Construction Of Low Immunogenicity Virus Vector With Super Antigen SEA Gene And Its Potential Anti Tumor Effect

Objective: 1.To construct the low-immunogenicity lentiviral vector carrying superantigen SEA gene.2.Investigate the expression of superantigen SEA gene in BIU-87 which transfected by lentiviral vector carrying superantigen.3.Investigate the anti-tumor effect of superantigen SEA gene translation on peripheral lymphocytes.Methods: Genebank search h TERT-CMV promoter and SEA gene sequence,designed oligonucleotide primers,based on the original adenovirus vector as template by polymerase chain reaction(Polymerase Chain Reaction,PCR)for promoter and gene fragments.The obtained gene was constructed into the lentiviral vector,and the gene was sequenced and transferred into the competent cells.The shuttle plasmid and packaging plasmid were used to transfect 293 FT cells to obtain the positive plaque and extract the genome of adenovirus.The viral supernatant was collected by the method of amplification of virus and ultracentrifugation.BIU-87 cells were transfected with different concentrations of virus supernatant,SEA gene sequences were confirmed by PCR and the SEA protein was detected by Western-blot sequencing.Observe the proliferation of lymphocytes and tumor cells under the reverse-microscope.and the cytokine secretion was detected by enzyme linked immunosorbent assay(ELISA).Results: 1.Detection of gene sequences confirmed that the SEA gene of the lentiviral vector was connected with the promoter correctly.The plasmid size was 10458 bp,and there was no obvious mutation.The titer of virus was detected(4.30±2)×109TU/m L.Detection of gene sequences the enzyme product confirmed that lentivirus vector is correctly.2.Western-blot assay showed that SEA gene could be translated into superantigen sea protein in tumor cells.3.Lymphocytes and tumor cells co cultured in 24,48,96 h experimental group was significantly less than the control group,the expression of cytokines in the experimental group was significantly higher than that in the control group.Conclusion: 1.The recombinant lentiviral vector carrying SEA gene was successfully constructed,and the recombinant lentivirus genome was sequenced and the toxicity was detected.2.The recombinant lentiviral vector has been used to study the anti-tumor effect of bladder cancer cell line BIU-87.