Construction and identification of human growth factor receptor binding protein 2 associated binding 1 gene recombinant lentivirus RNA interference vector

Abstract

Objective To construct and identify a lentiviral vector harboring RNA interference (RNAi)sequence targeting human growth factor receptor-binding protein 2 associated binding protein 1 (Gab1)gene and investigate its silenced effect on Gab1 gene in EA.hy926 cells. Methods Five small interfering RNAs(siRNAs) based on the sequence of Gab1 mRNA in the Genbank were designed and synthesized.The designed and synthesized single-stranded primers were annealed to double-stranded oligo sequences and subcloned into linear GV118 lentiviral plasmid digested by enzyme Hpa Iand Xho I to produce Gab1-RNAi lentiviral vectors.After the positive clone was chosen and confirmed by polymerase chain reaction(PCR) and DNA sequencing, Gab1-RNAi lentiviral vectors with p Helper 1. 0 and p Helper 2. 0 were co-transfected into 293T cells to package lentiviral particles.The recombinant lentivirus interfering vectors were transfected into EA.hy926 cells. Transfection efficiency was evaluated with expression of green fluorescent protein(GFP) determined by fluorescent microscope. The expression of Gab1 in EA.hy926 cells was detected using reverse transcription-polymerase chain reaction(RT-PCR). Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting the human Gab1 gene was successfully inserted into the lentiviral vector, and the titer of vitrus was 3×109 TU/mL.The transfection ratio of EA.hy926 cells was up to 70%.The expression level of Gab1 mRNA(0. 088±0. 003)was significantly decreased in the Gab1-shGab1 transfected groups as compared with the control groups (0. 947±0. 087, P<0. 01)and the blank groups(0. 999±0. 067, P<0. 01). Conclusion The recombinant lentivirus RNAi vectors expressing human Gab1 gene were successfully constructed and could effectively inhibit the expression of Gab1 gene in EA.hy926 cells. Key words: Growth factor receptor binding protein 2 associated binding 1; RNA interference; Lentiviral vector; Gene recombination