An Endophytic Fungus From Camptotheca Acuminate That Produce CPT And Research Of Biology Of CPT

A total of 80 isolates of endophytic fungi were isolated from the Camptothecaacuminate in Cartagena territory Gui Zhou province in this study. Through sterilizing thesurface of the tissue of Camptotheca acuminate, we screen endophytic fungi frombranch, leaf, fruit, root. Most of them from upground tissue are abundant in kinds.Compound CPT was identified by means of TLC, HPLC and electric spray massspectrometry methods. Among the 80 strains endophytic fungi, one strain S19 producedthe anticancer drug lead compound CPT. We describe the feature of S19 based on themorphological characteristics of the colony and conidia of the fungus. The S19 wasbelonged to Fusarium family. Total DNA was isolated from the mycelial mass.ThoughAmplification and Clone, we give the final alignment. S19 belongs to the specie ofFusarium Solani, through indification of feature and molecular methods.The cultured hypha was moved to liquid PDA and cultured at 29℃. The maximumyield of camptothecin was 7.4±0.48ug/lg of dry cell mass on day 4 in shake flasks. Themaximum yield of camptothecin was 8.96±0.34mg in per liter liquid fungal culture onday 5.We have researched CPT from S19 for cells. After 24 hours, the typical apoptoticmanifestations observed in adherent cells including cell shrinkage and membraneblebbing were evaluated. So we guass the CPT can make the vero cells apoptotised.Fragmentation of cell nuclei in response to camptothecin treatment. Cells were stainedwith AO/EB. The apopotosis rate were 13%. Time and concentration-dependent apoptoticratio in VERO cells following incubation with campothecin.To investigate the apoptotic mechanisms, we used antibody Caspase-3 forWestenblot after extracting the expression protein from PC3 cells which treated withCPT in 48 hours. The Caspase-3 about 35KD did have a significant expression inWestenblot assay. So we can concluded that apoptotic mechanisms of CPT from S19to PC3 cells for apoptosis was follow the way of Caspase-3.