Abstract: To construct a eukaryofic expression plasmid pIRESRb94 and investigate its effect on expression of SphK in lung adenocarcinoma A549 cell line. MethodsA primer of Rb94 was designed and composed based on the Rb94 sequence． The Rb94 gene was cloned by PCR and was sequenced. The eukaryofic expression plasmid plRKSRb94 was correctly constructed and transfected into the A549 cells by lipofectamine 2O00, then the stably transfected pIRESRb94 cell line was selected through G418. Expression of the Rb94 gene was detected by real time RTPCR and Western blotting. ResultsThe recombination expression vector containing the Rb94 gene was successfully constructed and a new cell line with stable and high expression of Rb94 was established． Real time RTPCR and Western Blotting analysis demonstrated that the Rb94 gene could be effectively expressed in the A549 cells. Moreover, expression of SphK1 was downregulated and of SphK2 was upregulated. ConclusionThe pIRESRb94 expression plasmid was successfully constructed and could be effectively transfected into A549 cells. After transfection, Rb94 can efficiently inhibit expression of SphK1, and enhances expression of SphK2 in the A549 cell line.

Key words: Genes, Sphingomyelins, Plasmids, Heterozygote detection, A549 cells