Construction Of Lentiviral Vector Of Superantigen SEB Gene And SEB~+ AKR Mice Esophageal Cancer Cell

Background and Objective: China is a high-incidence area of esophageal cancer.The majority of patients are diagnosed in middle or advanced stages,usually owing to typical symptoms mainly including dysphagia or chest pain.During the past decades,great achievements have been made in surgical treatment,radio-chemotherapy for esophageal cancer.However,5-year survival rate of esophageal cancer patients remains at about 20-30%.Most patients still died of local tumor recurrence and/or distant metastasis soon or later after surgery.Therefore,it is of great significance to explore new therapeutic approaches for esophageal cancer.Because of great achievements in researches of immune mechanism during tumorigenesis and development,and the increasingly mature regulation of gene expression,tumor immune gene therapy has become one of the foci in cancer research lately.Although there are still many challenges and obstacles to be resolved before clinical application,immune gene therapy is one of the most promising and expectable novel therapy for esophageal cancer.The tumor antigens originate from the host itself,and its antigenicity is relatively weak.The tumor antigen expression in many cases are often reduced or absent during tumor development,and it is difficult to provoke an effective immune response.One of the main strategies of immune-therapy for cancer is to enhance immunogenicity of tumor cells,so that effective immune responses can be aroused to recognize and kill tumor cells.Superantigens are powerful activators of T cell proliferation originating from pathogenic microorganisms.They have broad application prospects in tumor immunotherapy.Staphylococcal enterotoxins(SEs)are the superantigens that were discovered earliest and applied most frequently in cancer researches.In past animal experiments and clinical trials,SEs have been proved to be certain effective in treatment for some malignant tumors,including kidney cancer and lung cancer,by means of intratumoral injection or intra-thoracic injection.However,the direct application of SE has insuperable deficiencies.For advanced patients with distant metastasis,the local injection of super-antigen has low anti-cancer effects,especially for metastatic lesions,while systemic use of antigen would inevitably cause a series of immune disorders,some of which maybe severe,or even fatal.Therefore,in recent years,super-antigen gene therapy has become the focus of research on superantigen tumor therapy.Researchers attempted to introduce super-antigen gene into tumor cells,so that tumor cells could express super-antigen,and consequently provoke immune response that is anti-tumor effective.Present study is intended to construct SEB lentiviral vector,and infect mice esophageal cancer AKR cells with SEB lentivirus.Then,SEB+ AKR cells are to be screened,using puromycin resistance gene in vector.The aim of present study is to pave the way and provide researching materials for further research on SEB immune gene therapy for esophageal cance.Methods: 1.SEB gene is artificially synthesized,according to c DNA sequence of SEB in gene bank.Codon of SEB gene are optimized for better possibility of its expression in eukaryote cells.2.SEB gene is inserted into lentiviral vector plasmid p Lent-EF1a-FH-CMV-GP,and SEB lentivirus is packed in human embryonic kidney cells 293 T.3.Mice esophageal cancer AKR cells are infected with SEB lentivirus,and SEB+ AKR cells are screened with puromycin of optimal concentration.4.RT-PCR and western blot are performed to investigate SEB expression in SEB+ AKR cells at m RNA and protein levels.Results:1.The length of SEB gene synthesized in this study was 801 bp.It was identified by agarose gel electrophoresis and DNA sequencing.The size and sequence of the gene were completely consistent with the expected results.2.A SEB lentiviral vector was successfully constructed;3.AKR cells were infected with SEB lentiviral vector and SEB+AKR cells were screened with 5μg/ml puromycin;4.RT-PCR and Western blot verified that SEB exactly expressed in SEB+ AKR cells at m RNA and protein levels.Conclusion:1.SEB gene could be introduced and express in mice esophageal cancer cells AKR;2.We successfully established SEB+ AKR cells in present study.