Construction Of Eukaryotic Expression Vector Of CD80-pIRES-SART3

Objective: Construction of eukaryotic expression vector of CD80-pIRES-SART3.Methods: Firstly,by using RT-PCR technolgy amplify the whole fragment of CD80 gene of mouse and to add EcoR I restriction enzyme cutting site on the both ends of the gene fragment.Then purify the purpose extract of CD80 fragment after agarose gel electrophoresis and gel extraction.Next,insert the CD80 gene fragment into EcoR I restriction enzyme cutting site of the linearized eukaryotic expression vector of pIRES by homelogens DNA technology. Afterward, get the recombinant vector into competence cells and choose positive cloning strains. Aμgment the positive cloning strains and extract the plasmid from the strains. Detect the recombinant vector by PCR technology,enzyme digest and gene sequence analysis. Meanwhile,by using PCR technolgy amplify the whole fragment of SART3 gene and to add Xba I restriction enzyme cutting site on the both ends of the gene fragment. Then purify the purpose extract of SART3 fragment after agarose gel electrophoresis and gel extraction. Next,insert the SART3 gene fragment into Xba I restriction enzyme cutting site of the linearized eukaryotic expression vector of CD80-pIRES by homelogens DNA technology.Finally, get the recombinant vector into competence cells and choose positive cloning strains. Aμgment the positive cloning strains and extract the plasmid from the strains. Detect the recombinant vector by PCR technology,enzyme digest and gene sequence analysis.ResμLts: 1.SuccessfμLly extract the RNA of mouse's spleen and reverse transcribe the RNA into cDNA.Using synthetized primer amplify the whole fragment of CD80 gene. 2. SuccessfμLly insert the CD80 gene fragment into eukaryotic expression vector of pIRES and construct the CD80-pIRES vector.3. By using PCR technolgy amplify the whole fragment of SART3 gene.4. SuccessfμLly insert the SART3 gene fragment into eukaryotic expression vector of CD80- pIRES and construct the CD80-pIRES-SART3 vector.Conclusion: SuccessfμLly construct the CD80-pIRES-SART3 vector.This will lay a foundation for further study of gene immunotherapy of osteosarcoma,also lay a foundation for further study of gene immunotherapy of interrelated tumour and provide a referable strategy for gene immunotherapy of tumour.