1.Preparation Of Strain D2 Protease From Perinereis Aibuhitensis Grube Digestive Tract And Preliminary Study On Its Anti-tumor Activity 2.Construction Of Recombination Plasmid PEGFP-N3-c-myc

Objective The present study was conducted to cultivate protease-hyperproducing strain from the digestive tract of Perinereis aibuhitensis Grube,and to purify the sought-for microbial extracellular protease as well as analyze its anti-tumor activity.Methods The fermentation broth of strain D2 was used to separate and purify protease.Sought-for protease was purified to homogeneity by a stepwise procedure including centrifugation,ammonium sulfate precipitation,ion-exchange chromatography and size-exclusion chromatography.RPMI 1640 was utilized to dissolve the purified protease.Five concentration gradients were prepared:0.2mg/L, 2mg/L,20mg/L,200mg/L and 2000mg/L.0.0365~365mg/L of paclitaxel/RPMI 1640 were used as positive control.The target tumor cells were mouse breast cancer cell line EMT6 cells.And the human embryonic lung cells were normal control. EMT6 or HELC cells without D2 Protease or PTX were used as negative group. Solution D2 protease and paclitaxel were added to target cells and normal control cells respectively for five different times and to observe their effects on the cells. MTT assay was used to detect OD and the rates of cell proliferation inhibition were calculated.Then the rates were compared between the exprimental group and the positve group or the normal group.Results The four-step purification protocol resulted in a 4.9 fold purification with 33%activity recovery.The ODs of the experiment group,positive group and normal cells group were all lower than that of the negative control group.Compared with the experiment group,the cell proliferation inhibition rates of the normal control group were lower(P<0.05) in dose-dependent and time-dependent manner.Conclusion D2 protease has inhibition effect on proliferation of EMT6 cells and shows antineoplastic activity in some degree. Objective To construct recombination plasmid pEGFP-N3-c-myc,which can express c-myc with fluorescent labeling EGFP after transfecting eukaryocyte,for the basis of following study;Methods The oncogene c-myc was obtained from SKOV3 by RT-PCR,and the recombination plasmid pEGFP-N3-c-myc was constructed with the combination of c-myc with expression vector pEGFP-N3 that underwent restriction digest.pEGFP-N3-C-myc was identified by sequencing.Results Agarose gel eletrophoresis:the length of the PCR product was in accordance with the target gene.Recombinant plasmid pEGFP-N3 was validated by restriction digestion with EcoRâ… and BamHâ… .DNA sequencing showed that point mutation happened in the c-myc segment,but the function of the c-myc protein was formal.Conclusion We construct the recombination plasmid of pEGFP-N3-c-myc,which can express c-myc with fluorescent labeling EGFP after transfecting eukaryocyte.