Study On Transfected P～(35) Gene Insect Cell Lines

In order to generate excellent transfected p³⁵ gene cell lines,we have cloned the cells in single-cell cloning way,and the transfected cells was firstly selected in G418-containing medium,We obtained 45 clones from the first selection after cell clone,after several passages, 37 of cell clones were examined for their resistance to nutritional stress in PBS and AMD(1 u g/mL). 10 clones which appeared strong resistance to PBS or AMD and high products of OBs were obtained,finally one of the ten clones can support the replication of three viruses: TnNPV, MsNPV PiNPV and AcMNPV and was named as Tn5B-P35(Tn5B-35). The morphology of Tn5B-35, grown in TNM-FH medium, generally consisted of spindle cells with presence of some round cells,the combined size of these cells was 18.2 ×41.0 m. The Tn5B-35 cell line reached maximum density of 2.2 ×10⁶cells/mL at four days and the population doubling time is 22h. Wild-type AcMNPV infected the Tn5B-35 cells with an infection of 95% of the cells and the production of occlusion bodies (OBs) by it was high, average of 115 OBs/cell. Tn5B-35 cells showed high resistance to nutrient stress in comparison with parental cell line Tn5Bl-4 when cells were exposed to PBS,cell viability was 86% with Tn5B-35 after one day in PBS. 5 days later, 42% Tn5B-35 cells were survived but Tn5Bl-4 died within five days.When the cells were exposed to Actinomycin D concentrations of 0.31, 0.62, 1.25 and 2.5μg/mL for one hour, cell viability for Tn5B-35 was more than Tn5Bl-4 cells,at a concentration of 5μg/ml that all the Tn5Bl-4 cells were killed, but 52% of Tn5B-35 cells were still viable,it was demonstrated that Tn5B-35 cells were resistant to apoptosis induced by Actinomycin D. The first serial passage of AcMNPV, which was obtained from the hemolymph of infected larvae, was established following infection of the cells at a MOI=10. AcMNPV was replicated in Tn5B-35 cells for 25 passages continuously and there was no significant change in morphology of the occlusion bodies, OBs production and virus titer after serial passage of the virus. Bioassay was performed using OBs generated from different viral passage to examine the relationship between mortality and passage number. Under the same OBs concentration, the AcMNPV produced constant larval mortality. The cell line was transformed with p³⁵ using cellfecin reagent. Cell clones of transformed gene were obtained by screening with G418. To confirm the clones with p³⁵, PCR analysis was carried outthe result shew that PCR product of Tn5B-35 cell gene was anticipatory 0.7 kb instead of Tn5Bl-4 cell gene,as same as of pIZ/V5-His/p35.It proved that Tn5B-35 cell included the/5 gene.