JSRV-Env Eukaryotic Expression Products Interact With In Vitro Cells And The Impact On Key Enzyme In Signaling Pathways

Background:Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumour in sheep caused by jaagsiekte sheep retrovirus (JSRV). Env is an oncogene of JSRV, Hyaluronoglucosaminidase 2 (Hyal-2) is the receptor of JSRV. When Env transformed NIH 3T3 cells the cytoplasmic tail (CT) of Env directly activate the signaling pathways, However when Env transformed epithelial cells, at first it combined with Hyal-2, and then activate of downstream signaling pathways.Objective:Hyal-2 is the receptor of JSRV Env, so the research of interacte between Hyal-2 and Env interaction is significant for JSRV tumorigenic mechanism.Methods:1. The pDsRed-monomer-Nl-Hyal-2 eukaryotic expression vector was transfected TC-lcells to construct the cell line stable expressed ovine Hyal-2, which was screened and purified by G418. Use to verify the stability of Hyal-2 in the TC-1 cells by RT-PCR method and Western blot method. Determine the localization of Hyal-2 within cells by laser confocal microscopy.2. The pEGFP-C1-env eukaryotic expression vector was transfected TC-1 cells, TC-1-Hyal2 cells and NIH 3T3cells. Verify the expression of Env in cells by RT-PCR method and Western blot method. Determine the localization of JSRV Env within cells by laser confocal microscopy.3. After transfected by pEGFP-C1-env, detect the expression changes of AKT, ERK and TERT in cells.The results and conclusions:1. The ovine Hyal-2 stable expression in TC-1 cells, named TC-1-Hyal2.2. The pEGFP-C1-env eukaryotic expression vector was successfully transfected TC-1 cells, TC-1-HyaI2 cells and NIH 3T3cells.3. In TC-1 cells and TC-1-Hyal2 cells which were transfected by pEGFP-Cl-env vetors the expression quantity of AKT, ERK and TERT increased significantly compared with control group. Compared with the expression quantity in TC-1 cells the expression quantity in TC-1-Hyal2 cells increased more significantly.4. The ability of JSRV Env in transformation was promoted by ovine Hayl-2 in TC-1...