Abstract:

Objective To construct the eukaryotic cell translation initiation factor 4A3 （EIF4A3） short hairpin RNA （shRNA） lentiviral vector， and to establish the Neuro-2a-EIF4A3-shRNA stable transfection cell line. Methods The EIF4A3 gene sequence was retrieved from the National Center for Biotechnology Information （NCBI） database； the PCR identification primers were designed and synthesized， and connected to the lentiviral GV493 vector digested with EcoR I and Age I enzymes to construct the GV493-EIF4A3-shRNA lentiviral plasmid； PCR method was used to screen the positive clones， which were sequenced for the identification； the GV493 empty plasmid and GV493-EIF4A3-shRNA recombinant plasmid were transfected into the HEK293T cells， regarded as GV493 control lentivirus and GV493-EIF4A3-shRNA lentivirus， respectively. After 48 h of transfection， the lentiviruses were collected for packaging and the viral titer was determined. The Neuro-2a cells were divided into blank group， GV493 control group， and GV493-EIF4A3 shRNA group. The Neuro-2a cells in blank group were untreated， and the Neuro-2a cells in GV493 control group and GV493-EIF4A3 shRNA group were infected with the respective lentiviruses at a multiplicity of infection （MOI） of 100.The infected Neuro-2a cells were selected by 10 mg·L^-1 puromycin， and the growth status and green fluorescence expression of the Neuro-2a cells in various groups were observed under fluorescence microscope； real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of EIF4A3 mRNA and protein in the Neuro-2a cells in various groups. Results The PCR sequencing results showed that the gene sequence of the GV493-EIF4A3-shRNA recombinant plasmid was consistent with the designed EIF4A3-shRNA sequence， indicating successful construction of the GV493-EIF4A3 lentiviral vector. The fluorescence microscope observation results showed that there was strong fluorescence expression and good growth status in the HEK293T cells， confirming successful lentiviral packaging. The viral titers for GV493 control lentivirus and GV493-EIF4A3-shRNA lentivirus both were 2×10⁸ TU·mL^-1. The growth status of the Neuro-2a cells in GV493 control group and GV493-EIF4A3 shRNA group was good， and they expressed green fluorescence， indicating successful construction of the stable transfection cell line. The RT-qPCR results showed that compared with blank group and GV493 control group， the expression level of EIF4A3 mRNA in the cells in GV493-EIF4A3 shRNA group was significantly decreased （P<0.01）. The Western blotting results showed that the specific bands was at a relative molecular mass of 49 000， indicating successful EIF4A3 protein expression in the Neuro-2a cells. Compared with blank group and GV493 control group， the expression level of EIF4A3 protein in the cells in GV493-EIF4A3 shRNA group was significantly decreased （P<0.01）. Conclusion The GV493-EIF4A3-shRNA lentiviral vector is succfssfully constructed， and the Neuro-2a-EIF4A3-shRNA stable transfection cell line is established； the results provide the reference for the study of the effect of EIF4A3 on the intracranial atherosclerosis.

Key words: Eukaryotic translation initiation factor 4A3, Short hairpin RNA, Lentivirus, Stable transfection cell line, Neuro-2a cell