Construction Of Recombinant Oncolytic Vaccinia Virus And In Vitro Killing Effect On Pancreatic Cancer Cells

As early as a century ago, people have begun to try to use the virus to treat cancer. Oncolytic virus refers to targeting killing tumor cells without harming normal tissue virus. At the same time, the oncolytic viruses to the current use of antineoplastic therapy does not produce antagonistic effect, more suitable for combined drug use. Now often on genetically modified vaccinia virus construct targeting strong tumoricidal ability and strong safety oncolytic poxvirus.This whole study includes two parts.1. Construction of recombinant oncolytic vaccinia virus vvDD-GFP-DsRed and in vitro killing effect on pancreatic cancer cellsObjective To successfully construct thymidine kinase and vaccinia growth factor gene deletion while expressing double fluorescent DsRed and GFP oncolytic vaccinia virus,and to explore the effect of virus on the cell lysis of BxPC-3 and Patu8988 in vitro.Methods The DsRed gene and GFP gene were inserted into the TK gene of the pox virus by homologous recombination method, and cloned into the VGF gene deletion virus VSC20. Successfully constructed the VGF and TK double gene deletion of oncolytic vaccinia virus vvDD-GFP-DsRed. The lytic effect of recombinant virus on pancreatic cancer cells BxPC-3 and Patu8988 was detected by CCK-8 and Crystal violet stain.Results Using the method of homologous recombination and soft agar screening method to obtain high purity recombinant oncolytic vaccinia virus vvDD-GFP-DsRed. The results of CCK-8 method showed that the cell lytic effect of recombinant virus vvDD-GFP-DsRed on pancreatic cancer cells BxPC-3 and Patu8988 was positively correlated with MOI value and time (P<0.05). Compared with MOI=0, the survival rate of Patu8988 in MOI=0.01 was low (P<0.05), and there was an obvious plaque in the Crystal violet staining.Conclusion The successful construction of the wDD-GFP-DsRed recombinant oncolytic poxvirus has obvious lytic effect on BxPC-3 and Patu8988.2. Construction of recombinant oncolytic vaccinia virus vvDD-EphA2xCD3-DsRed and in vitro killing effect on pancreatic cancer cellsObjective To successfully construct carrying EphA2xCD3 gene while expressing fluorescent DsRed and thymidine kinase and vaccinia growth factor gene deletion oncolytic vaccinia virus,and to explore the effects of PBMC cell mediated virus on the cell lysis of BxPC-3 and Patu8988 in vitro.Methods The EphA2xCD3 gene and DsRed gene were inserted into the TK gene of the pox virus by homologous recombination method, and cloned into the VGF gene deletion virus VSC20. Successfully constructed the carrying EphA2xCD3 gene while expressing fluorescent DsRed, VGF and TK double gene deletion of oncolytic vaccinia virus vvDD-EphA2xCD3-DsRed. The expression of EphA2 in pancreatic cancer cells BxPC-3 and Patu8988 was detected by RT-PCR method; The lytic effect of PBMC cell mediated recombinant virus on pancreatic cancer cells BxPC-3 and Patu8988 was detected by CCK-8 and Crystal violet stain. At the same time, when MOI respectively 1,0.1,using ELISA kits to detect the expression of IL-2 and IFN-γ in supernatant fluid that were collceted by Infection of recombinant virus containing PBMC cells 24 h,48 h,72 h.Results Using the method of homologous recombination and soft agar screening method to obtain high purity recombinant oncolytic vaccinia virus vvDD-EphA2xCD3-DsRed. EphA2 were highly expressed in pancreatic cancer cells in BxPC-3 and Patu8988.The results of CCK-8 method showed that the cell lytic effect of recombinant virus vvDD-EphA2xCD3-DsRed on pancreatic cancer cells BxPC-3 and Patu8988 was positively correlated with MOI value and time (P<0.05). Compared with non PBMC cell group, the survival rate of BxPC-3 and Patu8988 infected with recombinant virus containing PBMC cells was low (P<0.05). Compared with MOI=0, the survival rate of Patu8988 in MOI=0.01 was low (P< 0.05), and there was an obvious plaque in the Crystal violet staining. The results of ELISA showed that PBMC cell co-cultured with Patu8988 cell,PBMC cell infected with wDD-EphA2xCD3-DsRed expressed IL-2 and IFN-γ, and PBMC cell infected wDD-GFP-DsRed did not express IL-2 and IFN-γ.Conclusion The successful construction of the vvDD-EphA2xCD3-DsRed recombinant oncolytic poxvirus has obvious lytic effect on BxPC-3 and Patu8988. And the lytic effect of recombinant virus on BxPC-3 and Patu8988 was increased after the addition of PBMC cells.