Construction and identification of alpha-fetoprotein antigen epitope modified interleukin-15 lentiviral expression vector

Abstract

Objective To construct and package alpha fetal protein (AFP) antigen epitope modified interleukin-15 (IL-15)-expression lentiviral vector,infect BJAB cells in vitro for effective expression of AFP158-166,in order to amplify specific T lymphocytes in vitro.Methods IL-15 gene was amplified by polymerase chain reaction (PCR) and part of the IL-2 signal peptide were replaced with AFP epitope using site-directed mutagenesis technique.AFP158-166 modified IL-15 gene was cloned into lentiviral plasmid pEZ-Lv201.The positive clones were verified by enzyme digestion and sequencing.The recombinant vector named CCS-afpIL15-Lv201 was then transfected with helper plasmids into 293T cells to package lentivirus.The expression of IL-15 and AFP epitope in lentiviral-infected BJAB cells was examined by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography-mass spectrometry (HPLC-MS).Results The recombinant plasmid was constructed successfully,which was demonstrated by enzyme digestion and sequencing.BJAB cells infected with AFP158-166 and IL-15-expressing lentivirus expressed IL-15 and AFP158-166 efficiently.The expression after being transfected 24h and 48h was respectively 892.7,1 232.6 ng/L.The relative molecular mass of expressed AFP158-166 was 1204.65.Conclusion The AFP158-166 and IL-15-expressing lentivirus was prepared successfully and was able to infect tumor cells,resulting efficient IL-15 and AFP158-166 expression.Resolving the difficulty of insufficient cells in adoptive cellular immunotherapy for Hepatic carcinoma can be expected. Key words: Tumor gene therapy; Interleukin-15; Alpha fetal protein; Lentiviral vector