 1) Species identification: WSK-Sf9 cells were detected using DNA barcoding, and the results showed that WSK-Sf9 cells were derived from Spodoptera frugiperda.

[0045] 2) Sterility test: Sterility test was performed using membrane filtration method, and the results showed that the growth of WSK-Sf9 cells was sterile.

[0046] 3) Mycobacterium test: Culture method was used to test for mycobacterium, and the results showed that mycobacterium was negative.

[0047] 4) Mycoplasma test: Mycoplasma was tested using culture method, indicator cell culture method, and touchdown PCR method, and the results showed that mycoplasma was negative.

[0048] 5) Spiroplasma test: Fluorescent PCR method was used to test for spiroplasma, and the results showed that spiroplasma was negative.

[0049] 6) Exogenous virus testing: (1) Using in vitro cell observation, hemadsorption testing, and hemagglutination inhibition testing, subcultures of different cells, including monkey-derived Vero cells, human MRC-5 cells, Sf9 cells, BHK-21 cells, mosquito-derived Aedes cells, and Drosophila-derived D. Mel cells, were tested. All cells showed normal morphology, and the tested cells were negative.

[0050] (2) Suckling mice, adult mice, and chicken embryos (5-6 day-old chicken embryos and 9-11 day-old chicken embryos) were inoculated by the in vivo method, and the results showed that they all met the requirements.

[0051] 7) Retrovirus testing: No virus-like particles were observed by transmission electron microscopy, HEK293 cell inoculation and serial infection tests, and chemical reagent-induced virus tests, and the results showed that all of these met the requirements.

[0052] 8) The cells tested for bovine and porcine viruses were negative.

[0053] 9) Other specific viruses were tested, including Baculovirus, T. ni Flock House Virus Variant (FHVvar), Rhabdovirus, Reoviridae, Togavirus, Flavivirus, Bunyaviridae, Asfarvirus, Ascoviridae, Iridoviridae, Poxviridae, Baculoviridae, Poldnaviridae, Parvoviridae, Birnaviridae, Reoviridae, Picornaviralses, Dicistrovitidae, Nodaviridae, and Tetraviridae, and all results were negative, indicating that the requirements were met.

[0054] 10) Tumorigenicity test: Experimental mice were inoculated with WSK-Sf9 cells, and the results showed that the cells were not tumorigenic but could meet the requirements.

[0055] In conclusion, the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 meets all safety testing requirements and cell matrix requirements for biologics production.

[0056] Embodiment 4: Exogenous recombinant proteins expressed by the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9

[0057] A baculovirus expression vector containing the RBD structural domain of SARS-CoV-2 was constructed and the recombinant protein-expressing baculovirus was packaged in WSK-Sf9 cells. Sf9 and WSK-Sf9 cells were cultured separately until the cell density reached 2.5 × 10 6 / ml, the viruses were separately infected at an MOI of 0.5.

[0058] Supernatants were collected preinfection (0 h) and postinfection (24, 48, 72, and 96 h) and detected by Western blot using an antibody against the His tag. The results showed that the expression level of the recombinant protein in WSK-Sf9 cells was upregulated compared to that in Sf9 cells. After production and purification in a GMP workshop and subsequent adjuvant formulation, the recombinant protein could be used to prevent SARS-CoV-2 infection. This demonstrates that the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 can be used to express and produce exogenous recombinant proteins, such as protein vaccines, and that the expression level is higher than that in Sf9 cells.

Claims

1. The rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 has CCTCC accession number C202246.

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

3. The use according to claim 2, including the application of the baculovirus expression system for the production of recombinant protein pharmaceuticals or vaccines.

4. The use according to claim 3, wherein the recombinant protein pharmaceutical comprises a cytokine, a hormone, a recombinant enzyme or an antibody.

5. 5. The use according to claim 4, wherein the cytokine comprises a recombinant human interleukin, a recombinant human epidermal growth factor, a recombinant human interferon, a recombinant human fibroblast growth factor, a recombinant human erythropoietin, or a recombinant human granulocyte-macrophage stimulating factor.

6. 5. The use of claim 4, wherein the hormone comprises recombinant human growth hormone, recombinant human insulin, or recombinant human follicle-maturing hormone.

7. The use according to claim 4, wherein the recombinant enzyme comprises recombinant human α-glucosidase or recombinant human pro-urokinase.

8. The use of claim 3, wherein the vaccine comprises a recombinant protein vaccine or a virus-like particle vaccine.

9. 9. The use according to claim 8, wherein the recombinant protein vaccine comprises a SARS-CoV-2 protein vaccine, an influenza virus protein vaccine, a syncytial virus recombinant protein vaccine, a hepatitis B virus protein vaccine, or a rabies virus protein vaccine.

10. The use of claim 9, wherein the recombinant protein vaccine is a SARS-CoV-2 protein vaccine.

11. 9. The use of claim 8, wherein the virus-like particle vaccine comprises a SARS-CoV-2 virus-like particle vaccine, a human papillomavirus-like particle vaccine, an influenza virus-like particle vaccine, a poliovirus-like particle vaccine, a respiratory syncytial virus-like particle vaccine, or a hand, foot, and mouth disease virus-like particle vaccine.

12. The use of claim 11, wherein the virus-like particle vaccine is a SARS-CoV-2 virus-like particle vaccine.

Images