Construction Of CTLA-4 Eukaryotic Expression Vector And Its Lentivirus Packaging

Objective: Pancreatic cancer is one of the most common cancers in the world,with high morbidity and mortality.The death toll from pancreatic cancer has been rising steadily over the past few decades.Unlike other digestive tumors,chemotherapy and radiotherapy do not improve the survival rate of patients with pancreatic cancer to a large extent.Therefore,gene targeting therapy may provide a new approach for pancreatic patients.As a member of immunoglobulin superfamily,CTLA-4 plays an important role in the occurrence and development of many tumors.Clinically targeting CTLA-4 has become the main treatment for melanoma and small cell lung cancer.Therefore,an in-depth study of the mechanism of CTLA-4 in diabetes-related pancreatic cancer may be helpful for the early diagnosis and treatment of the disease.The purpose of this study is to construct CTLA-4 eukaryotic expression vector and and its lentivirus packaging by molecular biological methods,so as to provide an effective biological tool to explore whether CTLA-4 plays a role in the occurrence and development of diabetes-related pancreatic cancer.Methods: Through Pub Med query and Luciferase primers designed by Primers5 software,the primers contained Eco RI and NOTI double restriction sites.After PCR amplification,the amplified products were agarose gel electrophoresis,and the gel electrophoretic patterns were observed on the gel imager.After the amplification of the target fragment and the vector were digested with double enzymes,the digested products were recovered,and then agarose gel electrophoresis was performed.After the double digestion products were recovered and purified,the target gene was directionally inserted into the vector with T4 DNA enzyme to construct the recombinant plasmid pCAG-3Flag-Luciferase-IRES-mcherry.The recombinant plasmid was transformed into competent bacteria DH5 ? and the plasmid was extracted by non-endotoxic plasmid extraction kit.The extracted recombinant plasmid was identified by colony PCR,double endonuclease digestion and gene sequencing to determine whether the target gene fragment was connected correctly.In the same way,the recombinant plasmid pCAG-CTLA4-3Flag-Luciferase-IRES-mcherry was constructed.The recombinant plasmids with correct sequencing were packaged with lentivirus and transfected into293T cells.The expression of the recombinant plasmids was observed by fluorescence microscope.Results: After designing Luciferase primers,PCR amplification primers and agarose gel electrophoresis showed that the target gene fragment was amplified by PCR,which was consistent with the size of the DNA fragment published by genebank,which indicated that the successfully amplified fragment was Luciferase gene fragment.PCAG-3Flag-Luciferase-IRES-mcherry was successfully constructed by Eco RI/NOT I double restriction enzyme digestion analysis,recombination,colony PCR and gene sequencing.PCAG-CTLA4-3 Flagly Luciferase Mu IRES was successfully constructed by the same method.Two kinds of recombinant plasmids were packaged by virus and transfected into 293T cells.The cells were observed by fluorescence microscope and red fluorescence was excited,which indicated that the packaging of lentivirus was successful.Conclusion: The eukaryotic expression vectors of pCAG-CTLA4-3Flag-LuciferaseIRES and pCAG-3Flag-Luciferase-IRES-mcherry were successfully constructed,and the lentiviral packaging of the two types of recombinant plasmids was completed.