Spodoptera frugiperda cell strain negative for rhabdovirus and screening, identification and application thereof

Abstract

The present application belongs to the field of genetic engineering and cell engineering, and particularly relates to a rhabdovirus-negative Spodoptera exigua insect cell strain and screening, identification and application thereof. The present application obtains the rhabdovirus-negative Spodoptera exigua insect cell strain WSK-Sf9 through screening and identification, and the preservation number is CCTCC NO: C202246. The cell strain is verified by various high-sensitivity detection methods such as nested PCR, second-generation transcriptome sequencing, fluorescent quantitative PCR and probe quantitative PCR, and finally the Sf-rhabdovirus-negative Spodoptera exigua insect cell strain WSK-Sf9 is screened. According to the requirements of the pharmacopoeia, the sterility, mycoplasma, exogenous virus and tumorigenicity of the cell are detected, and the results show that all indexes meet the requirements, and the baculovirus expression system can be used to produce recombinant proteins and for recombinant protein vaccine production.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and cell engineering technology, and relates to a new fall armyworm cell line, specifically a fall armyworm cell line that is negative for rhabdovirus and its screening, identification and application. Background Technology

[0002] Insect cell expression systems have been widely used for the production of recombinant proteins. Compared with other expression systems, they have many advantages, such as: insect baculoviruses specifically parasitize invertebrates, resulting in high safety; high recombinant protein expression levels; the ability to correctly fold and post-translational modify recombinant proteins to obtain biologically active proteins; adaptability to complex designs for multi-gene expression, such as virus-like particles; and suitability for large-scale serum-free culture.

[0003] Currently, several recombinant protein vaccines produced using insect cell expression systems have been approved for marketing worldwide, demonstrating good efficacy and safety. These include GSK's Cervarix cervical cancer vaccine, Dendreon's Provenge prostate cancer vaccine, and ProteinSciences' FluBlok influenza vaccine. In addition, many other recombinant protein vaccines in the preclinical trial stage have also shown promising application prospects.

[0004] Sf9 cells (Spodoptera frugiperda cells) are the most commonly used insect cells in insect baculovirus expression systems for expressing and producing 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 pupa, isolated and cultured in 1977. Fall armyworm rhabdovirus (Sf-rhabdovirus) is a novel negative-sense RNA virus discovered in 2014 by researchers at the US FDA in the Sf9 insect cell line and its parental Sf21. It contains genetic information encoding five structural proteins: N, P, M, G, and L. Additionally, an extra gene sequence X with an unknown function was detected between G and L. Although the Sf-rhabditis virus has not integrated into the host cell's genome, it has a complete genome and could potentially be packaged into complete viral particles, posing a potential risk to the production and use of recombinant proteins such as vaccines based on the use of Sf9 cell baculovirus expression systems. Summary of the Invention

[0005] This invention obtains the fall armyworm Sf9 cell line WSK-Sf9 (abbreviated as WSK-Sf9) through screening and identification. This cell line can produce recombinant proteins based on the baculovirus expression system and can be used for the production of recombinant protein vaccines.

[0006] The purpose of this invention is to provide the fall armyworm cell line WSK-Sf9, which is negative for rhabdoviruses and has the accession number CCTCC NO:C202246. It is named the fall armyworm Sf9 cell-derived line WSK-Sf9, and the accession date is February 16, 2022. The accession center is the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.

[0007] The fall armyworm cell line WSK-Sf9, which is rhabdovirus-negative, was obtained by screening a single clone using the limiting dilution method and verifying its efficacy through various highly sensitive detection methods, including nested PCR, next-generation transcriptome sequencing, quantitative real-time PCR, and probe-based quantitative PCR. Simultaneously, according to pharmacopoeia requirements, its sterility, mycoplasma, exogenous virus, and tumorigenicity were tested, and the results showed that all indicators met the requirements, making it suitable as a cell matrix for production.

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

[0009] The applications include the production of recombinant protein drugs or vaccines based on baculovirus expression systems.

[0010] The recombinant protein drug includes cytokines, hormones, recombinant enzymes, or antibodies.

[0011] 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.

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

[0013] The recombinant enzymes include recombinant human α-glucosidase or recombinant human prourokinase.

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

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

[0016] 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.

[0017] 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-mouth disease virus-like particle vaccine (EV71-VLP). Preferably, it is a novel coronavirus virus-like particle vaccine.

[0018] The technical solution for realizing the application of WSK-Sf9 in the production of recombinant proteins based on baculovirus expression systems or in the vaccine production process is as follows: using the Bac-to-Bac insect baculovirus expression system, baculovirus is packaged and produced, WSK-Sf9 cells are infected to express the target protein, and the target recombinant protein is obtained through affinity purification technology.

[0019] Beneficial Effects: This invention employs a limiting dilution method to screen single clones, which are then verified using various highly sensitive detection methods, including nested PCR, next-generation transcriptome sequencing, quantitative real-time PCR, and probe-based quantitative PCR, to obtain the fall armyworm cell line WSK-Sf9, which is negative for rhabdoviruses. Simultaneously, according to pharmacopoeia requirements, its sterility, mycoplasma, exogenous virus, and tumorigenicity were tested, and the results showed that all indicators met the requirements, making it suitable for producing recombinant protein products such as clinically used protein vaccines. Furthermore, this invention utilizes a Bac-to-Bac baculovirus expression system to package and produce baculoviruses, infect WSK-Sf9 cells to express the target protein, and obtain the target recombinant protein through affinity purification and other techniques.

[0020] The fall armyworm cell line WSK-Sf9, which was screened and identified by this invention and is negative for rhabdovirus, has the accession number CCTCC NO: C202246. The accession date was February 16, 2022. The accession center is the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The culture name and identification characteristics are WSK-Sf9, a cell-derived line of fall armyworm Sf9. Attached Figure Description

[0021] Figure 1Nested PCR agarose gel electrophoresis results for identifying WSK-Sf9 cells in this invention;

[0022] Figure 2 Morphological characteristics of the fall armyworm cell line WSK-Sf9, which was screened out by the present invention and is negative for rhabdovirus;

[0023] Figure 3 Karyotype analysis of WSK-Sf9, a fall armyworm cell line that was screened for by the present invention and is negative for rhabdovirus;

[0024] Figure 4 The growth curve of the fall armyworm cell line WSK-Sf9, which was screened out by the present invention, is shown.

[0025] Figure 5 The continuous passage culture curve of WSK-Sf9, a fall armyworm cell line that was screened out in this invention, is shown.

[0026] Figure 6 The temporal gradient detection of exogenous recombinant protein expression in the WSK-Sf9 cell line of fall armyworm, which was screened by the present invention, is used to detect the expression of exogenous recombinant protein. Detailed Implementation

[0027] The present invention will be explained below with reference to specific embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0028] The following implementation will further illustrate the screening, identification, and application of the Sf-rabbit virus-negative fall armyworm cell line WSK-Sf9. The invention will be further described below with reference to the accompanying drawings.

[0029] Example 1: Screening and identification of the Sf-rhabditis virus-negative fall armyworm cell line WSK-Sf9

[0030] The only commercially available Sf-rhabdovirus-negative Sf-RVN cell line (Sf-rhabdovirus-negativeSf9) was obtained by Professor Jarvis's team through limiting dilution combined with antiviral drug treatment from Sf9 (Maghodia AB, et al. Protein expression and purification. 2016; 122:45-55.). Its Sf-rhabdovirus negativity was verified by nested PCR targeting the L gene. This cell line belongs to GlycoBac (http: / / www.glycobac.com / sf-rvn-cells), which has partnered with Millipore / Sigma to distribute the cells.

[0031] According to 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 cellclones. Virology 2019; 536:125-33., the Sf9 cell line of the fall armyworm is a heterogeneous cell population containing both Sf-rhabdovirus-positive and Sf-rhabdovirus-negative cell populations. A single Sf-rhabdovirus-negative cell population can be obtained through limiting dilution. This invention utilizes limiting dilution to select single cell clones, and then uses nested PCR, transcriptome sequencing, quantitative real-time PCR (Q-PCR), and probe-based quantitative PCR to screen and identify Sf-rhabdovirus-negative cells.

[0032] 1) The Sf9 parental cell line was purchased from Thermo Fisher Scientific (Lot No: 2043331). The purchased cells were defined as passage P0, and the passage used in this screening experiment was passage P4. The cell culture medium was SIM SF serum-free medium (Beijing Yiqiao Shenzhou Technology Co., Ltd., MSF1). The suspension-cultured Sf9 cells were serially diluted 10-fold into well plates. The cell status was observed every few days. When a relatively clear single cell population was formed, it was transferred to a new well plate for expansion culture. Then, an appropriate amount of cells were collected, total RNA was extracted, and the candidate cells were preliminarily identified using nested PCR primers targeting the Sf-rhabditis virus-specific M gene (Table 1).

[0033] Table 1. Nested PCR primer sequence information targeting the Sf-bombyx mori specific M gene

[0034]

[0035] 2) Candidate Sf-Rhabdovirus-negative cells were serially passaged, and cell samples were collected and frozen at -80°C for later use. After 45 generations of continuous culture, nested PCR was used for detection. The results showed that one cell strain, named WSK-Sf9, was negative for the Sf-Rhabdovirus M gene in all P1 to P45 generations (e.g., Figure 1 ).

[0036] 3) Transcriptome sequencing: Collect 5×10⁵ samples each. 6 Transcriptome sequencing was performed on parental Sf9 cells and WSK-Sf9 cells. The results showed that Sf-Rhabdovirus RNA gene information was detected in the transcriptome of parental Sf9 cells (GenBank: KF947078.1), while it was not detected in WSK-Sf9.

[0037] 4) Quantitative real-time PCR: Two pairs of quantitative PCR primers targeting the Sf-Rhabdovirus M gene were designed (Table 2). Quantitative real-time PCR was performed using the Bio-Rad SsoFastEvaGreensupermix kit. The results showed that no fluorescent signal was detected at WSK-Sf9 P3 and P28, proving that the WSK-Sf9 cells were Sf-Rhabdovirus negative (Table 3).

[0038] Table 2. Specific Q-PCR primers for Sf-rhabditis virus M gene

[0039]

[0040] Table 3. Q-PCR experimental data

[0041]

[0042] 5) Quantitative PCR using probe method: TaqMan probes targeting the Sf-Rhabdovirus M gene were designed (Table 4). Quantitative PCR was used to detect the results, and the results showed that no fluorescent signal was detected in either the WSK-Sf9 master cell bank (MCB) or the working cell bank (WCB), which further proved that the WSK-Sf9 cells were Sf-Rhabdovirus negative (Table 5).

[0043] Table 4. Specific TaqMan probe primers targeting the Sf-rhabditis virus M gene

[0044]

[0045] Table 5. Q-PCR experimental data

[0046]

[0047] Example 2: Growth characteristics of the Sf-rhabditis virus-negative fall armyworm cell line WSK-Sf9

[0048] 1) WSK-Sf9 Culture Characteristics: This cell can adhere to or grow in suspension in serum-free medium at a culture temperature of 27℃. The average diameter during suspension growth is 16.28±0.34 mm. Figure 2 Morphological characteristics of WSK-Sf9.

[0049] 2) Karyotype analysis of WSK-Sf9 cells: Karyotype analysis of parental Sf9 cells showed that the number of chromosomes in 60 metaphase cells ranged mainly from 180 to 250, with an average of 215 chromosomes per metaphase cell. Karyotype analysis of WSK-Sf9 cells showed that the number of chromosomes in 60 metaphase cells ranged mainly from 191 to 538, with an average of 299 chromosomes per metaphase cell (e.g., ...). Figure 3 This also indicates that the Sf-rhabditis virus-negative WSK-Sf9 cell line is different from its parent Sf9 cells.

[0050] 3) WSK-Sf9 cell growth curve: WSK-Sf9 cells in the logarithmic growth phase were diluted to 1×10⁻⁶. 6 Approximately 100 ml of cells were passaged into 250 ml shake flasks with breathable caps, resulting in a culture volume of 100 ml. These flasks were then continuously cultured in a 27°C shaker, with cell density and viability counted every 24 hours. After three different batches of culture, the growth curves and viability were as follows: Figure 4 As shown, the cells grew to 1×10⁻⁶ after 96 hours. 7 The cell density remained at approximately 1.2 × 10⁹ / ml for 6 days, with a peak cell density approaching 1.2 × 10⁹ / ml. 7 / ml, starting from day 11, cell viability gradually decreased, and the average cell doubling time was about 23 hours.

[0051] 4) Continuous passage culture curve of WSK-Sf9 cells: WSK-Sf9 cells in logarithmic growth phase were diluted to 1×10⁻⁶. 6 Cells were cultured at approximately 100 ml / ml and passaged into 250 ml shake flasks with breathable caps. The flasks were then continuously cultured in a 27°C shaker. Cell concentration and viability were counted every 3 days, and the cells were passaged. Generally, after 3 days of culture, the cell concentration reached 6–8 × 10⁹ / ml. 6 / ml, viability is greater than 98%, continuous subculture curves are as follows Figure 5 As shown, the cell growth characteristics are stable after 100 generations of culture.

[0052] Example 3: Safety testing of the Sf-rhabditis virus-negative fall armyworm cell line WSK-Sf9

[0053] Based on the 2020 edition of the Chinese Pharmacopoeia, Volume III, the following series of tests were performed on WSK-Sf9 cells.

[0054] 1) Species identification: DNA barcoding analysis showed that the WSK-Sf9 cells originated from the fall armyworm.

[0055] 2) Sterility test: The membrane filtration method was used for detection, and the result showed no sterile growth;

[0056] 3) Mycobacterial examination: The culture method was used for detection, and the result showed that it was negative for mycobacteria;

[0057] 4) Mycoplasma test: Using culture method, indicator cell culture method and Touchdown PCR method, the results showed negative for mycoplasma;

[0058] 5) Spiroplasma test: The result of fluorescent PCR was negative for spiroplasm.

[0059] 6) Exogenous virus testing:

[0060] (1) Using in vitro cell culture observation method, red blood cell adsorption test and red blood cell adsorption test, different cell passage cultures such as monkey Vero cells, human MRC-5 cells, Sf9 cells, BHK-21 cells, mosquito Aedes cells and D. Mel cells were tested. All cells were morphologically normal and the test results were negative.

[0061] (2) In vivo inoculation of suckling mice, adult mice and chicken embryos (5-6 day old chicken embryos and 9-11 day old chicken embryos) showed that all met the requirements.

[0062] 7) Retrovirus detection: No virus-like particles were observed using transmission electron microscopy, HEK293 cell inoculation and passage infectivity assay, and chemical reagent induction method. The results met the requirements.

[0063] 8) Tests for both bovine and porcine viruses were negative;

[0064] 9) Other specific virus tests, including baculoviruses, T. ni horovirus variants (FHVvar), rhabdoviruses, arboviruses (Reoviridae), Togaviruses, Flaviviruses, Bunyaviruses, Asfaviruses, invertebrate viruses (Ascoviridae), Iridoviridae, poxviruses (… Tests were conducted on the following families: Poxviridae, Baculoviridae, Poldnaviridae, Parvoviridae, Birnaviridae, Reoviridae, Picornaviralses, Dicistrovitidae, Nodaviridae, and Tetraviridae. All results were negative and met the requirements.

[0065] 10) Tumorigenicity test: The experimental mice were inoculated with WSK-Sf9 cells, and the results showed that the cells were not tumorigenic and met the requirements.

[0066] In summary, the safety tests of the Sf-rabbit virus-negative fall armyworm cell line WSK-Sf9 all met the requirements and satisfied the requirements for cell substrates used in the production of biological products.

[0067] Example 4: Expression of exogenous recombinant protein in the Sf-rhabditis virus-negative fall armyworm cell line WSK-Sf9

[0068] A baculovirus expression vector containing the RBD domain of the novel coronavirus SARS-CoV-2 was constructed, and the baculovirus for recombinant protein expression was packaged using WSK-Sf9 cells. Sf9 and WSK-Sf9 cells were cultured separately until their concentrations reached 2.5 × 10⁻⁶. 6At a concentration of / ml, Sf9 and WSK-Sf9 cells were infected with the virus at an MOI of 0.5. Supernatants were collected before infection (0 hours) and at 24, 48, 72, and 96 hours post-infection. Western blotting was performed using an antibody targeting the His tag. The results showed that the expression level of the recombinant protein in WSK-Sf9 cells was upregulated compared to Sf9 cells. After production and purification in a GMP facility, and compounded with adjuvant, this recombinant protein can be used to prevent infection with the novel coronavirus. This indicates that the Sf-rhabditis elegans negative fall armyworm cell line WSK-Sf9 can be used to express exogenous recombinant proteins for the production of protein vaccines, with an expression level higher than that in Sf9 cells. sequence list <110> Chengdu Wesker Biomedical Co., Ltd. <120> Rhabdovirus-negative fall armyworm cell lines and their screening, identification and application <130> A220064K (Preface) <141> 2022-03-01 <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 aggagaactc caaagactca gc 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 aaaaggagtc cccactcagc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 ccacatctcc gctatcacca 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 aggagaagga gcggttgga 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 tgaaaacctt cgcacagcac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 cgagacccct ttggaccttt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 ggattgcacg gagcctatca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 tgcccaagct aaggaaaggg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 tgacatgtgg tctccaaccg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 gtatgcaggt ggttgaggct 20 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <400> 11 ctccttctcc tccacccaca t 21

Claims

1. The fall armyworm cell line WSK-Sf9, which is negative for rhabdovirus, has the accession number CCTCC NO:C202246.

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

3. The application as described in claim 2, characterized in that: The applications include the production of recombinant protein drugs or vaccines based on baculovirus expression systems.

4. The application as described in claim 3, characterized in that: The recombinant protein drug is selected from cytokines, hormones, recombinant enzymes, or antibodies.

5. The application as described in claim 4, characterized in that: The cytokines are selected from 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 application as described in claim 4, characterized in that: The hormones are selected from recombinant human growth hormone, recombinant human insulin, insulin analogs, or recombinant human follicle-stimulating hormone.

7. The application as described in claim 4, characterized in that: The recombinant enzymes are selected from recombinant human α-glucosidase or recombinant human prourokinase.

8. The application as described in claim 4, characterized in that: The antibody is selected from monoclonal antibodies, Fab antibodies, scFv antibodies, or nanobodies.

9. The application as described in claim 3, characterized in that: The vaccine is selected from recombinant protein vaccines or virus-like particle vaccines.

10. The application as described in claim 9, characterized in that: The recombinant protein vaccine is selected from novel coronavirus protein vaccine, hepatitis B virus protein vaccine or rabies virus protein vaccine.

11. The application as described in claim 9, characterized in that: The virus-like particle vaccine is selected from novel coronavirus-like particle vaccines, human papillomavirus-like particle vaccines, influenza virus-like particle vaccines, polio virus-like particle vaccines, respiratory syncytial virus-like particle vaccines, or hand-foot-mouth disease virus-like particle vaccines.

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