Characteristics Of Cell Lines Of Lepidopteran Insects And Expression Of Recombinant Protein By New Cell Clones Derived From Trichoplusia Ni (qb-tn9-4s)

The development of insect cell lines have been played a significant role in research and application of virology, medicine, agriculture, and biology since the first insect cell line was established in 1962. Lepidopteran insect cell lines are used broadly in insect virology research and recombinant protein expression. With the advent of the baculovirus-insect cell expression system a new exciting application for insect cells in biotechnology was realized. Insect cell lines have become more important as a new bioreactor for the production of virus biopesticides and recombinant proteins. In this study establishment of two new cell lines from two important agricultural pest insects, Helicoverpa armigera and Mythimna separata, identification of the biological characterization, screening of cell clones with higher production of virus and recombinant protein from Trichoplusia ni cell line QB-Tn9-4s were performed. These rerults can provide new resources in studies of apoptosis, biochemical mechanisms and virology, and the basis for potential application and development of insect cell lines.1. Two new cell lines, QB-Ha-E-1 and QB-Ha-E-5, were established from the embryonic tissue of Helicoverpa armigera (Lepidoptera: Noctuidae). The cell lines have been subcultured over 60 passages in TNM-FH medium supplemented with 10% FBS. Each cell line consists of two major morphological types: round cells and spindle-shaped cells. The analyses of DNA amplification fingerprinting (DAF) and random amplified polymorphic DNA (RAPD) showed similar DNA profiles between the two cell lines and the embryonic tissue of Helicoverpa armigera. Two cell lines had a typical lepidopteran chromosomes pattern. The cell doubling times of QB-Ha-E-1 and QB-Ha-E-5 were 63.7 h and 66.9 h at the 30th passage, respectively, and they can be infected by Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) with infection rates of 86.6% and 56.5%, respectively, at 4 d postinfection (p.i.). Approximately 15% of both cell lines were infected by Mamestra brassicae nucleopolyhedrovirus (MbNPV) at 7 d p.i.. Two cell lines had differential responses to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection. The results from the fluorescent staining with DAPI and electrophoresis of genomic DNA indicated that QB-Ha-E-5 cell line had typical apoptotic response following AcMNPV infection, but QB-Ha-E-1 cell line with AcMNPV infection rate of 55.3% appeared anti- apoptosis. Actinomycin D could induce apoptosis of the both cell lines at the concentrations of 1.25μg/mL.2. Four cell lines, QB-Ms-E-1, QB-Ms-E-2, QB-Ms-E-3, and QB-Ms-E-4,were established from the embryonic tissue of Mythimna separata (Lepidoptera: Noctuidae). The cell lines have been subcultured over 50 passages in TNM-FH medium supplemented with 10% fetal bovine serum (FBS). Four cell lines consist of two major morphological types: round cells and spindle-shaped cells. The DAF and RAPD analyses indicated similar DNA profiles between the four cell lines and the embryonic tissue of Mythimna separata. The four cell lines showed a typical chromosomes pattern of lepidopteran insect. The cell population doubling times of QB-Ms-E-1, QB-Ms-E-2, QB-Ms-E-3, and QB-Ms-E-4 were 44.9 h, 46.6 h, 46.5 h, and 47.1 h, respectively, and they can be infected by Mythimna separata nucleopolyhedrovirus (MsNPV) with infection rates of 56.6%, 53.6%, 49.3%, and 62.5%, respectively, at 7 d p.i.. Less than 5% of four cell lines were infected by Mamestra brassicae nucleopolyhedrovirus (MbNPV) at 10 d p.i.. Four cell lines were highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with infection rates of over 90% at 4 d p.i..3. Nine cell clones were successfully derived from the established Trichoplusia ni cell line QB-Tn9-4s and designated QB-Tn-Ⅰ, QB-Tn-Ⅱ, QB-Tn-Ⅲ, QB-Tn-Ⅳ, QB-Tn-Ⅴ, QB-Tn-Ⅵ, QB-Tn-Ⅶ, QB-Tn-Ⅷ, and QB-Tn-Ⅸ, respectively. RAPD analysis of the genomic DNA of these clones confirmed their genetic identity as their parent cell line QB-Tn9-4s. Each clone was highly susceptible to AcMNPV virus with infection rates of over 87% cells with production of around 70-87 OBs per cell. The clone QB-Tn-Ⅰproduced 86.7±1.5 OBs per cell, which was higher than BTI-Tn-5B1-4, QB-Tn9-4s and Sf-9. The expression ofβ-galactosidase (β-gal) was determined and compared at 6 d p.i., the clone QB-Tn-Ⅴshowed the highest expression level (3.06×104 IU/mL), closely followed by QB-Tn-Ⅰ(2.70×104 IU/mL), both cells were higher than BTI-Tn-5B1-4, QB-Tn9-4s and Sf-9 cells. The expression of secreted alkaline phosphatase (SEAP) at 7 d p.i. clone QB-Tn-Ⅴwas highest (2.96 IU/mL) among the cell lines, followed by QB-Tn-Ⅰ(2.41 IU/mL), both cells were higher than BTI-Tn-5B1-4, QB-Tn9-4s and Sf-9 cells.4. The cell line QB-Tn9-4s and its clones QB-Tn-Ⅰand QB-Tn-Ⅴwere successfully adapted to a serum-free medium, Sf-900Ш, but one clone QB-Tn-Ⅴwas successfully adapted to a serum-free medium, EX-CELL 420, only. Their morphology and size in serum-free medium differed greatly from serum-containing medium, the results of growth kinetics showed that their cell population doubling times in Sf-900Шwere shorter than the cultures in serum-containing medium TNM-FH, the densities of all cells in Sf-900Шwere higher also than the cultures in serum-containing medium. But the clone QB-Tn-Ⅴgrew slower in EX-CELL 420 medium, its cell population doubling time was longer, and the density was lower than in serum-containing medium. The cell population doubling times of QB-Tn9-4s and its clones QB-Tn-Ⅰand QB-Tn-Ⅴin serum-containing medium were 27.4 h, 28.0 h, and 27.0 h, respectively, and in Sf-900ШSFM were 24.1 h, 26.1 h, 25.4 h, respectively, the doubling times of QB-Tn-Ⅴin EX-CELL 420 medium was 28.8 h. The maximum densities of QB-Tn9-4s and its clones QB-Tn-Ⅰand QB-Tn-Ⅴin Sf-900ШSFM were higher than in serum-containing medium (TNM-FH) by factors of 1.8, 1.8, and 1.7-fold, respectively.The virus infection rate of QB-Tn-Ⅰwas 80.3% with production of 33.9 OBs per cell in Sf-900ШSFM, the remainder of the cell lines were highly susceptible to AcMNPV with an infection rates of over 90% cells with production of around 81-87 OBs per cell, no significant difference were observed between in both Sf-900ШSFM and serum-containing medium TNM-FH.The expression ofβ-galactosidase (β-gal) of QB-Tn9-4s, QB-Tn-Ⅰ, QB-Tn-Ⅴand BTI-Tn5B1-4 in Sf-900ШSFM medium was higher than in serum-containing cultures, the clone QB-Tn-Ⅴexpressed the highest level (3.27×104 IU/mL) among these cell lines in Sf-900ШSFM medium at 6 d p.i.. The expression of secreted alkaline phosphatase (SEAP) of QB-Tn9-4s, QB-Tn-Ⅰ, QB-Tn-Ⅴand BTI-Tn5B1-4 in Sf-900ШSFM medium was higher than in serum-containing cultures also, BTI-Tn5B1-4 showed the highest expression level (4.43 IU/mL) in Sf-900ШSFM medium at 7 d p.i., closely followed by QB-Tn-Ⅴ(4.30 IU/mL) in Sf-900ШSFM medium. Whereas the expression levels ofβ-gal and SEAP of QB-Tn-Ⅴin EX-CELL 420 medium were lower than in Sf-900ШSFM and serum-containing medium TNM-FH.