Construction of weak immunogenicity virus vector with super antigen staphylococcal enterotoxin A gene

Abstract

Objective To construct a weak immunogenicity lentiviral vector expressing the staphylococcal enterotoxin A (SEA) gene in order to exert a potent antitumor effect on activated T cells. Methods The human telomerase reverse transcriptase (hTERT)-CMV promoter sequence of 851 bp and the SEA gene of 774 bp were obtained by polymerase chain reaction (PCR). The target genes were constructed into lentiviral vector plasmids. After sequencing, they were transferred into DH5α competent cells and amplified. Viral supernatants were collected by co-transfection of 293FT cells with shuttle plasmids and packaging plasmids, and virus titer was determined by fluorescence titer. Different concentrations of virus supernatant were transfected into BIU-87 cell line. The target gene was sequenced and the expression of SEA gene was detected by Western blotting. Results The size of the vector was 10 458 bp, and the gene sequence was correct. No obvious mutation was found. The titer of the virus titer was (4.30±2.00)×109 TU/ml. PCR product sequencing of the target gene confirmed that the target gene was correct, the total RNA were 742、803、726 mg/L respectively by spectrophotometric assay. Western blotting revealed that SEA gene was successfully expressed. Conclusion The weak immunogenicity virus carrying the superantigen SEA gene was successfully constructed. Key words: Lentivirus; Staphylococcal enterotoxin A; Tumor; Super antigen