Construction And Eukaryotic Expression Of Minicircle Vector Containing Porcine MRPS18Gene

Mitochondrial ribosomal protein S18-2could immortalize B-cells afterinfection by interfering with normal cellular pathways. Overexpression of the humanMRPS18-2could lead to immortalization of primary rat embryonic fibroblasts. In recentyears, Various methods have been used to induce cell immortalization, and the commonlyused method is that importing the immortalized exogenous gene into the cells. Immortalizedcell lines can be used to prepare proteins for a long time. the vector is the tool to importgenes into cells. Minicircle expression vector is an efficient eukaryotic expression vector. Ithas not bacterial origin of replication and resistance genes, can express green fluorescentprotein, easy track response, has high transfection efficiency, low immunogenicity, also canexpress vivo for a long time.In this study,MRPS18gene was cloned from the Liver ofporcine by RT-PCR and inserted into Minicircle plasmid.The Minicircle-MRPS18vectorwas transfected into Hela cells to investigate MRPS18expression and effects onimmortalization. The results indicated that the MRPS18gene which is a800bp length regionwas completely cloned. It is Encoded by258amino acids. Protein molecular weight is67.39kDa, Isoelectric point is4.84. It is distributed in heart, brain, heart, liver, kidney, lung,fat, skin, ileum tissue,etc. The distribution of kidney,lung and fat was higher, the distributionof brain and rectal tissues was lower. We also constructed Minicircle expression vectorMinicircle-MRPS18.The vector was transfected into Hela cells. MRPS18wasOverexpression By Real-time Quantitative PCR detection; The duration of GFP expressionof recombinant Minicircle-MRPS18vector was22days; The experiment of adriamycinstimulated cell aging, we detected the activity of β-galactosidase,then we discovered thatMRPS18can reduce the aging degree of cells in some extent;MRPS18can promote theexpression of TERT;MRPS18could increase the activity of telomerase.