Rhabdovirus-free genetically modified insect cell strain, construction method and application thereof

Abstract

This invention relates to genetically modified insect cell lines comprising nucleotide sequences encoding baculoviruses IE0 / IE1, Orf146, Lef2, and Orf91, and expressing baculoviruses IE0, IE1, Orf146, Lef2, and Orf91. The invention also relates to methods for screening and acclimating said genetically modified insect cell lines to cell lines free from rhabdovirus contamination, and to the rhabdovirus-free genetically modified insect cell lines obtained by said method. The rhabdovirus-free genetically modified insect cell lines obtained by said method exhibit low oxygen and low sugar consumption characteristics during fermentation culture, which can be used to reduce production costs and safety risks of insect cell-based bioproducts in the production of recombinant proteins or viral vectors such as AAV viral vectors.

Description

Technical Field

[0001] This invention relates to the technical field of producing recombinant proteins and viral vectors. Specifically, it relates to genetically modified insect cell lines free from rhabdovirus contamination for AAV production and protein expression, and methods for constructing such cell lines. The genetically modified insect cell lines free from rhabdovirus contamination obtained by the method of this invention exhibit low oxygen and sugar consumption during fermentation culture, enabling large-scale production of recombinant proteins or viral vectors such as recombinant AAV viral vectors. Furthermore, the resulting recombinant AAV viral vectors possess increased VP1 content and biological activity. Background Technology

[0002] The baculovirus expression vector system (BEVS) is one of the most powerful, robust, and widely used eukaryotic expression systems available. It utilizes baculoviruses as vectors to introduce foreign genes into insect cells or the insect body, achieving efficient expression of foreign proteins. Given its rapid development and versatility in expressing multiple protein families, BEVS offers numerous advantages for protein production in various applications.

[0003] The most commonly used baculovirus vector for recombinant protein production in industrial and research laboratories is based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV), using Sf9 or Sf21 insect cells, High Five cells derived from Trichoplusiani, and whole Trichoplusiani larvae as suitable expression hosts (Silvia Gómez-Sebastián et al., “Significant Productivity Improvement of the Baculovirus Expression Vector System by Engineering a Novel Expression Cassette”, PLOSONE, 2014, 9(5)e96562: p1-10).

[0004] The Sf9 cell line is a clone isolated from the Spodoptera frugiperda (Sf) 21 cell line. It is a common cell line used in baculovirus expression vector systems and is widely used in the production of recombinant proteins and viral vectors such as AAV viral vectors.

[0005] Although the baculovirus-Sf9 cell system has been successfully used for AAV production at various scales, it has been observed that the rAAV produced in insect cells of this system has a lower VP1 content (approximately 1:1:30 to 1:1:60 VP1:VP2:VP3 content in rAAV compared to approximately 1:1:10 in wild-type AAV), and the rAAV produced by Sf9 cells has reduced potency compared to rAAV produced by mammalian cells (Yoko Marwidi et al., “A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsidratios and potency”, Molecular Therapy: Methods & Clinical Development, 2024, 32: p1-16).

[0006] In addition, rhabdovirus (Sf-) is usually detected in the culture supernatant of commercially sourced Sf9 cells during culture. R habdo v Sf9 cells contaminated with rhabdovirus (also known as Sf-RV) pose a potential safety risk to bioproducts made from Sf9 cells contaminated with rhabdovirus.

[0007] There is a need in the art to use genetically modified Sf9 cells with improved performance and free from rhabdovirus contamination for the production of recombinant proteins and viral vectors such as AAV viral vectors. Summary of the Invention

[0008] This invention provides genetically modified insect cell lines free from rhabdovirus contamination for AAV production and protein expression. These genetically modified insect cell lines exhibit high viable cell density and viability, low oxygen and sugar consumption during fermentation culture, making them suitable for large-scale production of target proteins or viral vectors such as AAV vectors. Furthermore, the resulting recombinant AAV viral vectors possess enhanced VP1 content and biological activity.

[0009] Therefore, in a first aspect, the present invention provides a genetically modified insect cell line comprising nucleotide sequences encoding baculoviruses IE0 / IE1, Orf146, Lef2, and Orf91.

[0010] In some embodiments, the nucleotide sequences encoding baculovirus IE0 / IE1, Orf146, Lef2, and Orf91 are independently derived from the nucleotide sequences encoding IE0 / IE1, Orf146, Lef2, and Orf91 of baculoviruses selected from Alpha baculovirus, Betabaculovirus, Gamma baculovirus, and Delta baculovirus.

[0011] In some embodiments, the nucleotide sequences encoding baculovirus IE0 / IE1, Orf146, Lef2, and Orf91 are effectively linked to regulatory elements that express the nucleotide sequences in insect cells. For example, one or more nucleotide sequences encoding baculovirus IE0 / IE1, Orf146, Lef2, and Orf91 are under the control of the baculovirus IE0 promoter, the baculovirus gp64 promoter, or the silkworm actin 3 (A3) cell promoter.

[0012] In some embodiments, the insect cell line is derived from lepidopteran insects, such as the grass cutworm (Spodoptera frugiperda), the white-spotted cutworm (Trichoplusia ni), or the silkworm (Bombyxmori).

[0013] In some embodiments, the genetically modified insect cell line is a genetically modified Sf9 cell, a genetically modified Sf21 cell, or a genetically modified High Five cell.

[0014] In some embodiments, the genetically modified insect cell lines express baculoviruses IE0, IE1, Orf146, Lef2, and Orf91.

[0015] In a second aspect, the present invention provides a method for screening genetically modified insect cell lines free from rhabdovirus contamination, characterized by comprising the following steps:

[0016] 1) Preparation of a genetically modified insect cell suspension of the first aspect of the present invention contaminated with rhabdovirus: For example, a cell suspension is prepared by culturing genetically modified insect cells of the first aspect of the present invention contaminated with rhabdovirus in a basal serum-free insect cell culture medium supplemented with 10% FBS, wherein the basal serum-free insect cell culture medium is selected from ESF AF. TMInsect cell culture medium, Grace insect cell culture medium, ESF 921 medium, Sf 900 II SFM medium and Sf 900 III SFM medium

[0017] 2) Preparation of monoclonal screening culture medium:

[0018] Monoclonal screening culture was prepared using a first basic serum-free insect cell culture medium of 60% supplemented with whey protein hydrolysate and Yeastolate, a second basic serum-free insect cell culture medium of 20% supplemented with FBS and 20% supplemented with FBS. The first basic serum-free insect cell culture medium supplemented with whey protein hydrolysate and Yeastolate was Grace supplemented insect cell culture medium, and the second basic serum-free insect cell culture medium was Sf 900 II SFM medium.

[0019] 3) Dilution and plating of cell suspensions

[0020] The cell suspension obtained in step 1 was centrifuged and washed multiple times with basal serum-free insect cell culture medium to remove culture medium containing rhabdovirus; then, the cells were diluted to 0.1 × 10⁻⁶ using the freshly prepared monoclonal selection culture medium from step 2. 4 1 x 10⁻¹ ml 6 Cells / ml, then serially diluted to 25-100 cells / ml, seeded at 1-10 cells / well in culture plates, and incubated statically in a 27-28°C incubator; or

[0021] The cell suspension obtained in step 1 was centrifuged and washed multiple times with basal serum-free insect cell culture medium to remove culture medium containing rhabdovirus; then, the freshly prepared monoclonal selection culture medium from step 2 was taken and an antiviral compound was added to achieve a final concentration of 10-50 ng / ml, and the cells were diluted to 0.1 × 10⁻⁶. 4 1 x 10⁻¹ ml 6 The cells were diluted to 25-100 cells per milliliter and then serially diluted to 1-10 cells per well. The cells were then seeded into culture plates and incubated at 27-28°C. The antiviral compound was ribavirin.

[0022] 4) Cell culture, rhabdovirus detection, and cell passage

[0023] From the second to the tenth day after plating, the wells were observed daily to identify single-cell wells. The growth of the monoclonal cells obtained through limiting dilution was observed regularly, and rhabdovirus was detected. Based on the cell growth and rhabdovirus detection results, the medium in the wells was changed or expanded for culture and passage. The medium was changed to the monoclonal selection medium corresponding to the cells in each well or monoclonal selection medium with the addition of the antiviral compound ribavirin at a final concentration of 10-50 ng / ml.

[0024] 5) Expand the culture of cells free from rhabdovirus contamination.

[0025] Cells that were initially free of rhabdovirus contamination obtained in step 4 were cultured in monoclonal screening medium corresponding to the cells in each well plate, or monoclonal screening medium with ribavirin added to a final concentration of 10-50 ng / ml; rhabdovirus detection was performed to obtain genetically modified insect cell lines free of rhabdovirus contamination.

[0026] In some implementations, in step 3, serially diluted cells are seeded into 96-well culture plates at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cells per well and incubated statically in an incubator at 27°C, 27.5°C, or 28°C.

[0027] In some embodiments, the rhabdovirus detection is performed using bioinformatics techniques selected from polymerase chain reaction (PCR), reverse transcription (RT), reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR combined with nested PCR, quantitative PCR (qPCR), RT-PCR combined with quantitative PCR (quantitative RT-PCR or RT-qPCR), multiple probe hybridization techniques, electron microscopy, antibody-based detection techniques (e.g., ELISA assay), plaque assay and cytopathic effect (CPE), and BLAST search.

[0028] In a third aspect, the present invention provides a method for domesticating genetically modified insect cell lines free from pneumovirus contamination obtained by the screening method of the second aspect of the present invention, characterized in that the genetically modified insect cell lines free from pneumovirus contamination obtained by the screening method of the second aspect of the present invention are cultured until the cell adhesion reaches more than 60%, and then subjected to trypsin digestion to prepare a cell suspension; then, a cell suspension is prepared using a solution containing Pluronic acid. TMThe F-68 serum-free insect cell culture medium was used for suspension culture in a shaking incubator at 27-28°C and 100-130 rpm. The suspension-cultured cells were then stably cultured for 4-20 generations to obtain a serum-independent, rhabdovirus-free, genetically modified suspension insect cell line, for example, one containing Pluronic acid. TM The basic serum-free insect cell culture medium for F-68 is Vigor-S101 medium.

[0029] In a fourth aspect, the present invention provides genetically modified insect cell lines free from bullet virus contamination, which are obtained by the screening method of the second aspect of the present invention and the domestication method of the third aspect of the present invention.

[0030] In a fifth aspect, the present invention provides the use of a genetically modified insect cell line free from bullet virus contamination, as described in the fourth aspect of the present invention, for the production of recombinant proteins or AAV viral vectors. Attached Figure Description

[0031] Figure 1 The image shows a map of the MD768 plasmid. In the image, "Neo" represents the G418 resistance gene, used for pressure selection of Sf9 cells transfected with the MD768 plasmid.

[0032] Figure 2 The growth curves of Sf9-MD768-3C7 (abbreviated as "MD768-3C7"), Sf9-MD768-1E4 (abbreviated as "MD768-1E4"), and Sf9-MD768-2H12 (abbreviated as "MD768-2H12") cell lines compared to the rhabdovirus-contaminated control cells Sf9-MD768-Rha (abbreviated as "MD768-Rha") are shown in 125 ml Erlenmeyer flasks. In the figure, "VCD" represents the viable cell density. V iable c ell d "ensity", "via" means cell viability.

[0033] Figure 3 The figure shows the changes in intracellular and supernatant recombinant AAV production after infection with recombinant baculoviruses in Sf9-MD768-1E4 (abbreviated as "1E4"), Sf9-MD768-2H12 (abbreviated as "2H12"), and Sf9-MD768-3C7 (abbreviated as "3C7") cell lines. In the figure, 3 dpi represents day 3 after cell infection with recombinant baculoviruses; 4 dpi represents day 4 after cell infection with recombinant baculoviruses.

[0034] Figure 4 The figures show the intracellular and culture supernatant recombinant AAV production after infection of Sf9-MD768-3C7 (abbreviated as "MD768-3C7") and control Sf9-MD768-Rha (abbreviated as "MD768-Rha") cells with recombinant baculovirus. In the figures, 3 dpi represents day 3 after cell infection with recombinant baculovirus; 4 dpi represents day 4 after cell infection with recombinant baculovirus.

[0035] Figure 5 The figures show the viable cell density (VCD) and cell viability (via) of the Sf9-MD768-3C7 cell line when cultured in a 2L fermenter, compared to the control cell line Sf9-MD768-Rha (abbreviated as "MD768-Rha" in the figure) contaminated with rhabdovirus. In the figure, dpi represents the number of days after Sf9 cells were infected with recombinant baculovirus.

[0036] Figure 6 The figure shows the glucose (Glc) and oxygen (O2) consumption of the Sf9-MD768-3C7 cell line (abbreviated as "MD768-Rha") when cultured in a 2L fermenter, compared to the control cell line Sf9-MD768-Rha (abbreviated as "MD768-Rha") contaminated with rhabdovirus. In the figure, dpi represents the number of days after cell infection with recombinant baculovirus.

[0037] Figure 7 The figure shows the intracellular and supernatant recombinant AAV yield of Sf9-MD768-3C7 cell line compared to the control cell line Sf9-MD768-Rha (abbreviated as "MD768-Rha" in the figure) when cultured in a 2L fermenter. In the figure, dpi represents the number of days after Sf9 cells were infected with recombinant baculovirus.

[0038] Figure 8 The SDS-PAGE results of recombinant AAV product (BZ2024061001) from the Sf9-MD768-3C7 cell line and recombinant AAV product (BZ2024062401) from the control Sf9-IE1 cell line (the cell line of patent application US20240352482A1) are shown (inset A); and the gray values ​​of VP1, VP2, and VP3 proteins obtained by grayscale scanning are shown (inset B).

[0039] Figure 9The image shows the expression levels of Gluc protein in HEK-293 and BHK21 cells after infection with recombinant AAV product (BZ2024061001) from the Sf9-MD768-3C7 cell line and recombinant AAV product (BZ2024062401) from the control Sf9-IE1 cell line (patent application US20240352482A1). The values ​​on the ordinate represent the luminescence intensity per mg of expressed Gluc protein, i.e., relative light units (RLU).

[0040] Figure 10 The images show the expression levels of Gluc protein measured in serum (inset A) and in the heart, liver, lung, kidney, brain, and spinal cord (inset B) of 6–8-week-old C57BL / 6J male rats 3 weeks after a single tail vein injection of recombinant AAV product (BZ2024061001) from the Sf9-MD768-3C7 cell line and recombinant AAV product (BZ2024062401) from the control Sf9-IE1 cell line (patent application US20240352482A1). The values ​​on the ordinate represent the fluorescence intensity RLU value per mg of expressed Gluc protein, and FF5 indicates the name of the solvent reagent used, which is a formulated PBS buffer. Detailed Implementation

[0041] Unless otherwise defined below, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0042] I. Definition

[0043] In this document, the term “about” when used in conjunction with a numerical value means to cover a range of numerical values ​​having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value. The term is also intended to cover values ​​within ±1%, ±0.5%, or ±0.1% of the specified numerical value. Unless otherwise specified, “%” in this document refers to volume percentages.

[0044] In this document, the expression “and / or” is used to refer to any one of the listed related items, or any and all possible combinations of multiple listed related items.

[0045] In this document, the terms "comprising" or "including" mean including the stated elements, integers, or steps, or groups of elements, integers, or steps, but do not exclude any other elements, integers, or steps, or other groups of elements, integers, or steps. When the terms "comprising" or "including" are used herein, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps. For example, when referring to a polypeptide / protein that "comprising" a specific sequence, it is also intended to cover polypeptides / proteins consisting of that specific sequence.

[0046] In this article, the use of terms such as "first" and "second" to modify elements does not imply any priority or order of one element relative to another element or the chronological order of the implementation of methods and actions, but is merely used as a label to distinguish one element with a specific name from another element with the same name.

[0047] A "cell line" refers to cells that have proliferated after the first passage of primary cells; it can also be called a cell line or subclone. As cells are passed down, cells with strong growth capacity will become dominant, leading to a certain degree of homogeneity in the cell population in terms of genotype and phenotype.

[0048] A "cell strain" is a culture obtained from a primary culture or cell line through selection or cloning. A monoclonal cell strain is a cell strain obtained by expanding a single cell.

[0049] The terms "culture medium" and "culture solution" are used interchangeably. They refer to the fundamental substances that provide nutrients to cells and promote cell proliferation, as well as the environment in which cells grow and proliferate. There are many types of culture media, and selecting the appropriate medium based on different cell types and experimental requirements is crucial for successful cell culture.

[0050] In this article, the terms "serum-free media (SFM)" or "serum-free insect cell culture medium" are used interchangeably, referring to media that do not contain any animal-derived serum components. Serum-free media typically require the addition of specific chemical additives to mimic the effects of serum. Several serum-free media are currently available on the market, such as serum-free hybridoma cell culture medium, serum-free CHO cell culture medium, serum-free Vero cell culture medium, and serum-free Sf cell culture medium. Serum-free media usually contain added cell growth-promoting components, such as hormones and growth factors, low-molecular-weight nutrients, and transferrin, generally including insulin, progesterone, sodium selenate, putrescine, and transferrin.

[0051] The terms "basal serum-free medium" or "basal serum-free insect cell medium" are used interchangeably and, in this document, refer to liquid or solid culture media that support Sf9 cell growth and maintain the essential nutrients required for Sf9 cell growth. Examples include Grace insect cell medium (Gibco), ESF 921 medium (Expression Systems), and ESF AF. TM Insect cell culture medium (Expression Systems), IB907d medium (YSK Bio, https: / / www.yskbio.com / Insect-Cell-Serum-Free-Medium.html), Sf 900II SFM medium (GIBCO), Sf 900III SFM medium, etc.

[0052] A basal serum-free medium allows for the growth and expansion of Sf9 cells under specific conditions by adding additional chemicals and / or cytokines. In some embodiments, the addition of whey protein hydrolysate and yeastolate to the basal serum-free medium facilitates the rapid growth and expansion of monoclonal Sf9 cells. In some embodiments, the addition of... F-68 can increase the viable cell density (VCD) of Sf9 cells in suspension culture.

[0053] "Viable cell density," also known simply as "cell density," refers to the number of cells per unit volume of liquid.

[0054] Cell viability refers to the percentage of live cells in the total cell count. Measurement methods include staining exclusion methods, such as trypan blue staining dead cells blue while live cells remain unstained; aniline black staining dead cells black while live cells remain unstained; the eosin Y method; fluorescence exclusion methods, where viable cells emit strong yellow-green fluorescence, weakly viable cells emit weak fluorescence, and dead cells show no fluorescence; and the MTT assay, where succinate dehydrogenase breaks down MTT to produce blue crystalline formazan granules that accumulate inside and around cells, with the amount directly proportional to the cell number and cell viability.

[0055] Primary cell culture is the culture of cells, tissues, or organs obtained directly from an organism before the first subculture. Cells proliferate in the culture when they are placed in a growth medium under conditions conducive to cell growth and / or division, resulting in a larger cell population. The rate of cell proliferation is measured by the amount of time required for the cell number to double during this proliferation process. This is called the cell doubling time.

[0056] Primary cells are cultured once or multiple times. Each round of subculture is called passage. After cells have been subcultured, they are called passaged cells. Sometimes the number of passages is used to refer to or characterize a particular cell population. For example, a cell population that has been passaged 10 times may be called a P10 culture. The primary culture, i.e., the first generation culture after cells are isolated from the tissue, is called P0. After the first subculture, the cells are called the second generation culture (P1 or generation 1). After the second subculture, the cells become the third generation culture (P2 or generation 2), and so on. Those skilled in the art will understand that there may be multiple population doublings during passage; therefore, the population doubling number of a culture is greater than the number of passages. The expansion of cells (i.e., the population doubling number) during the period between passages depends on many factors, including but not limited to seeding density, culture medium, growth conditions, and the time between passages.

[0057] The Baculoviruses family comprises double-stranded DNA viruses that specifically infect arthropods. It includes four genera: Alpha baculovirus, Beta baculovirus, Gamma baculovirus, and Delta baculovirus. Baculoviruses cannot replicate in mammalian or other vertebrate cells.

[0058] The baculovirus genome consists of a circular double-stranded DNA molecule ranging in size from 80 to 180 kbp, containing approximately 100-200 open reading frames (ORFs). These ORFs encode proteins involved in various functions, including baculovirus replication, transcription, assembly, and host regulation. Baculovirus ORFs are typically named according to their location in the genome, such as ORF6, ORF14, ORF91, ORF146, and ORF147. Some important ORFs are also named according to their function; for example, ORF6 corresponds to the lef-2 gene, which encodes late expression factor 2; ORF147 corresponds to the immediate early gene ie1; and ORF128 corresponds to the gp64 gene, which encodes a glycoprotein mediating viral binding to the cell surface.

[0059] The terms "IE0 protein" and "IE0" are used interchangeably in this document; similarly, "IE1 protein" and "IE1" are used interchangeably. Baculoviruses, such as AcMNPV, express two immediate-early genes, ie1 and ie0, early in the infection process, producing an unspliced ​​1.9kb transcript and a spliced ​​2.1kb transcript, encoding the IE1 and IE0 proteins, respectively. The IE0 protein contains 54 additional amino acids at its amino terminus that are absent in the IE1 protein. The IE1 protein is expressed in both early and late infection, while the IE0 protein is expressed early in infection. They promote viral DNA replication by regulating viral gene expression.

[0060] Orf146 is a key factor in the budding of baculovirus particles. Enhancing Orf146 can help baculoviruses mature and bud, increasing the titer of baculoviruses in the culture medium supernatant. The reverse DNA sequence of Orf146 is also an important component of the IE0 / 1 promoter and is an element that activates gene expression.

[0061] "Lef2" is an essential gene for baculoviruses, and its biological function is to unwind double-stranded DNA. With the unwinding effect of Lef2, the baculovirus genome and the host cell genome can more easily activate DNA replication and transcription.

[0062] "Orf91" is a gene sequence located on the genome of a natural baculovirus, and its main physiological function is not yet fully understood. The inventors compared the baculosome genome and the baculovirus genome using next-generation sequencing (NGS) and found that, compared with recombinant baculoviruses, the NGS matching depth of the Orf91 region in the baculosome genome was poor, indicating partial deletion of Orf91 (Note: the mean coverage of the Orf91 region was 6597.8, while the mean coverage of the control gene segment (GP64) was 8976.2, p < 0.001). Therefore, the inventors believe that Orf91 may affect the baculovirus maturation process.

[0063] Recombinant baculoviruses are obtained by inserting a foreign gene (e.g., one or more genes from other species such as humans, other vertebrates, plants, bacteria, and viruses) into the genome of a parent baculovirus. Once inside insect cells, the recombinant baculovirus utilizes the cell's mechanisms to express the foreign gene, thereby producing recombinant proteins. The foreign gene is placed under the control of a viral promoter (e.g., a polyhedrin gene promoter) or a cellular promoter. The promoter allows the foreign gene to be transcribed into messenger RNA, which is then translated into protein in the insect cells infected with the recombinant baculovirus.

[0064] "Recombinant viral vectors" refer to vectors based on recombinant viruses. These vectors are created by genetically modifying the viral genome to carry exogenous target genes and related viral elements, which are then packaged into viral particles. After infecting a host, the recombinant viral vector expresses the carried exogenous gene within the host. Examples of recombinant viral vectors include, for instance, recombinant baculovirus vectors and recombinant adeno-associated virus (AAV) vectors.

[0065] An expression cassette is a DNA segment designed to achieve gene expression in a host cell. It typically contains several key elements, including a promoter, the coding sequence of the target gene, and a terminator. In some cases, the expression cassette may also contain other regulatory elements, such as enhancers and silencers, to finely regulate the expression level of the target gene.

[0066] "Operationally linked," also known as "effectively linked" or "functionally linked," refers to a relationship in which two or more polynucleotide (e.g., DNA) segments are positioned to allow them to function in the intended manner. For example, if a promoter sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, then the promoter sequence is effectively linked to that coding sequence. Generally, promoters effectively linked to a transcribed sequence are contiguous with that transcribed sequence; that is, they are cis-acting. However, some transcriptional regulatory sequences (such as enhancers and 5'UTR introns) do not need to be physically adjacent to or closely proximate to the coding sequence that enhances transcription.

[0067] II. Genetically modified insect cells

[0068] This invention provides genetically modified insect cell lines. The genetically modified insect cell lines of this invention are characterized by containing nucleotide sequences encoding baculoviruses IE0 / IE1, Orf146, Lef2, and Orf91.

[0069] In some embodiments, the nucleotide sequence encoding baculovirus IE0 / IE1 is a single nucleotide sequence encoding baculovirus IE0, which includes a nucleotide sequence encoding baculovirus IE1. In some embodiments, the nucleotide sequence encoding baculovirus IE0 / IE1 is two nucleotide sequences, one encoding baculovirus IE0 and the other encoding baculovirus IE1.

[0070] In some embodiments, the genetically modified insect cell lines of the present invention comprise nucleotide sequences encoding baculoviruses IE0 / IE1, Orf146, Lef2, and Orf91, wherein the nucleotide sequence encoding baculovirus IE0 / IE1 comprises the nucleotide sequence of SEQ ID NO:1, or comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; the nucleotide sequence encoding baculovirus Orf146 comprises the nucleotide sequence of SEQ ID NO:3, or comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; the nucleotide sequence encoding baculovirus Lef2 comprises the nucleotide sequence of SEQ ID NO:4, or comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4; and the nucleotide sequence encoding baculovirus Orf91 comprises the nucleotide sequence of SEQ ID NO:4. The nucleotide sequence of NO:5, or a nucleotide sequence containing at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5.

[0071] In some embodiments, the nucleotide sequences encoding baculoviruses IE0 / IE1, Orf146, Lef2, and Orf91 are efficiently linked to regulatory elements that express these nucleotide sequences in insect cells. "Regulatory elements" refer to all elements required to transcribe a specific gene into messenger RNA (mRNA) and to translate mRNA into protein. Among the elements required for transcription, the promoter is of particular importance. Promoters can be constitutive or regulatory. Promoters can be baculovirus-derived or arthropod-derived (e.g., insect-derived) promoters. Importantly, the selected promoter is suitable for expressing the foreign gene in insect cells, for example, the foreign gene encoding baculoviruses IE0 / IE1, Orf146, Lef2, and / or Orf91. Examples of promoters include polyhedromic protein promoters, P10 promoters, synthetic promoters derived from polyhedromic protein promoters and P10 promoters, baculovirus CfMNPV IE1 promoters, baculovirus LdMNPV IE1 promoters, baculovirus OpMNPV gp64 promoters, and silkworm actin 3 (A3) cellular promoters. In some specific embodiments, one or more genes encoding baculovirus IE0 / IE1, Orf146, Lef2, and Orf91 are placed under the control of the baculovirus IE1 promoter, the baculovirus IE0 promoter, the baculovirus gp64 promoter, or the silkworm actin 3 (A3) cellular promoter.

[0072] In some embodiments, the genetically modified insect cell lines of the present invention produce baculovirus proteins IE0, IE1, Orf146, Lef2, and Orf91, wherein the baculovirus belongs to a species selected from Alphabaculovirus, Beta baculovirus, Gamma baculovirus, and Delta baculovirus. Alfalfa silver-striped moth multinucleate polyhedrovirus (AcMNPV) is a species of the genus Alphabaculovirus.

[0073] In some embodiments, the genetically modified insect cell lines of the present invention are derived from lepidopteran insects, such as the grass armyworm (Spodoptera frugiperda), the white armyworm (Trichoplusia ni), or the silkworm (Bombyx mori). For example, the genetically modified insect cell lines are genetically modified Sf9 cells, genetically modified Sf21 cells, or genetically modified High Five cells.

[0074] In some embodiments, the genetically modified insect cell lines of the present invention produce baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins. For example, the baculovirus IE0 comprises or is composed of the following amino acid sequence:

[0075] (a) The amino acid sequence of SEQ ID NO:6;

[0076] (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:6 and having baculovirus IE0 activity;

[0077] The baculovirus IE1 contains or is composed of the following amino acid sequence:

[0078] (a) The amino acid sequence of SEQ ID NO:7;

[0079] (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:7 and having baculovirus IE1 activity;

[0080] The baculovirus Orf146 comprises or is composed of the following amino acid sequence:

[0081] (a) The amino acid sequence of SEQ ID NO:8;

[0082] (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:8 and having the activity of baculovirus Orf146;

[0083] The baculovirus Lef2 contains or is composed of the following amino acid sequence:

[0084] (a) The amino acid sequence of SEQ ID NO:9;

[0085] (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:9 and having the activity of baculovirus Lef2;

[0086] The baculovirus Orf91 contains or is composed of the following amino acid sequence:

[0087] (a) The amino acid sequence of SEQ ID NO:10;

[0088] (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:10 and having the activity of baculovirus Orf91.

[0089] In some embodiments, the genetically modified insect cell lines of the present invention produce baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins, wherein the baculovirus IE0 comprises or consists of the amino acid sequence of SEQ ID NO:6.

[0090] In some embodiments, the genetically modified insect cell lines of the present invention produce baculovirus IE0, IE1, Orf146, Lef2 and Orf91 proteins, wherein the baculovirus IE1 comprises or is composed of the amino acid sequence of SEQ ID NO:7.

[0091] In some embodiments, the genetically modified insect cell lines of the present invention produce baculovirus IE0, IE1, Orf146, Lef2 and Orf91 proteins, wherein the baculovirus Orf146 comprises or is composed of the amino acid sequence of SEQ ID NO:8.

[0092] In some embodiments, the genetically modified insect cell lines of the present invention produce baculoviruses IE0, IE1, Orf146, Lef2, and Orf91 proteins, wherein the baculovirus Lef2 comprises or consists of the amino acid sequence of SEQ ID NO:9.

[0093] In some embodiments, the genetically modified insect cell lines of the present invention produce baculovirus IE0, IE1, Orf146, Lef2 and Orf91 proteins, wherein the baculovirus Orf91 comprises or is composed of the amino acid sequence of SEQ ID NO:10.

[0094] In some embodiments, the genetically modified insect cell lines of the present invention are obtained by introducing one or more expression frames containing nucleotide sequences encoding baculoviruses IE0 / IE1, Orf146, Lef2, and / or Orf91 into a parental insect cell line, culturing the cells in a medium containing a selector, and isolating the genetically modified insect cells. In some embodiments, the selector is an antibiotic, for example, selected from neomycin, bleomycin, hygromycin, blastomycin, puromycin, and G418.

[0095] Insect cells were infected with a recombinant baculovirus vector, which assembled the recombinant baculovirus within the cells, ultimately leading to severe cell damage. Apoptosis associated with baculovirus infection is one of the most significant cytopathic effects induced by baculovirus in insect cells. In unmodified parental insect cells, viable cell density and viability peaked 48–72 hours after recombinant baculovirus infection, subsequently declining sharply, which is detrimental to the expression of more exogenous target proteins.

[0096] Compared with unmodified parental insect cell lines that do not express baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins, genetically modified insect cell lines expressing baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins showed significantly increased viable cell density and cell viability on days 4 and 5 after infection with recombinant baculovirus. In some implementations, genetically modified insect cell lines expressing baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins have a viable cell density of approximately 1E+6 cells / ml to approximately 4E+6 cells / ml on day 5 post-infection with recombinant baculovirus, with a cell viability of approximately 50% to 70%; for example, viable cell densities of approximately 1E+6 cells / ml, approximately 2E+6 cells / ml, approximately 3E+6 cells / ml, and approximately 4E+6 cells / ml, with cell viability of approximately 50%, approximately 55%, approximately 60%, approximately 65%, and approximately 70%, respectively.

[0097] Compared with unmodified parental insect cell lines that do not express baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins, genetically modified insect cell lines expressing these proteins showed significantly reduced glucose and oxygen consumption in the culture medium during cell culture.

[0098] When producing recombinant AAV vectors using a baculovirus-insect cell system, compared to unmodified parental insect cell lines that do not express baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins, the recombinant AAV vectors packaged in genetically modified insect cell lines expressing baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins are more distributed intracellularly than in the cell culture supernatant; they also exhibit high recombinant AAV vector titers, preferably approximately 1E+11v. The concentration of the recombinant AAV vector is approximately g / mL to about 2E+11vg / mL, for example, about 1E+11vg / mL, about 1.2E+11vg / mL, about 1.4E+11vg / mL, about 1.6E+11vg / mL, about 1.8E+11vg / mL, and about 2E+11vg / mL; it has a significantly increased VP1 protein ratio, for example, the VP1:VP2:VP3 content is about 1:1:10, and the recombinant AAV vector has enhanced biological activity, for example, enhanced biological activity in serum, heart, liver, lung, brain and spinal cord, and reduced nephrotoxicity.

[0099] III. Screening methods for genetically modified insect cell lines free from rhabdovirus contamination

[0100] During the culture process, rhabdovirus contamination is commonly detected in the culture supernatant of commercially sourced parent insect cells. This invention provides a method for preparing the aforementioned "II. Genetically Modified Insect Cells" from commercially sourced parent insect cells, and then screening for genetically modified insect cell lines free from rhabdovirus contamination. The screening method includes the following steps:

[0101] 1) Preparation of cell suspension:

[0102] Genetically modified insect cells prepared from the above-mentioned "II. Genetically Modified Insect Cells" contaminated with rhabdovirus were cultured in a basal serum-free insect cell culture medium supplemented with 10% FBS to prepare a cell suspension. The basal serum-free insect cell culture medium was selected from ESF AF. TM Insect cell culture medium, Grace insect cell culture medium, ESF 921 medium, Sf 900II SFM medium and Sf 900III SFM medium;

[0103] 2) Preparation of monoclonal screening culture medium:

[0104] Monoclonal screening culture was prepared using a first basic serum-free insect cell culture medium of 60% supplemented with whey protein hydrolysate and Yeastolate, a second basic serum-free insect cell culture medium of 20% supplemented with FBS and 20% supplemented with FBS. The first basic serum-free insect cell culture medium supplemented with whey protein hydrolysate and Yeastolate was Grace supplemented insect cell culture medium, and the second basic serum-free insect cell culture medium was Sf 900II SFM medium.

[0105] 3) Dilution and plating of cell suspensions

[0106] The cell suspension obtained in step 1 was centrifuged and washed multiple times with basal serum-free insect cell culture medium to remove culture medium containing rhabdovirus; then, the cells were diluted to 0.1 × 10⁻⁶ using the freshly prepared monoclonal selection culture medium from step 2. 4 1 x 10⁻¹ ml 6 Cells / ml, then serially diluted to 25-100 cells / ml, seeded at 1-10 cells / well in culture plates, and incubated statically in a 27-28°C incubator; or

[0107] The cell suspension obtained in step 1 was centrifuged and washed multiple times with basal serum-free insect cell culture medium to remove culture medium containing rhabdovirus; then, the freshly prepared monoclonal selection culture medium from step 2 was taken and an antiviral compound was added to achieve a final concentration of 10-50 ng / ml, and the cells were diluted to 0.1 × 10⁻⁶. 4 1 x 10⁻¹ ml 6 The cells were diluted to 25-100 cells per milliliter and then serially diluted to 1-10 cells per well. The cells were then seeded into culture plates and incubated at 27-28°C. The antiviral compound was ribavirin.

[0108] 4) Cell culture, rhabdovirus detection, and cell passage

[0109] From the second to the tenth day after plating, the wells were observed daily to identify single-cell wells. The growth of the monoclonal cells obtained through limiting dilution was observed regularly, and rhabdovirus was detected. Based on the cell growth and rhabdovirus detection results, the medium in the wells was changed or expanded for culture and passage. The medium was changed to the monoclonal selection medium corresponding to the cells in each well or monoclonal selection medium with the addition of the antiviral compound ribavirin at a final concentration of 10-50 ng / ml.

[0110] 5) Expand the culture of cells free from rhabdovirus contamination.

[0111] Cells that were initially free of rhabdovirus contamination obtained in step 4 were cultured in monoclonal screening medium corresponding to the cells in each well plate, or monoclonal screening medium with ribavirin added to a final concentration of 10-50 ng / ml; rhabdovirus detection was performed to obtain genetically modified insect cell lines free of rhabdovirus contamination.

[0112] In some implementations, commercially sourced Sf9 cells are typically contaminated with rhabdovirus (Sf-) in the culture supernatant during culture. R habdo v Irus (also abbreviated as "Sf-RV"). This invention exemplifies a method for preparing the above-mentioned "II. Genetically Modified Insect Cells" from commercially sourced Sf9 cells, and then screening out genetically modified Sf9 cell lines free from Sf-RV contamination.

[0113] In some embodiments, the above-mentioned "II. Genetically Modified Insect Cells" are prepared from commercially sourced Sf9 cells to obtain genetically modified Sf9 cells expressing baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins. Then, genetically modified Sf9 cell lines free from Sf-RV contamination are screened. The screening method includes the following steps:

[0114] 1) Recovery and restoration of cell growth status of genetically modified Sf9 cells expressing baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins, prepared from commercially sourced Sf9 cells.

[0115] Genetically modified Sf9 cells expressing baculovirus IE0, IE1, Orf146, Lef2, and Orf91 proteins, prepared from commercially sourced Sf9 cells, were removed from the liquid nitrogen container. The cells were then transferred to a basal serum-free insect cell culture medium (e.g., ESF AF). TM Complete culture media were prepared by supplementing fetal bovine serum (FBS) with insect cell culture media (purchased from Expression Systems, such as Grace insect cell culture medium (Gibco), ESF 921 medium, Sf-900 II SFM medium, Sf-900 III SFM medium, etc.) (e.g., supplementing with 5%–20% FBS, e.g., 10% FBS). The genetically modified Sf9 cells were statically cultured in culture flasks or plates at approximately 27–28°C, e.g., approximately 27°C, approximately 27.5°C, or approximately 28°C. When the genetically modified Sf9 cells adhered and achieved an adhesion rate of over 60%, they were digested with trypsin and passaged. The cells were passaged 2–3 times to restore their optimal growth state. A cell suspension was then prepared.

[0116] 2) Preparation of monoclonal screening culture medium

[0117] A first basal serum-free insect cell culture medium is prepared by supplementing a first basal serum-free insect cell culture medium with whey protein hydrolysate and yeastolate, for example, by supplementing whey protein hydrolysate to 2000 mg / L–5000 mg / L, preferably 3000 mg / L, 3500 mg / L, 4000 mg / L, or 4500 mg / L; and by supplementing yeastolate to 2000 mg / L–5000 mg / L, preferably 3000 mg / L, 3500 mg / L, 4000 mg / L, or 4500 mg / L. This first basal serum-free insect cell culture medium can be any basal serum-free insect cell culture medium (e.g., ESF AF). TM Insect cell culture medium (purchased from Expression Systems), Grace insect cell culture medium (Gibco), Sf-900 II SFM medium, Sf-900 III SFM medium, etc.

[0118] Whey protein hydrolysate is an enzymatic hydrolysate of whey protein and contains high levels of essential amino acids.

[0119] There are no particular limitations on the whey protein hydrolysate of this invention, as long as it is obtained by degrading whey protein using an enzymatic enzyme. Commercially available whey protein hydrolysates can be used and added in the amounts recommended in the manufacturer's instructions. For example, Merck's commercially available whey protein hydrolysate can be used.

[0120] Yeastolate is a yeast extract obtained by autolysis of yeast cells of *Saccharomyces cervisiae*. There are no particular limitations on the Yeastolate used in this invention; commercially available Yeastolate can be used and added in the amounts recommended in the manufacturer's instructions. For example, commercially available Yeastolate from USBiological or Sartorius can be used.

[0121] In some implementations, the primary serum-free insect cell culture medium supplemented with whey protein hydrolysate and Yeastolate is Grace-supplemented insect cell culture medium (Gibco).

[0122] Monoclonal selection culture was prepared using a first basal serum-free insect cell medium supplemented with whey protein hydrolysate and Yeastolate (30%-60%), a second basal serum-free insect cell medium supplemented with whey protein hydrolysate and Yeastolate (20%-35%), and FBS (10%-35%). The first basal serum-free insect cell medium (i.e., basal serum-free insect cell medium without supplementation of whey protein hydrolysate and Yeastolate) can be any basal serum-free insect cell medium (e.g., ESF AF). TM Insect cell culture medium (purchased from Expression Systems), such as Grace insect cell culture medium (Gibco), Sf-900 II SFM medium, Sf-900 III SFM medium, etc., and the second basal serum-free insect cell culture medium can be any basal serum-free insect cell culture medium (e.g., ESF AF). TM Insect cell culture media (purchased from Expression Systems, including Grace insect cell culture medium (Gibco), Sf-900 II SFM medium, Sf-900 III SFM medium, etc.). The first and second basic serum-free insect cell culture media can be the same or different; for example, the first and second basic serum-free insect cell culture media can be independently selected from ESF AF. TM Insect cell culture medium (purchased from Expression Systems), including commercially available serum-free insect cell culture media such as Grace insect cell culture medium (Gibco), Sf-900II SFM medium, and Sf-900 III SFM medium, for culturing Sf9 cells.

[0123] In some implementations, Grace-supplemented insect cell culture medium (GIBCO), Sf 900II SFM medium (GIBCO), and FBS are mixed in a volume ratio of 3:1:1 to obtain a monoclonal screening culture medium.

[0124] In some implementations, Grace-supplemented insect cell culture medium (Pronosai), Sf900 III SFM medium (GIBCO), and FBS are mixed in a volume ratio of 1:1:1 or 3:2:1.

[0125] The monoclonal screening culture medium prepared in this invention functions to screen genetically modified monoclonal Sf9 cells and support the growth and expansion of genetically modified monoclonal Sf9 cells.

[0126] 3) Dilution and plating of cell suspensions

[0127] Centrifuge the cell suspension obtained in step 1) and wash the cells multiple times with basal serum-free insect cell culture medium; then take the freshly prepared monoclonal selection culture medium from step 2) and dilute the cell suspension prepared in step 1) to 0.1 × 10⁻⁶. 4 Cells / ml - 1×10 6 cells / ml, for example 1×10 5 Cells / ml, then serially diluted with monoclonal selection medium to 25-100 cells / ml, and seeded into culture plates, for example, 96-well culture plates, at 1-10 cells / well, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells / well, and placed in a constant temperature incubator at about 27-28°C, for example, about 27.5°C for static culture;

[0128] or

[0129] Centrifuge the cell suspension obtained in step 1) and wash the cells multiple times with basal serum-free insect cell culture medium; then take the freshly prepared monoclonal selection culture medium from step 2) and add at least one antiviral compound to a final concentration of 10-50 ng / ml, and dilute the cells to 0.1 × 10⁻⁶. 4 1 x 10⁻¹ ml 6 cells / ml, for example, 1×10 5 Cells / ml, then serially diluted to 25-100 cells / ml, and seeded into culture plates, for example, 96-well plates, at 1-10 cells / well, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells / well, and incubated statically in an incubator at approximately 27-28°C, such as approximately 27°C, approximately 27.5°C, or approximately 28°C.

[0130] Antiviral compounds added to the culture medium are used to inhibit rhabdovirus replication. In some embodiments, diluted and plated single cells or several cells are cultured in monoclonal selection medium supplemented with at least one antiviral compound to inhibit rhabdovirus replication.

[0131] Exemplary antiviral compounds include, but are not limited to, nucleoside analogs, interferons, and virus-specific antibodies, such as monoclonal or polyclonal antibodies that neutralize viruses. Non-limiting examples of nucleoside analogs include ribavirin, 6-azauridine, vidarabine, acyclovir, 9- / 3-D-arabinofuranosyladenine (Ara-A), cytosine arabinose, adenine arabinoside, and guanine 7-N-oxide (G-7-Ox). In some embodiments, at least one antiviral compound comprises 6-azauridine. In some embodiments, the antiviral compound is selected from ribavirin, 6-azauridine, vidarabine, acyclovir, 9- / 3-D-arabinofuranosyladenine (Ara-A), cytosine arabinose, adenine arabinoside, and guanine 7-N-oxide (G-7-Ox). In some embodiments, the antiviral compound is 6-azauridine. In some implementations, the antiviral compound is ribavirin.

[0132] 4) Cell culture, Sf-RV detection and cell passage

[0133] From day two to day ten after plating (e.g., day two to day seven after plating), observe the wells daily, identifying and labeling the wells containing single-celled cells obtained through limiting dilution. Regularly observe cell growth in the corresponding wells of the 96-well plate, simultaneously performing Sf-RV detection. Based on cell growth and Sf-RV results, selectively change the medium or expand the culture and passage the cells in the wells. This medium change involves replacing the medium with the specific single-clone selection medium corresponding to the cells in each well, or with a single-clone selection medium supplemented with at least one antiviral compound at a final concentration of 10-50 ng / ml.

[0134] Collect the cell culture supernatant for Sf-RV virus detection.

[0135] Many known detection techniques exist in this field for detecting Sf-RV virus, including polymerase chain reaction (PCR), reverse transcription (RT), reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR combined with nested PCR, quantitative PCR (qPCR), RT-PCR combined with quantitative PCR (quantitative RT-PCR or RT-qPCR), various probe hybridization techniques, electron microscopy, and various antibody-based detection techniques known in this field, such as ELISA assays. Detection techniques also include, but are not limited to, plaque assays and cytopathic effect (CPE) observation, as well as bioinformatics techniques such as BLAST retrieval.

[0136] In one implementation, viral RNA is extracted from cell culture supernatant using a virus extraction kit, and Sf-RV virus is detected using RT-PCR.

[0137] 5) Expand the culture of cells free from Sf-RV virus contamination.

[0138] Single cells initially found to be free of Sf-RV virus contamination in 96-well plates were expanded using monoclonal selection culture medium corresponding to the cells in each well, or monoclonal selection culture medium supplemented with at least one antiviral compound at a final concentration of 10-50 ng / ml, for example, in 24-well plates, 6-well plates, T25 flasks, T75 flasks, and T225 flasks, and then subjected to Sf-RV virus detection to obtain genetically modified Sf9 cell lines free of Sf-RV virus contamination.

[0139] In some implementations, the Sf-RV virus-free cell line is the cell line obtained after culturing using only monoclonal screening culture medium after performing steps 3) and 4).

[0140] In some implementations, the Sf-RV virus-free cell line is a cell line obtained after culturing in steps 3) and 4) using a monoclonal screening culture medium supplemented with at least one antiviral compound at a final concentration of 10-50 ng / ml.

[0141] Compared with using only monoclonal screening medium to perform steps 3) and 4), performing steps 3) and 4) with monoclonal screening medium supplemented with at least one antiviral compound at a final concentration of 10-50 ng / ml can obtain genetically modified Sf9 cell lines free of Sf-RV virus contamination more quickly, for example, 15-30 days faster.

[0142] In some embodiments, the antiviral compound is selected from ribavirin, 6-azauridine, vidarabine, acyclovir, 9- / 3-D-arabinofuranosyladenine (Ara-A), cytosine arabinose, adenine arabinoside, and guanine 7-N-oxide (G-7-Ox). In some embodiments, the antiviral compound is 6-azauridine. In some embodiments, the antiviral compound is ribavirin.

[0143] IV. Domestication methods for genetically modified insect cell lines free from rhabdovirus contamination

[0144] The genetically modified insect cell lines free from rhabdovirus contamination obtained by the screening method of the present invention are adherent cells adapted to culture in serum-containing media. To meet the needs of large-scale production of recombinant proteins or viral vectors such as AAV viral vectors, it is important to establish stable, rhabdovirus-free, genetically modified, serum-free suspension insect cell lines and to scale up suspension cell culture on a large scale.

[0145] The fully suspended culture system is simple to operate, and can complete the passage without trypsin digestion of adherent cells. It has a high cell growth density, saves culture costs, and makes the culture process easier to scale up.

[0146] This invention provides a method for the domestication of genetically modified insect cells free from rhabdomyovirus contamination. The method involves serum-free culture domestication and suspension domestication of genetically modified insect cell lines obtained by screening genetically modified insect cell lines free from rhabdomyovirus contamination using the screening method of this invention.

[0147] In some implementations, the domestication method for genetically modified insect cell lines free from rhabdovirus contamination includes: culturing genetically modified insect cell lines free from rhabdovirus contamination obtained through the screening method described in "III. Method for Screening Genetically Modified Insect Cell Lines Free from Rhabdovirus Contamination" until the cell adhesion reaches 60% or more, performing trypsin digestion to prepare a cell suspension; and then using a suspension supplemented with Pluronic acid. TM The F-68 serum-free insect cell culture medium was used for suspension culture in a shaking incubator at 27-28°C and 100-130 rpm. The suspension-cultured cells were then stably cultured for 4-20 generations to obtain a serum-independent, rhabdovirus-free, genetically modified suspension insect cell line, for example, one containing Pluronic acid. TM The basic serum-free insect cell culture medium for F-68 is Vigor-S101 medium.

[0148] In some embodiments, the present invention provides a method for acclimatizing genetically modified Sf9 cells free from Sf-RV contamination, which involves acclimatizing and suspension-acclimatizing Sf9 cells free from Sf-RV contamination obtained by the screening method of the present invention for genetically modified Sf9 cell lines free from rhabdovirus contamination.

[0149] In some implementations, the acclimatization method for Sf9 cell lines free from Sf-RV contamination includes: adhering culture of the genetically modified Sf9 cells free from Sf-RV virus contamination obtained in step 5) above; when the cell adhesion reaches more than 60%, trypsin digestion is performed to prepare a cell suspension, and then the suspension is prepared at approximately 1×10⁻⁶ cells / mL. 5 1 x 10⁻¹ ml6 Inoculate per ml into Erlenmeyer flask, using a solution containing Pluronic acid. TM The serum-free culture medium of F-68 was used for suspension culture in a shaking incubator at approximately 27-28°C and 100-130 rpm. The suspension cultured cells were stably cultured for at least 3 passages, for example, 4-20 passages, to obtain serum-independent, rhabdovirus-free suspension genetically modified Sf9 cell lines. A cell bank was established and named.

[0150] The time required for the genetically modified Sf9 cells obtained in step 5) to adapt to serum-free culture conditions will vary depending on the specific growth characteristics of the Sf9 cells. If cell viability drops below 80% or doubling time is significantly prolonged during serum-free acclimatization, the acclimatization process may have been too rapid. Cells need to be passaged several times at the previous cell density before restarting continuous serum-free acclimatization.

[0151] In some implementations, the basal serum-free insect cell culture medium is commercially available, such as ESF AF. TM Insect cell culture media (purchased from Expression Systems), including Grace insect cell culture medium (Gibco), Sf-900 II SFM medium, and Sf-900 III SFM medium. In some embodiments, Pluronic acid is added to the basal serum-free insect cell culture medium. TM F-68, for example, with Pluronic TM F-68 is available in concentrations from 500 mg / L to 2000 mg / L, preferably 600 mg / L, 800 mg / L, 1000 mg / L, or 1500 mg / L.

[0152] In some implementations, Pluronic is added. TM The basic serum-free insect cell culture medium for F-68 is Vigor-S101 medium (Beianji Biotechnology Co., Ltd.).

[0153] In some embodiments, the present invention has obtained genetically modified cell lines named Sf9-MD768-3C7, Sf9-MD768-1E4, and Sf9-MD768-2H12, respectively, free from Sf-RV virus contamination.

[0154] The Sf9-MD768-3C7 cell line (classification name: Spodopterafrugiperda Ovary Cell) has been deposited at the China General Microbiological Culture Collection Center (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China), accession number CGMCC No. 46339, deposit date: April 2, 2025.

[0155] V. Use of the genetically modified insect cells of the present invention that are free from rhabdovirus contamination

[0156] This invention relates to the use of genetically modified insect cell lines free from rhabdovirus contamination obtained by the screening method and optionally domestication method of the present invention, for use in fermenter production of recombinant proteins and fermenter production of viral vectors such as AAV viral vectors.

[0157] In some embodiments, the cell lines obtained by the screening and optionally acclimatization methods of the present invention for Sf9 cell lines free from Sf-RV contamination are low-oxygen and low-glucose cell lines, providing greater convenience for future large-scale fermenter production of recombinant proteins and viral vectors such as AAV viral vectors. Preferably, this reduces oxygen consumption and glucose consumption in the culture medium, thereby saving production costs and reducing the safety risks of Sf9-produced bioproducts.

[0158] In some embodiments, the present invention provides a method for screening genetically modified Sf9 cell lines with low oxygen and low glucose consumption, comprising:

[0159] Multiple genetically modified Sf9 cell lines free of Sf-RV contamination, obtained by the screening method and optional domestication method of the present invention, are inoculated into fermenters for culture, for example, into 1L-5L fermenters, for example, using serum-free insect cell culture medium, preferably using Pluronic acid-containing medium. TM F-68 cells were cultured in serum-free insect culture medium. Two days later, they were infected with recombinant baculovirus to produce recombinant proteins or viral vectors such as AAV viral vectors. Cell counts were performed regularly, glucose concentrations in the culture medium were measured, and oxygen consumption was recorded to obtain genetically modified Sf9 cell lines with low oxygen and glucose consumption.

[0160] In some implementations, Pluronic is added. TMThe basic serum-free insect cell culture medium for F-68 is Vigor-S101 medium (Beianji Biotechnology Co., Ltd.).

[0161] In some embodiments, the genetically modified Sf9 cell line free of Sf-RV contamination of the present invention exhibits significantly increased viable cell density and cell viability on days 4 and 5 after infection with the recombinant baculovirus vector; the produced rAAV titer is higher and the distribution of rAAV within the cells is better; the proportion of VP1 protein in the prepared rAAV is significantly increased, thereby enhancing the biological activity of the AAV vector.

[0162] Example

[0163] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, all reaction reagents involved in the embodiments can be purchased through commercial channels.

[0164] Example 1: Construction of a plasmid to improve the performance of Sf9 cell line and preparation of Sf9 cells transfected with the plasmid.

[0165] In this embodiment, the MD768 plasmid was designed to improve the overall performance of the Sf9 cell line. The MD768 plasmid contains the following expression boxes:

[0166] 1) IE0 / coIE1 expression box (Note: "co" stands for "codon optimized"). c odon o (abbreviation of "ptimized"), containing the sequence shown in SEQ ID NO:1, used to express the baculovirus IE0 protein (SEQ ID NO:6) and IE1 protein (SEQ ID NO:7);

[0167] 2) Orf146 expression cassette, containing the sequence shown in SEQ ID NO:3, used to express the baculovirus Orf146 protein (SEQ ID NO:8);

[0168] 3) opLef2 expression box (Note: "op" also means "codon optimized"). op (abbreviation of "timized"), containing the sequence shown in SEQ ID NO:4, used to express the baculovirus Lef2 protein (SEQ ID NO:9);

[0169] 4) An Orf91 expression cassette, containing the sequence shown in SEQ ID NO:5, for expressing the baculovirus Orf91 protein (SEQ ID NO:10). Optionally, taking advantage of the polycistronic nature of viruses, the baculovirus Lef2 gene and the Orf91 gene can be combined into an expression cassette, named: opLef2-Orf91 expression cassette. Within this opLef2-Orf91 expression cassette, the upstream baculovirus Lef2 regulatory element drives and expresses both the baculovirus Lef2 protein and the Orf91 protein; and

[0170] 5) NeoR expression box.

[0171] Specifically, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the IE0 gene sequence shown in SEQ ID NO: 1, the Orf146 gene sequence shown in SEQ ID NO: 3, the opLef2 gene sequence shown in SEQ ID NO: 4, and the Orf91 gene sequence shown in SEQ ID NO: 5. Using the pUC57 vector as the starting material, the IE0 gene sequence shown in SEQ ID NO: 1, the Orf146 gene sequence shown in SEQ ID NO: 3, the opLef2 gene sequence shown in SEQ ID NO: 4, and the Orf91 gene sequence shown in SEQ ID NO: 5 were cloned into the pUC57 vector to obtain a plasmid vector named pUC57-IE0 / 1-opLef2-Orf91 (delivered by Sangon Biotech (Shanghai) Co., Ltd.).

[0172] Using pUC57-IE0 / 1-opLef2-Orf91 as a template, the following primers were synthesized to amplify the IE1 promoter sequence by PCR:

[0173] KZ-IE1PRO-F:TTCAATCATGGTGGCATGAACGTCAATTTAT(SEQ ID NO:11)

[0174] KZ-IE1PRO-R: CGCAGAAGCGCGTCGACCGATGTCT (SEQ ID NO: 12)

[0175] PCR conditions were: Tm: 58℃, extension 20s; cycle number 33;

[0176] A fragment of the IE1 launcher sequence with a length of approximately 0.6kb was obtained.

[0177] Using pEGFP-C1 plasmid (Nova Lifetech / Affigen, AFG-NVF-092925) as a template, the following primers were synthesized to amplify the NeoR and SV40 polyA sequences via PCR:

[0178] KZ-NEO-F: GAGGGGATCGAGCCCCTTCCTTGCTGTACTGGTACCC (SEQ ID NO: 13)

[0179] KZ-NEO-R: GCCACCATGATTGAACAAGATGG (SEQ ID NO: 14)

[0180] PCR conditions were: Tm: 59℃, extension 20s; cycle number 33;

[0181] Fragments of NeoR and SV40 polyA sequences, each approximately 1.0 kb in length, were obtained.

[0182] The pUC57-IE0 / 1-opLef2-Orf91 plasmid vector was linearized using SalI restriction enzyme. The amplified IE1 promoter sequence fragment, along with the amplified NeoR and SV40 polyA sequences, were ligated using the Fusion-In cloning method. The resulting plasmid was named MD768. Figure 1 As shown.

[0183] The obtained MD768 plasmid was aseptically extracted and purified, and then transfected into Sf9 cell lines using PEI transfection reagent. Specifically, the Sf9 cell line was purchased from the National Biomedical Experimental Cell Resource Bank (Resource No. 1101INS-PUMCO00117). Sf9 cell lines were cultured in six-well plates at 28°C using Sf9-specific medium (CELL-WISE, catalog number: BWCXD0401). PEI transfection reagent (Yisheng Biotechnology, 40816ES01) was added to each 100 μL of transfection medium with 4 μg of MD768 plasmid, and the cells were transfected into the six-well plates. The medium was changed 24 h after transfection, and then replaced with medium containing 400 μg / mL G418 after 48 h for continuous culture. The medium was changed during culture according to cell death and fragmentation. After culturing at 400 μg / mL G418 pressure for 3–4 weeks, the G418 pressure was reduced to 200 μg / mL and then increased for another 2 weeks. Finally, a G418-resistant positive Sf9 cell line was obtained, named Sf9-MD768 cell line, and placed in cryovials and stored in liquid nitrogen.

[0184] Example 2: Screening of Sf9-MD768 cell lines free from Sf-RV contamination

[0185] In this embodiment, Sf9-MD768 cell lines free from Sf-RV contamination were screened. The specific screening method is as follows:

[0186] 1. Cell resuscitation: The Sf9-MD768 cell cryovials prepared in Example 1 were removed from liquid nitrogen, thawed in a 37°C water bath, centrifuged at 400g for 5 min, the cell supernatant was discarded, and the culture medium was replaced with Sf9 cell culture medium, specifically ESF AF. TM Insect cell culture medium (purchased from Expression Systems) + 10% FBS (purchased from AusGeneX) (the culture medium using serum is also referred to as "complete medium" in this article) was used to resuspend cells. After resuspending, the cells were placed in T25 square flasks and incubated statically in a biochemical incubator (Yatai Kelon, model: SPX-150B) at 28°C. The cell culture supernatant was tested for rhabdovirus using qPCR, and rhabdovirus contamination (also referred to as "Sf-RV contamination" in this article) was detected. The Sf9-MD768 cells contaminated with Sf-RV are also referred to as "Sf9-MD768-Rha" cells, or simply "MD768-Rha" cells in this article.

[0187] 2. Cell Culture: When the adhesion of Sf9-MD768-Rha cells from step 1 reaches more than 60%, passage and expansion culture are performed. Passage is repeated 2-3 times until the cell condition is restored to the optimal level. The Sf9-MD768-Rha cells that have been restored to the optimal state are then cultured in cell culture plates or flasks. Thus, Sf9-MD768-Rha cells are used as a routine maintenance population of polyclonal Sf9-MD768 adherent cells.

[0188] 3. Cell passage: After culturing Sf9-MD768-Rha cells for 2-3 days, aspirate the cell supernatant into sterile centrifuge tubes; wash the cell culture plate or flask containing the Sf9-MD768-Rha cells twice with DPBS, add 0.25% trypsin for digestion, and after digestion, add complete culture medium (ESF AF) to the cell culture plate or flask. TM Insect cell culture medium (purchased from Expression Systems) + 10% FBS (purchased from AusGeneX) was used to terminate trypsin digestion. Cells were dispersed and mixed, then transferred to sterile centrifuge tubes containing cell supernatant. The cells were centrifuged at 800 rpm for 5 min. Sf9-MD768-Rha cells were resuspended in complete culture medium and cultured and passaged at a ratio of 1:3 to 1:5. The cells were then incubated statically in a biochemical incubator at 27.5°C.

[0189] 4. Cell Screening: Using the limited cell culture method, cells were seeded at 150 μl / well at 5 cells / well × 4 96-well plates and 10 cells / well × 4 96-well plates. Before seeding, the cells were washed 3-6 times to remove all the original culture medium containing rhabdovirus. Fresh monoclonal selection culture medium was added, and the cells were incubated statically at 27.5°C in a biochemical incubator. The monoclonal selection culture medium was prepared by mixing Grace-supplemented insect cell culture medium (GIBCO, catalog number: 11605094), Sf 900II SFM medium (GIBCO), and FBS at a volume ratio of 3:1:1.

[0190] In addition, the following experiments were performed in parallel. Using the limited cell culture method, cells were seeded at 5 cells / well × 4 96-well plates and 10 cells / well × 4 96-well plates, with 150 μl / well. Before seeding, the cells were washed 3–6 times to remove all original culture medium containing rhabdovirus. Fresh monoclonal selection culture medium was added, and ribavirin was added to a final concentration of 10, 20, 30, 40, or 50 ng / ml. The cells were then incubated statically in a 27.5°C biochemical incubator. The monoclonal selection culture medium was prepared by mixing Grace-supplemented insect cell culture medium (GIBCO, catalog number: 11605094), Sf 900II SFM medium (GIBCO), and FBS at a volume ratio of 3:1:1.

[0191] From day 2 to 7 after plating, the wells were observed daily to identify and label the wells containing single-celled cells obtained through limiting dilution. Cell growth in the corresponding wells of the 96-well plates was monitored periodically, and rhabdovirus was detected in the cell culture supernatant using qPCR. Based on cell growth and rhabdovirus detection results, the medium was selectively changed, expanded, or passaged. The medium change involved replacing the culture medium with the specific single-clone selection medium for each well or adding ribavirin to a final concentration of 10-50 ng / ml for single-clone selection.

[0192] Control cell wells were set up. As a comparison, limited cell culture was used, with cells seeded at 5 cells / well × 4 96-well plates and 10 cells / well × 4 96-well plates, 150 μl / well. Cells were washed 3-6 times before seeding to remove all original culture medium containing rhabdovirus. No monoclonal selection medium or ribavirin was added; instead, ESF AF medium was added. TMCells were cultured on insect cell culture medium (purchased from Expression Systems) or IB907d medium (YSK Bio, https: / / www.yskbio.com / Insect-Cell-Serum-Free-Medium.html), with or without 2%-10% FBS. Results were obtained from observing the corresponding wells on days 2-7, with no single-cell wells obtained.

[0193] 5. Cell expansion culture and passage: After culturing single cells in 96-well plates, the cells were sequentially expanded and passaged to 24-well plates, 6-well plates, T25 square bottles, T75 square bottles, and T225 square bottles.

[0194] Specifically, after the single cells in the 96-well plate formed obvious cell clusters, the cells were dispersed by pipetting and directly transferred to the 24-well plate at 750 μL / well and incubated statically at 27.5℃.

[0195] Once the cell confluence in the 24-well plate reaches 30-60%, the cells are amplified and dispersed by pipetting. The cell suspension is then transferred to a centrifuge tube and centrifuged at 800 rpm for 5 min. The old culture medium is discarded, and the cells are resuspended in fresh, appropriate culture medium (i.e., the appropriate monoclonal selection culture medium or monoclonal selection culture medium with a final concentration of 10-50 ng / ml ribavirin). The cells are then amplified and transferred to 6-well plates at 3 mL / well and incubated statically in a biochemical incubator at 27.5°C.

[0196] Once the cell confluence in the 6-well plate reaches 30-60%, proceed with cell amplification. Disperse the cells using pipetting, transfer the cell suspension to centrifuge tubes, centrifuge at 800 rpm for 5 min, discard the old culture medium, and resuspend the cells in fresh, appropriate culture medium (i.e., the corresponding monoclonal selection culture medium or monoclonal selection culture medium with a final concentration of 10-50 ng / ml ribavirin). Transfer the cells to T25 square flasks, 5 mL / well, and incubate statically in a biochemical incubator at 27.5℃.

[0197] Diffusion in T75 and T225 flasks: Diffusion should be performed in T25 flasks when the cell confluence reaches 30-60%. For cells that adhere firmly, digestion is required: After rinsing the T25 flask twice with DPBS, add 0.25% trypsin digestion solution to fully wet the cells, then aspirate the digestion solution. Place the T25 flask in a 27.5℃ biochemical incubator for 5-10 minutes for digestion. After digestion, add monoclonal selection culture medium to stop digestion, disperse the cells by pipetting, centrifuge, and transfer to the appropriate culture medium (i.e., the appropriate monoclonal selection culture medium or monoclonal selection culture medium with a final concentration of 10-50 ng / ml ribavirin) for continued culture. If the cell line does not adhere firmly, the cells can be dispersed by pipetting, centrifuged, and transferred to the appropriate culture medium (i.e., the appropriate monoclonal selection culture medium or monoclonal selection culture medium with a final concentration of 10-50 ng / ml ribavirin) for continued culture. The culture volumes were 12 mL for T75 square bottles and 30 mL for T225 square bottles.

[0198] 6. Cell acclimatization: Once the cells have been expanded and passaged to a certain scale and the rhabdovirus test result is negative, a certain number of cells are frozen for later use. At the same time, the cells are digested, expanded, and passaged into Erlenmeyer flasks for suspension acclimatization. The culture medium used is Vigor-S101 medium (Beianji Biotechnology Co., Ltd.). Culture conditions: 15-30 ml of cell suspension is cultured in a 125 ml shake flask at 100-120 rpm in a biochemical incubator at 27-28℃ for about 20-30 days, with the medium changed every 3-5 days during this period.

[0199] During suspension acclimatization, samples were taken and counted daily, and acclimatization conditions were adjusted in real time based on the cell count results.

[0200] When cell growth is slow, centrifuge at 800 rpm for 5 minutes and then change the medium. When cell aggregation is severe (aggregation rate exceeds 40%), increase the shaker speed to 130-140 rpm. When cell viability is low, decrease the shaker speed to 80-90 rpm.

[0201] In contrast, Vigor-S101 medium was replaced with unsupplemented Pluronic acid. TM F-68 cells were subjected to suspension acclimatization using serum-free Sf-900 II SFM medium, and suspension cells were eventually obtained. However, the acclimatization time was prolonged to about 40 days, which increased the time required to obtain suspension-acclimatized cells. Furthermore, cell metabolism and the number of cells obtained were lower than when using Vigor-S101 medium.

[0202] 7. Cell cryopreservation: Take the acclimatized cells, count them using an automated cell counter, and determine the cell viability to ensure that the cell viability is greater than 95%. Centrifuge the cells at 800 rpm at room temperature for 5 minutes, resuspend the cell pellet in cell cryopreservation solution, and maintain a cell density of 2E+07 cells / ml. Aliquot the cells into cryovials, and after gradient cooling, transfer them to a liquid nitrogen tank for long-term storage.

[0203] Multiple Sf9-MD768 cell banks free of Sf-RV contamination were established after expansion culture and domestication of monoclonal cells free of Sf-RV contamination. The Sf9-MD768 cell banks free of Sf-RV contamination were named Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7, respectively. All of them were obtained by using monoclonal selection culture medium supplemented with a final concentration of 10 ng / ml ribavirin in steps 4 and 5, followed by domestication in step 6.

[0204] Example 3: Detection of Sf-RV virus in Sf9-MD768 cell line

[0205] In the process of obtaining Sf-MD768 cell lines free of Sf-RV contamination in Example 2, the cell culture supernatants harvested in step 4 "cell screening" and step 5 "cell expansion culture and passage" were detected by quantitative PCR. The culture supernatants of Sf9-MD768-1E4 cell line, Sf9-MD768-2H12 cell line, and Sf9-MD768-3C7 cell line obtained after 2-6 passages of the finally determined cell lines were monitored. The specific methods are as follows.

[0206] The design for quantitative PCR detection of rhabdovirus genome is as follows:

[0207] 1. Comparative analysis of genome sequences of rhabdoviruses derived from insect cells

[0208] Using the NCBI database, the genome sequences of seven rhabdoviruses derived from insect cells were obtained, with sequence numbers MH926031.1, MH926030.1, MF536978.1, MH926029.1, KF947078.1, MF536979.1, and NC_025382.1. The rhabdovirus sequence reported in patent publication number WO2015051255A1 (SEQ ID NO.1 in WO2015051255A1) was also used. Sequence alignment using Vector NTI11.0 software revealed high homology between these eight rhabdovirus genome sequences and the RNA-dependent RNA polymerase gene.

[0209] 2. Selection of homologous sequences

[0210] Based on sequence alignment results, sequences identical to those in the RNA-dependent RNA polymerase gene sequences of these eight rhabdoviruses were selected as target sequences for probe primer design. Specific sequence information is as follows.

[0211] 5'TTTATGGTGAGTTTGGATTTTTCCAAGTGGAACACCAATATGAGAGCCCCAGACAC ACAGCCATTTTACCACACTATAGATACGATGTTTGGTTTGGAAAATTGTTTTACCAGGACACATGAAATGTTCTACAATTCCTTTTTGTACCTTATAGACGGTTCTTATCTCCCAACAATAGTTGATGATGGGTTCAAAACAGATATTGGATGTTGGCGACATCATCTTGGGGGAATCGAAGGTCTCAGACAAAAAGGATGGACTCTGTGGACAGTTATGTTGATCAGGCTAGTTGCGGAAAAATATATTTTCAATATGTCTATCATGGGACAGGGGGACAATCAAATGCTACTTCTAACTTTCGATTCTAATA3' (SEQ ID NO.15), the sequence is named the Sf-RV RNA-dependent RNA polymerase gene common sequence.

[0212] 3. Probe and primer design based on homologous sequences

[0213] Using the common sequence of the Sf-RV RNA-dependent RNA polymerase gene as the target sequence, TaqMan-MGB probes and primers were designed using Primer express 3.0 software, as follows:

[0214] GQP-4-F TCGAAGGTCTCAGACAAAAAGGA(Tm:59)(SEQ ID NO.16)

[0215] GQP-4-R TTCCGCAACTAGCCTGATCA(Tm:58)(SEQ ID NO.17)

[0216] GQP-4-P FAM-ACTCTGTGGACAGTTATG-MGB(Tm:69)(SEQ ID NO.18)

[0217] The amplified fragment size is 64bp.

[0218] 4. Preparation of standards for quantitative PCR detection based on homologous sequences

[0219] A single-stranded RNA of 85 nt in length was synthesized as a quantitative PCR standard. Since rhabdoviruses are negative-sense RNA viruses, the synthesized sequence for the standard was negative-sense RNA. Details are as follows:

[0220] 5'aaauauauuuuuccgcaacuagccugaucaacauaacuguccacagaguccauccuuuuugucugagaccuucgauuccc ccaag3' (SEQ ID NO.19)

[0221] The results are shown in Table 1 below. No rhabdovirus was detected in the culture supernatant of Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7 cell lines.

[0222] Table 1. Detection of SfRV virus in the culture supernatant of each Sf9-MD768 cell.

[0223] Sample number Sample Information Quantity of samples submitted Rhabdovirus residual vg / mL 1 Culture supernatant of Sf9-MD768-Rha cells 50μl 7.40E+08 2 Culture supernatant of Sf9-MD768-1E4 cell line 50μl Not detected 3 Culture supernatant of Sf9-MD768-2H12 cell line 50μl Not detected 4 Culture supernatant of Sf9-MD768-3C7 cell line 50μl Not detected 5 Positive control 50μl 5.90E+08 6 negative control 50μl Not detected

[0224] Note: Sf9-MD768-Rha cells: Sf9-MD768 cell populations with rhabdovirus detected in cell culture supernatant before selection of Sf9-MD768 cell lines free from Sf-RV contamination; Positive control: Culture supernatant of Sf9-V432A cells contaminated with rhabdovirus (Sf9-V432A cells were preserved by Beijing Jinlan Gene Technology Co., Ltd., and tested positive for rhabdovirus by the China National Institutes for Food and Drug Control); Negative control: Monoclonal selection culture medium without ribavirin.

[0225] Example 4: Study on growth curves of various cell lines

[0226] The Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7 cell lines selected and tested in Examples 2 and 3 were seeded into 125ml Erlenmeyer flasks at a rate of 2.0E+06 cells / ml, 30ml / flask, and cultured at 27-28℃ on a shaker at 120rpm using Vigor-S101 serum-free insect cell medium (Shanghai Bei'anji). Cells were sampled and counted daily, and cell growth curves were plotted to determine cell growth status.

[0227] The results are as follows Figure 2As shown in the figure. Viable cell density analysis revealed that on day 5 of culture, compared to the rhabdovirus-contaminated control cells Sf9-MD768-Rha, the Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7 cell lines all exhibited higher viable cell densities. On day 4 of culture, compared to the rhabdovirus-contaminated control cells Sf9-MD768-Rha (9.1E6 cells / ml), the Sf9-MD768-3C7 cell line had an even higher viable cell density of 1.1E7 cells / ml.

[0228] Example 5: Study on the growth and metabolism of various cell lines in shake flasks

[0229] To further screen cell lines at the metabolic level, the three Sf9-MD768 cell lines without Sf-RV contamination (i.e., Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7 cell lines) and the control cell Sf9-MD768-Rha were seeded into 125 ml shake flasks for culture. Two days later, they were infected with recombinant baculovirus for AAV recombination.

[0230] Specifically, three Sf9-MD768 cell lines free of Sf-RV contamination (i.e., Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7) and control cells Sf9-MD768-Rha were seeded at 2.0E+6 cells / mL into 125 mL shake flasks. The culture medium used was Vigor-S101 serum-free insect cell medium (Shanghai Beiyanji), and the cells were cultured at 27-28℃ in a biochemical incubator at 120 rpm. After 2 days of culture, the cells were diluted to 4.0E+06 cells / mL and then infected with recombinant baculovirus as follows to prepare the recombinant AAV viral vector AAV9-EGFP (EGFP: enhanced green fluorescent protein). As described in CN111088284, Sf9-MD768 cells were co-infected with two recombinant baculoviruses to package and produce rAAV9. One recombinant baculovirus carried the rep2 and cap9 gene expression cassettes of AAV, while the other carried the ITR sequence of the rAAV vector and the gene expression cassette for expressing the EGFP protein. The Bac-to-AAV system is a patent of Virovek, Inc., USA. After co-infecting each Sf9-MD768 cell line (i.e., Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7) or the control cell line Sf9-MD768-Rha with the two recombinant baculoviruses, the cells were cultured for 72-96 hours. Cell viability was measured, and the recombinant AAV viral vector AAV9-EGFP was harvested. AAV genomic titer was detected, and the results were statistically analyzed as follows: Figure 3and Figure 4 As shown.

[0231] Depend on Figure 3 As can be seen, on day 3 after infection of the Sf9-MD768 cell lines with recombinant baculovirus, the intracellular recombinant AAV genome titers of Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7 were 1.60E+10 vg / mL, 5.18E+10 vg / mL, and 9.33E+10 vg / mL, respectively. On day 4 after infection of the Sf9-MD768 cell lines with recombinant baculovirus, the intracellular recombinant AAV genome titers of Sf9-MD768-2H12 and Sf9-MD768-3C7 were 6.22E+10 vg / mL and 1.15E+11 vg / mL, respectively. The Sf9-MD768-3C7 cell line produced the highest intracellular recombinant AAV yield. In the production process, the recombinant AAV in cells is collected and concentrated, which is beneficial for subsequent purification because there are basically no impurities such as baculoviruses distributed in the culture supernatant.

[0232] Depend on Figure 4 It is evident that, after infection with recombinant baculovirus, the Sf9-MD768-3C7 cell line produced a higher amount of recombinant AAV compared to Sf9-MD768-Rha cells. On day 4 post-infection with recombinant baculovirus, the Sf9-MD768-3C7 cell line exhibited superior intracellular distribution of recombinant AAV, at 1.15E+11 vg / mL, indicating that with prolonged culture time, the Sf9-MD768-3C7 cell line experienced less cell lysis, which is conducive to the production of more recombinant AAV.

[0233] Example 6: Study on the growth and metabolism of various cell lines in fermenters

[0234] To further screen cell lines at the fermenter and metabolic levels, the three Sf9-MD768 cell lines without Sf-RV contamination (i.e., Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7) and the control cell Sf9-MD768-Rha were seeded into 2L fermenters for culture. Two days later, they were infected with recombinant baculovirus for AAV recombination.

[0235] Specifically, three Sf9-MD768 cell lines free from Sf-RV contamination (i.e., Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7 cell lines) and control cells Sf9-MD768-Rha were inoculated into a 2L fermenter at a rate of 2.0E+6 cells / mL. The culture medium used was Vigor-S101 serum-free insect cell medium (Shanghai Beiyanji). After inoculation, the fermenter parameters were set as follows: 250 rpm, 35% DO, 28.0℃, with Sparger O2 and dissolved oxygen (DO) linked, and the maximum aeration rate was 500 mL PM (the unit of the flow meter used in the equipment, equivalent to 0.5 L / min). The glucose concentration in the culture medium was measured by sampling and counting. On day 2 of culture, the cell density was diluted to 4.0E+06 cells / mL and then infected with recombinant baculovirus to prepare the recombinant AAV viral vector AAV9-EGFP (EGFP: enhanced green fluorescent protein). As described in CN111088284, Sf9 cells were co-infected with two recombinant baculoviruses to package and produce rAAV9. One recombinant baculovirus carried the rep2 and cap9 gene expression cassettes of AAV, and the other recombinant baculovirus carried the ITR sequence of the rAAV vector and the gene expression cassette expressing the EGFP protein. The Bac-to-AAV system is a patent of Virovek, Inc. Two recombinant baculoviruses were used to co-infect various Sf9-MD768 cell lines (i.e., Sf9-MD768-1E4, Sf9-MD768-2H12, and Sf9-MD768-3C7) or control cells Sf9-MD768-Rha, followed by culturing for 96-120 hours. Cell viability was measured, and the recombinant AAV viral vector AAV9-EGFP was harvested. Cells were counted daily, and glucose levels in the culture medium and oxygen consumption were recorded. The results are summarized below. Figure 5 and Figure 6 As shown.

[0236] Depend on Figure 5 It is evident that during cell culture in the fermenter, Sf9-MD768-3C7 cells infected with recombinant baculovirus maintained a high cell density and cell viability in the late stage of AAV recombination (day 5 after cell infection with recombinant baculovirus), which is beneficial for improving the yield and quality of recombinant AAV.

[0237] Depend on Figure 6 It is evident that, during cell culture in the fermenter, the control cell line Sf9-MD768-Rha, which was contaminated with rhabdovirus, consumed significantly more glucose and oxygen in the culture medium than the Sf9-MD768-3C7 cell line without rhabdovirus contamination.

[0238] Because Sf9 cells are highly oxygen-consuming and intolerant of shear forces, their oxygen demand increases during large-scale fermenter production. However, the aeration rate cannot be increased indefinitely (as this would generate numerous bubbles and cause cell damage). Therefore, reducing the high oxygen consumption of Sf9 cells is a pressing issue. This invention, while ensuring the genetic modification and removal of rhabdovirus from commercially available Sf9 cells, further screens them in a fermenter. The results show that a low-oxygen and low-glucose cell line was obtained, providing greater convenience for future large-scale production of recombinant proteins or viral vectors such as AAV viral vectors.

[0239] Depend on Figure 7 It is evident that the Sf9-MD768-3C7 cell line maintained a high level of recombinant AAV production (1.54E+11 vg / mL) on day 5 post-infection with recombinant baculovirus. In contrast, the control cells Sf9-MD768-Rha contaminated with rhabdovirus showed a recombinant AAV production of 7.74E+10 vg / mL on day 3 post-infection with recombinant baculovirus, but from day 4 onwards, due to cell lysis, the recombinant AAV production became undetectable.

[0240] Example 7: Next-generation sequencing of the Sf9-MD768-3C7 cell line

[0241] A sequencing library was constructed using the Sf9-MD768-3C7 cell line, and next-generation sequencing was performed.

[0242] RNA-seq analysis workflow: Total RNA was extracted from Sf9-MD768-3C7 cell line using the TIANGEN kit (DP424) and sent to a sequencing company (Beijing Berry Genomics Co., Ltd.) for sequencing using the Trussq mrna-seq (EU) platform. The sequencing data was analyzed using software such as Hisat2, Samtools, R, and Python. The results are as follows.

[0243] 1. Test Subjects / Results

[0244] Data analysis software: Hisat2, Samtools

[0245] Data analysis methods: Hisat2, Samtools depth, Samtools sort, Samtools view

[0246] Sequencing data read volume:

[0247] RNAseq-7: 24,211,323reads; 7,263,396,900Nucleotides

[0248] RNAseq-8: 25,399,075reads; 7,619,722,500Nucleotides

[0249] RNAseq-9: 19,268,949reads; 5,780,684,700Nucleotides

[0250] Post-filter data quality standard: Clean Q30 Bases Rate ≥ 90%

[0251] Average fragment length: 150bp; GC% and AT% are uniformly distributed.

[0252] Reference sequences: MH926031.1, MH926030.1, MF536978.1, MH926029.1, KF947078.1, MF536979.1, NC_025382.1, and Sf-RV.

[0253] 2. Results and Analysis

[0254] The Q30 of the Sf9-MD768-3C7 RNA-seq sequencing data is greater than 90%, indicating that the sequencing data quality is qualified and the data analyzed in this study is reliable.

[0255] Based on the data analysis results, the following conclusions are drawn:

[0256] (1) No rhabdovirus homologous sequences were found in the Sf9-MD768-3C7 cell line.

[0257] The Sf9-MD768-3C7 (RNAseq-7~RNAseq-9) cell line does not contain a complete Sf-RV* genome, with 0% coverage in both 1X and 4X models. Therefore, it is determined that the Sf9-MD768-3C7 cell line does not contain rhabdovirus homologous sequences.

[0258] (2) The complete genome of rhabdovirus was not found in the Sf9-MD768-3C7 cell line product.

[0259] Sequencing data from the Sf9-MD768-3C7 cell line were compared with the genomes of rhabdoviruses MH926031.1, MH926030.1, MF536978.1, MH926029.1, KF947078.1, MF536979.1, and NC_025382.1. 1X coverage (only RNAseq-8 samples had aligned sequences, and all were below 10%) and 4X coverage (all were 0%).

[0260] Therefore, it was determined that the complete genome of the rhabdovirus was not present in the Sf9-MD768-3C7 cell line.

[0261] Example 8: Study on the efficacy of Sf9-MD768-3C7 cell line in packaging AAV5

[0262] To compare the differences between the Sf9-MD768-3C7 cell line and existing Sf9 cell lines, AAV5-Gluc products were prepared using different Sf9 cells, and the purity and proportion of different VP proteins were analyzed by SDS-PAGE. Furthermore, cell infection experiments were conducted to investigate the differences in in vitro expression levels of different batches of AAV5-Gluc products.

[0263] The recombinant baculovirus and recombinant AAV particles were prepared and packaged according to the method described in Haifeng Chen, “Intron Splicing–mediated Expression of AAVRep and Cap Genes and Production of AAV Vectors in Insect Cells”, Molecular Therapy vol.16, No.5, 924-930, 2008. In short, by transforming *E. coli* DH10Bac competent cells, donor plasmids carrying the target genes encoding AAV Rep and Cap were recombined with baculoviruses in the cells. Blue-and-white screening was performed to obtain recombinant baculoviruses, which were then transfected into Sf9 cells to obtain recombinant baculovirus Bac-ipRepCap. Following a similar method, recombinant baculovirus Bac-Gluc carrying the Gaussian luciferase (GLuc) target gene (ITR-CA-Gluc-BGH-ITR) with the AAV2 ITR sequence flanking it was obtained, where CA represents the cytomegalovirus immediate early promoter and BGH represents bGH polyA. Sf9 cells were co-infected with recombinant baculovirus Bac-ipRepCap and recombinant baculovirus Bac-Gluc to prepare recombinant AAV5 virus particles. Cells were collected 72 hours after co-infection, and crude AAV particles were obtained by chloroform extraction and purification. The ratios of VP1, VP2, and VP3 were compared by SDS-PAGE electrophoresis.

[0264] A recombinant AAV5-Gluc product, named BZ2024061001, was prepared using the Sf9-MD768-3C7 cell line. A recombinant AAV5-Gluc product, named BZ2024062401, was prepared using the Sf9-IE1 cell line (patent application US20240352482A1) as a control cell line. The purity of recombinant AAV particles and the VP protein ratio were determined by performing SDS-PAGE on AAV5 products produced by different Sf9 cell lines. Gray-scale analysis was used to calculate the recombinant AAV particle purity and the VP protein ratio of the AAV5 products produced by different Sf9 cell lines.

[0265] The results are as follows Figure 8 As shown. Figure 8 SDS-PAGE electrophoresis analysis of small figure A shows that the capsid protein bands of the AAV5-Gluc product are clear, the VP1, VP2, and VP3 proteins are correctly positioned, and the bands become darker with increasing sample loading. The particle purity of different sample loading amounts and different products is above 98%. The gray values ​​of VP1, VP2, and VP3 proteins for different sample loading amounts and different products are shown below. Figure 8 As shown in inset B, the proportion of VP1 protein in the recombinant AAV product (BZ2024061001) from the Sf9-MD768-3C7 cell line was significantly higher than that in the recombinant AAV product (BZ2024062401) from the control Sf9-IE1 cell line.

[0266] HEK-293 and BHK21 cells were infected with different recombinant AAV products (BZ2024061001 and BZ2024062401) for 72 hours. The cell culture supernatant was then analyzed for Gluconiosis. The results are as follows: Figure 9 As shown in the results, the expression levels of Gluc protein in HEK-293 and BHK21 cells infected with product BZ2024061001 were significantly higher than those infected with product BZ2024062401. Specifically, the Gluc protein expression level in HEK293 cells infected with BZ2024061001 was approximately 1.80 times that of BZ2024062401, and the Gluc protein expression level in BHK21 cells was approximately 2.83 times that of BZ2024062401. This indicates that the Sf9-MD768-3C7 cell line has a better ability to enhance the biological activity of the recombinant AAV vector.

[0267] To further verify the characteristic behavior of recombinant AAV products in vivo, we selected 6-8 week old C57BL / 6J male mice and administered a single tail vein injection of recombinant AAV products (BZ2024061001 and BZ2024062401) prepared using Sf9-MD768-3C7 cell line and Sf9-IE1 control cell line. The injection dose was 5E+13 vg / kg, which was quantified by qPCR titer. Blood and tissue samples were collected from mice 3 weeks after administration to detect Glucose levels.

[0268] The results are as follows Figure 10 As shown. Three weeks after administration of the recombinant AAV product, the levels of Gluc in serum, heart, liver, lung, brain, and spinal cord samples prepared from the Sf9-MD768-3C7 cell line (batch number BZ2024061001) were higher than those in the control Sf9-IE1 cell line (batch number BZ2024062401).

[0269] Furthermore, the rAAV product prepared from the Sf9-MD768-3C7 cell line (batch number BZ2024061001) only caused a slight decrease in renal transduction capacity. Considering that AAV gene therapy often causes kidney damage, the rAAV product prepared from the Sf9-MD768-3C7 cell line is positive and effective in preventing potential kidney toxicity.

[0270] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0271] Exemplary sequence

[0272] The nucleotide sequence of IE0 (1911 bp):

[0273]

[0274] The nucleotide sequence of IE1 (1749 bp):

[0275]

[0276] Nucleotide sequence of Orf146 (606 bp):

[0277] Atgaacgtcaatttatactgccctaatggcgaacacgataacaatatttcttttattatgccctctaaaaccaacgcggttatcgtttatttattcaaattagatatagaacatccgccgacatacaatgttaatgcaaaaacgcgtttggtgagcggatacgaaaacagtcggccgataaacattaatctgaggtcgataacaccgtccttgaacggaacacgaggagcgtacgtgatcagctgcattcgcgcgccgcgcctttatcgagatttatttgcatacaacaagtacactgcgccgttgggatttgtggtaacgcgcacacatgcagagctgcaagtgtggcacattttgtctgtgcgcaaaacctttgaagccaaaagtacgaggtccgttacgggcatgctagcgcacacggacaatggacccgacaaattctacgccaaggatttaatgataatgtcgggcaacgtatccgttcattttatcaataacctacaaaaatgtcgcgcgcatcacaaagacatcgatatatttaaacatttatgtcccgaactgcaaatcgataatagtgttgtgcaactggaaacccgctcttcatag(SEQ ID NO:3)

[0278] Nucleotide sequence of Lef2 (633 bp):

[0279] ATGGCTAACGCATCATATAATGTTTGGTCACCCCTGATACGGGCCTCTTGTCTTGATAAAAAGGCGACATACTTGATTGATCCGGATGACTTCATCGACAAACTGACCCTCACCCCTTACACTGTTTTCTATAATGGAGGAGTATTAGTTAAGATAAGCGGCTTGCGATTATATATGCTCCTTACGGCGCCTCCGACAATCAATGAAATAAAAAATAGTAATTTTAAGAAGCGATCCAAAAGGAATATATGCATGAAAGAGTGTGTCGAGGGTAAGAAGAATGTCGTAGATATGCTCAACAACAAAATTAATATGCCGCCATGTATCAAAAAGATTCTAAATGACTTAAAGGAAAACAACGTCCCAAGAGGAGGGATGTACAGGAAGCGCTTTATCCTCAACTGCTACATAGCAAATGTCGTATCGTGCGCCAAATGCGAGAACCGTTGCCTGATTAAAGCTTTGACGCACTTCTATAACCACGACTCTAAGTGCGTGGGGGAAGTAATGCATCTACTTATTAAAAGCCAGGACGTGTATAAACCCCCCAACTGTCAAAAAATGAAGACTGTGGATAAGCTTTGTCCATTTGCAGGTAATTGTAAAGGCCTAAACCCTATCTGTAACTACTGA(SEQ ID NO:4)

[0280] Nucleotide sequence of Orf91 (672 bp):

[0281] ATGTGGTACATCATTGTCATCATCATCATAATTATCCTCAATCTATTTATAATAATACTATTAACATTAAAGATCCTGGATGACATCCAAGAACTTTACCCAAATCCCCCGATTGTGCCACCACCCACAAGCCCGCCTATTGTTCCCTTACCAACTCCCCCGCCTACTCCTACCCCCAGTCCACCCTCTCCGACACCTCCACCCACACCAATACCACCAACGCCTACGCCGACGCCCCCTCCGACCCCTATACCGCCGACCCCGACGCCCACACCGCCACCGTCGCCCATTCCTCCAACACCAACGCCCTCCCCGCCGCCGAGCCCGATTCCTCCCACGCCCACTCCTTCCCCACCCCCATCGCCTATTCCTCCAACCCCTACCCCTAGCCCGCCTCCATCACCCTCTCCACTGGGGGAACCTATGTATTACCCCTCTAACATAGACACTAATGAGGCTCTCATCAACTTCTTGCGGCCTCTCTGCGCAACTGATCGAACCAGGGCTACTTATGCGGTTCCCTGGAACTGTAACGGTATTTTCCACTGTAACTACTTTTCAATACCGTTCTATATACTGTCCTGTCATAACAATGCCTACTCATTCGTAAATGACGGCTGCATTGATTTTAATAGTTCGGACTGCCCGCTTTATCCCTTGAATGTCCTTTGA(SEQID NO:5)

[0282] Amino acid sequence of IE0:

[0283] MIRTSSHVLNVQENIMTSNCASSPYSCEATSACAEAQQLQVDTGGDKIVNNQVTMTQINFNASYTSASTPSRASFDNSYSEFCDKQPNDYLSYYNHPTPDGADTVISDSETAAASNFLASVNSLTDNDLVECLLKTTDNLEEAVSSAYYSESLEQPVVEQPSPSSAYHAESFEHSAGVNQPSATGTKRKLDEYLDNSQGVVGQFNKIKLRPKYKKSTIQSCATLEQTINHNTNICTVASTQEITHYFTNDFAPYLMRFDDNDYNSNRFSDHMSETGYYMFVVKKSEVKPFEIIFAKYVSNVVYEYTNNYYMVDNRVFVVTFDKIRFMISYNLVKETGIEIPHSQDVCNGETAAQNCKKCHFVDVHHTFKAALTSYFNLDMYYAQTTFVTLLQSLGERKCGFLLSKLYEMYQDKNLFTLPIMLSRKESNEIETASNNFFVSPYVSQILKYSESVQFPDNPPNKYVVDNLNLIVNKKSTLTYKYSSVANLLFNNYKYHDNIASNNNAENLKKVKKEDGSMHIVEQYLTQNVDNVKGHNFIVLSFKNEERLTIAKKNKEFYWISGEIKDVDVSQVIQKYNRFKHHMFVIGKVNRRESTTLHNNLLKLLALILQGLVPLSDAITFAEQKLNCKYKKFEFN(SEQ ID NO:6)

[0284] Amino acid sequence of IE1:

[0285] MTQINFNASYTSASTPSRASFDNSYSEFCDKQPNDYLSYYNHPTPDGADTVISDSETAAASNFLASVNSLTDNDLVECLLKTTDNLEEAVSSAYYSESLEQPVVEQPSPSSAYHAESFEHSAGVNQPSATGTKRKLDEYLDNSQGVVGQFNKIKLRPKYKKSTIQSCATLEQTINHNTNICTVASTQEITHYFTNDFAPYLMRFDDNDYNSNRFSDHMSETGYYMFVVKKSEVKPFEIIFAKYVSNVVYEYTNNYYMVDNRVFVVTFDKIRFMISYNLVKETGIEIPHSQDVCNGETAAQNCKKCHFVDVHHTFKAALTSYFNLDMYYAQTTFVTLLQSLGERKCGFLLSKLYEMYQDKNLFTLPIMLSRKESNEIETASNNFFVSPYVSQILKYSESVQFPDNPPNKYVVDNLNLIVNKKSTLTYKYSSVANLLFNNYKYHDNIASNNNAENLKKVKKEDGSMHIVEQYLTQNVDNVKGHNFIVLSFKNEERLTIAKKNKEFYWISGEIKDVDVSQVIQKYNRFKHHMFVIGKVNRRESTTLHNNLLKLLALILQGLVPLSDAITFAEQKLNCKYKKFEFN(SEQ ID NO:7)

[0286] Amino acid sequence of Orf146:

[0287] MNVNLYCPNGEHDNNISFIMPSKTNAVIVYLFKLDIEHPPTYNVNAKTRLVSGYENSRPININLRSITPSLNGTRGAYVISCIRAPRLYRDLFAYNKYTAPLGFVVTRTHAELQVWHILSVRKTFEAKSTRSVTGMLAHTDNGPDKFYAKDLMIMSGNVSVHFINNLQKCRAHHKDIDIFKHLCPELQIDNSVVQLETRSS(SEQ ID NO:8)

[0288] Amino acid sequence of Lef2:

[0289] MANASYNVWSPLIRASCLDKKATYLIDPDDFIDKLTLTPYTVFYNGGVLVKISGLRLYMLLTAPPTINEIKNSNFKKRSKRNICMKECVEGKKNVVDMLNNKINMPPCIKKILNDLKENNVPRGGMYRKRFILNCYIANVVSCAKCENRCLIKALTHFYNHDSKCVGEVMHLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY(SEQ ID NO:9)

[0290] Amino acid sequence of Orf91:

[0291] MWYIIVIIIIIILNLFIIILLTLKILDDIQELYPNPPIVPPPTSPPIVPLPTPPPTPTPSPPSPTPPPTPIP

[0292] PTPTPTPPPTPIPPTPTPTPPPSPIPPTPTPSPPPSPIPPTPTPSPPPSPIPPTPTPSPPPSPSPLGEPMY

[0293] YPSNIDTNEALINFLRPLCATDRTRATYAVPWNCNGIFHCNYFSIPFYILSCHNNAYSFVNDGCIDFNSSDCPLYPLNVL(SEQ ID NO:10)

[0294] Primer sequences for amplifying the IE1 promoter:

[0295] KZ-IE1PRO-F: TTCAATCATGGTGGCATGAACGTCAATTTAT(SEQ ID NO:11)

[0296] KZ-IE1PRO-R: CGCAGAAGCGCGTCGACCGATGTCT(SEQ ID NO:12)

[0297] Primer sequences for amplifying the NeoR sequence and SV40 polyA sequence:

[0298] KZ-NEO-F: GAGGGGATCGAGCCCCTTCCTTGCTGTACTGGTACCC(SEQ ID NO:13) KZ-NEO-R: GCCACCATGATTGAACAAGATGG(SEQ ID NO:14)

[0299] Sf-RV RNA-dependent RNA polymerase gene common sequence

[0300] 5'TTTATGGTGAGTTTGGATTTTTCCAAGTGGAACACCAATATGAGAGCCCCAGACACAC AGCCATTTTACCACACTATAGATACGATGTTTGGTTTGGAAAATTGTTTTACCAGGACACATGAAATGTTCTACAATTCCTTTTGTACCTTATAGACGGTTCTTATCTCCCAACAATAGTTGATGATGGGTTCAAAACAGATATTGGATGTTGGCGAC ATCATCTTGGGGGAATCGAAGGTCTCAGACAAAAAGGATGGACTCTGTGGACAGTTATGTTGATCAGGCTAGTTGCGGAAAAATATATTTTCAATATGTCTATCATGGGACAGGGGGACAATCAAATGCTACTTCTAACTTTCGATTCTAATA3'(SEQ ID NO.15)

[0301] Primers and probes for amplifying the common sequence of the Sf-RV RNA-dependent RNA polymerase gene: GQP-4-FTCGAAGGTCTCAGACAAAAAGGA (forward primer) (SEQ ID NO.16)

[0302] GQP-4-R TTCCGCAACTAGCCTGATCA (reverse primer) (SEQ ID NO.17)

[0303] GQP-4-P FAM-ACTCTGTGGACAGTTATG-MGB (probe) (SEQ ID NO.18)

[0304] Negative strand RNA sequence:

[0305] 5'aaauauauuuuuccgcaacuagccugaucaacauaacuguccacagaguccauccuuuuugucugagaccuucgauuccccccaa g3' (SEQ ID NO. 19).

Claims

1. Genetically modified insect cell lines containing nucleotide sequences encoding baculoviruses IE0 / IE1, Orf146, Lef2, and Orf91. For example, the baculovirus mentioned belongs to the baculoviruses selected from Alpha baculovirus, Beta baculovirus, Gamma baculovirus, and Deltabaculovirus. For example, the nucleotide sequence encoding baculovirus IE0 / IE1 comprises the nucleotide sequence of SEQ ID NO:1, or comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; the nucleotide sequence encoding baculovirus Orf146 comprises the nucleotide sequence of SEQ ID NO:3, or comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; the nucleotide sequence encoding baculovirus Lef2 comprises the nucleotide sequence of SEQ ID NO:4, or comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4; the nucleotide sequence encoding baculovirus Orf91 comprises the nucleotide sequence of SEQ ID NO:5, or comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4; NO:5 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity; Preferably, the nucleotide sequences encoding baculovirus IE0 / IE1, Orf146, Lef2, and Orf91 are effectively linked to regulatory elements that express the nucleotide sequences in insect cells. For example, one or more nucleotide sequences encoding baculovirus IE0 / IE1, Orf146, Lef2, and Orf91 are under the control of the baculovirus IE1 promoter, the baculovirus IE0 promoter, the baculovirus gp64 promoter, or the silkworm actin 3 (A3) cell promoter.

2. The genetically modified insect cell line according to claim 1, wherein the insect cell line is derived from lepidopteran insects, such as Spodoptera frugiperda, Trichoplusia ni, or Bombyx mori, for example, the genetically modified insect cell line is a genetically modified Sf9 cell, a genetically modified Sf21 cell, or a genetically modified High Five cell.

3. The genetically modified insect cell line according to claim 1 or claim 2, wherein the genetically modified insect cell line expresses baculoviruses IE0, IE1, Orf146, Lef2, and Orf91. For example, the baculovirus IE0 comprises or is composed of the following amino acid sequence: (a) The amino acid sequence of SEQ ID NO:6; (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:6 and having baculovirus IE0 activity; The baculovirus IE1 contains or is composed of the following amino acid sequence: (a) The amino acid sequence of SEQ ID NO:7; (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:7 and having baculovirus IE1 activity; The baculovirus Orf146 comprises or is composed of the following amino acid sequence: (a) The amino acid sequence of SEQ ID NO:8; (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:8 and having the activity of baculovirus Orf146; The baculovirus Lef2 contains or is composed of the following amino acid sequence: (a) The amino acid sequence of SEQ ID NO:9; (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:9 and having the activity of baculovirus Lef2; The baculovirus Orf91 contains or is composed of the following amino acid sequence: (a) The amino acid sequence of SEQ ID NO:10; (b) An amino acid sequence having at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with SEQ ID NO:10 and having the activity of baculovirus Orf91.

4. A method for screening genetically modified insect cell lines free from rhabdovirus contamination, characterized in that, Includes the following steps: 1) Preparation of cell suspension: A cell suspension is prepared after culturing genetically modified insect cells according to any one of claims 1 to 3 contaminated with a Rhabdovirus in a basal serum-free insect cell culture medium supplemented with 10% FBS, wherein the basal serum-free insect cell culture medium is selected from the group consisting of ESF AF TM Grace's insect cell culture medium, ESF 921 medium, Sf900 II SFM medium and Sf 900 III SFM medium; 2) Preparation of monoclonal screening culture medium: Monoclonal screening culture was prepared using a first basic serum-free insect cell culture medium of 60% supplemented with whey protein hydrolysate and Yeastolate, a second basic serum-free insect cell culture medium of 20% supplemented with FBS and 20% supplemented with FBS. The first basic serum-free insect cell culture medium supplemented with whey protein hydrolysate and Yeastolate was Grace supplemented insect cell culture medium, and the second basic serum-free insect cell culture medium was Sf 900II SFM medium. 3) Dilution and plating of cell suspensions The cell suspension obtained in Step 1 was centrifuged and the cells were washed several times with basal serum-free insect cell culture medium to remove the culture fluid containing the rhabdovirus; then the freshly prepared monoclonal screening culture fluid in Step 2 was taken and the cells were diluted to 0.1 x 10 4 6 cells / ml - 1 x 10 6 6 cells / ml, and then gradient diluted to 25 - 100 cells per milliliter, inoculated in the culture plate at 1 - 10 cells per well, and placed in a constant temperature incubator at 27 - 28°C for static culture. The cell suspension obtained in Step 1 was centrifuged and the cells were washed several times with basal serum-free insect cell culture medium to remove the culture fluid containing the rhabdovirus; then fresh monoclonal selection culture fluid prepared in Step 2 was taken and the antiviral compound was added to a final concentration of 10-50 ng / ml, the cells were diluted to 0.1 x 10 4 6 cells / ml - 1 x 10 6 6 cells / ml, then further diluted in gradient to 25-100 cells per ml, seeded in culture plates at 1-10 cells per well and left to stand in an incubator at 27-28°C, wherein the antiviral compound is ribavirin; 4) Cell culture, rhabdovirus detection, and cell passage From the second to the tenth day after plating, the wells were observed daily to identify single-cell wells. The growth of the monoclonal cells obtained through limiting dilution was observed regularly, and rhabdovirus was detected. Based on the cell growth and rhabdovirus detection results, the medium in the wells was changed or expanded for culture and passage. The medium was changed to the monoclonal selection medium corresponding to the cells in each well or monoclonal selection medium with the addition of the antiviral compound ribavirin at a final concentration of 10-50 ng / ml. 5) Expand the culture of cells free from rhabdovirus contamination. Cells that were initially free of rhabdovirus contamination obtained in step 4 were cultured in monoclonal screening medium corresponding to the cells in each well plate, or monoclonal screening medium with ribavirin added to a final concentration of 10-50 ng / ml; rhabdovirus detection was performed to obtain genetically modified insect cell lines free of rhabdovirus contamination.

5. The method for screening genetically modified insect cell lines free from rhabdovirus contamination according to claim 4, wherein, In step 3, the serially diluted cells are seeded into 96-well culture plates at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells per well and placed in a constant temperature incubator at 27°C, 27.5°C or 28°C for static culture.

6. The method for screening genetically modified insect cell lines free from rhabdovirus contamination according to claim 4, wherein, The rhabdovirus detection is performed using a method selected from reverse transcription-polymerase chain reaction, RT-PCR combined with nested PCR, quantitative PCR, RT-PCR combined with quantitative PCR, probe hybridization, electron microscopy, antibody-based detection, plaque assay and cytopathic effect assays, and bioinformatics techniques.

7. A method for domesticating genetically modified insect cell lines free from rhabdovirus contamination, characterized in that, The genetically modified insect cell lines free from rhabdovirus contamination, obtained by the screening method for rhabdovirus-free genetically modified insect cell lines according to any one of claims 4-6, are cultured until the cell adhesion reaches 60% or more, then subjected to trypsin digestion to prepare a cell suspension; then, a suspension is prepared using a solution supplemented with Pluronic acid. TM The F-68 serum-free insect cell culture medium was used for suspension culture in a shaking incubator at 27-28°C and 100-130 rpm. The suspension-cultured cells were then stably cultured for 4-20 generations to obtain a serum-independent, rhabdovirus-free, genetically modified suspension insect cell line, for example, one containing Pluronic acid. TM The basic serum-free insect cell culture medium for F-68 is Vigor-S101 medium.

8. The method for domesticating genetically modified insect cell lines free from rhabdovirus contamination according to claim 7, wherein, Suspension culture was carried out at 27℃, 27.5℃ or 28℃ and 100-130 rpm; the suspended cells were cultured stably for 4-20 generations to obtain a genetically modified suspension insect cell line that was independent of serum and free from rhabdovirus contamination.

9. A genetically modified insect cell line free from rhabdovirus contamination, obtained by the screening method for genetically modified insect cell lines free from rhabdovirus contamination as described in any one of claims 4-6 and the domestication method for genetically modified insect cell lines free from rhabdovirus contamination as described in claim 7 or 8. Preferably, it is a genetically modified Sf9 cell line free from rhabdovirus contamination. Most preferably, it is named Sf9-MD768-3C7 cell line, with the biological accession number CGMCC No. 46339.

10. The genetically modified insect cell line free from rhabdovirus contamination according to claim 9, having one or more of the following characteristics: (1) On day 5 after infection with recombinant baculovirus, maintain a high cell density and cell viability, for example, a cell density of about 1E+6 cells / ml to about 4E+6 cells / ml and a cell viability of about 50% to 70%. (2) It has low oxygen consumption and low sugar consumption during cell culture; (3) When used in baculovirus-insect cell systems, the packaged recombinant AAV vector is more distributed intracellularly, has a high titer, for example, 1E+11vg / mL to 2E+11vg / mL, has an increased VP1 protein ratio, for example, VP1:VP2:VP3 content of about 1:1:10, and has enhanced biological activity, for example, enhanced biological activity in serum, heart, liver, lung, brain and spinal cord, and reduced nephrotoxicity.

11. The use of the genetically modified insect cell line free from rhabdovirus contamination according to claim 9 or 10, characterized in that, Used for recombinant protein production or AAV viral vector production.

12. The use of the genetically modified insect cell line free from rhabdovirus contamination according to claim 9 or 10, characterized in that, In insect cell-baculovirus expression vector systems, preferably, the produced recombinant AAV vector is more widely distributed within cells, has a high titer, for example, 1E+11vg / mL to 2E+11vg / mL, has an increased VP1 protein ratio, for example, VP1:VP2:VP3 content of approximately 1:1:10, and has enhanced biological activity, for example, enhanced biological activity in serum, heart, liver, lung, brain and spinal cord, and reduced nephrotoxicity.

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