The Research Of Tumor Vaccine Targeting Dll4Protein Mediated By Baculovirus

Dll4is a ligand of VEGF,which play a significant role in angiogenesis. It is report that Dll4have be detected over expression in majority kinds of cancer，such as breast cancer，gastriccancer，lung cancer. When blockade Dll4function results in hyper sprouting due to the loss oflateral inhibition of the endothelial tip cell phenotype mediated through Notch1. As aconsequence, an excessive, but nonproductive, angiogenic response is mounted, characterized bypoor perfusion that results in hypoxia. The research aim to use baculovirus induce to produceanti-Dll4to blockade Notch Dll4pathway and finally inhibate tumor growth.Generate five kinds of baculovirus including:Bmgp64Dll4,Bmcmv-Dll4-VSVG,BmCMV-Dll4-DAF，BmCMV-EGFP-VSVG，BmCMV-EGFP-DAF. We amplified human Dll4gene and signaol Pepitide, transmembrane area of gp64with PCR then spliced with overlapextension PCR. We clone the SP-Dll4-TM into pFastBac1between BamH1and Xhol1clonesite,Transformating pPastBac1gp64Dll4into DH10Bac,choosing white spot culture andextracting Bacmid genome, using liposome transfect into BmN cell. After six days, cell loatobviously. extract viral genome, PCR identification. Then transfer the virus in the bottle ofsilkworm cell, after three days,12000RPM centrifugal30min, collect the cells and supernatant,50000RPM speed centrifugal45min, with PBS suspending precipitation,30%and60%sucrosedensity gradient centrifugation, collecting zone, take off sugar, take a small amount of samplepreparation, Western Bloting appraisal. After taking off sugar, quantitate each100μ g proteinimmune c57bl/6mice every seven days, continuous immune three times.the second day afterthe last immune. Inoculate lewis lung cancer cells in mouse. Every two day inspecte and recordtumor growth situation. After tumor12days later, on laboratory mice eyes take blood and pullneck put to death, occlusion, measuring volume will be for the serum with elisa detection Dll4antibody titer after testing, in10μ g/ml antigen bag is under the condition of the vaccine miceserum dll4valence for1:3200comparing with control group, the vaccine immune group oftumor size is about half of the control group.Bm-CMV-Dll4-VSVG, Bm-CMV-Dll4–DAF, Bm-CMV-EGFP-VSVG, BmCMV-EGFP-VSVG constructed with similar method of Bmgp64Dll4。 We use the BmCMV- Dll4-VSVG, BmCMV-EGFP–DAF three virus in vitro transducte mammalian cells293T,all can be expressed in mammalian cells Dll4protein. Which use BmCMV-EGFP-DAFtransduct293T cells in different titer. The baculovirus of transduction efficiency is proportionalto the multiplicity of infection, when multiplicity of infection more than100can also generategood transduction effect.