Ding Wei-Feng, Liu Zhi-Gang, Sun Na, Zhang Xin, Li Xian, Feng Ying
Research Institute of Resource Insects, Chinese Academy of Forestry, The Key Laboratory of Cultivating and Utilization of Resource Insects of State Forestry Administration, Kunming, 650233, Yunnan, China.
In Vitro Cell Dev Biol Anim. 2020 Jun;56(6):444-451. doi: 10.1007/s11626-020-00484-z. Epub 2020 Jul 31.
In this paper, four established cell lines derived from newly hatched larvae of Papilio demoleus Linnaeus and 57 single-cell clones derived from the 3 lines were used as test materials. Recombinant β-galactosidase baculovirus AcMNPV-Gal was used to infect the P. demoleus L. cell lines and the single-cell clones, and recombinant protein expression in each cell line was detected and compared. Three clonal cell lines, RIRI-PaDe-1-C1, RIRI-PaDe-2-C6 and RIRI-PaDe-3-C52, which showed significantly higher β-galactosidase expression levels than those of the parental cell lines, were selected. Five types of commercial serum-free media for insect cells, Express Five SFM, Ex-Cell 405, Sf-900III SFM, Sf-900II SFM and HyClone Serum-Free Media, were used to adapt RIRI-PaDe-2-C6 cells and RIRI-PaDe-3-C52 cells to serum-free culture conditions, and the growth characteristics of the cells and the exogenous protein expression characteristics before and after adaptation were compared. The results showed that RIRI-PaDe-2-C6 cells could stably proliferate in Ex-Cell 405, RIRI-PaDe-3-C52 cells could stably proliferate in Express Five SFM and Ex-Cell 405, and the rate of proliferation of and the level of expression of β-galactosidase in RIRI-PaDe-3-C52 cells were significantly increased in Express Five SFM.
本文以4种来源于柑橘凤蝶初孵幼虫的已建细胞系及其中3个细胞系的57个单细胞克隆为试验材料。用重组β-半乳糖苷酶杆状病毒AcMNPV-Gal感染柑橘凤蝶细胞系和单细胞克隆，检测并比较各细胞系中重组蛋白的表达情况。筛选出3个克隆细胞系RIRI-PaDe-1-C1、RIRI-PaDe-2-C6和RIRI-PaDe-3-C52，其β-半乳糖苷酶表达水平显著高于亲本细胞系。用5种昆虫细胞商业无血清培养基Express Five SFM、Ex-Cell 405、Sf-900III SFM、Sf-900II SFM和HyClone无血清培养基对RIRI-PaDe-2-C6细胞和RIRI-PaDe-3-C52细胞进行无血清培养条件适应性培养，比较细胞适应前后的生长特性和外源蛋白表达特性。结果表明，RIRI-PaDe-2-C6细胞能在Ex-Cell 405中稳定增殖，RIRI-PaDe-3-C52细胞能在Express Five SFM和Ex-Cell 405中稳定增殖，且RIRI-PaDe-3-C52细胞在Express Five SFM中的增殖速率和β-半乳糖苷酶表达水平显著提高。
