The Study Of Establishing Rubripes Reprogramming Factors Oct4 And Sox2 Prokaryotic Expression Vectors, Transfering And Detecting The Prokaryotic Expression Proteins Into Testis Cells

Oct4 and Sox2, as transcription factors, play an important role in inducing pluripotent stem cells (iPS cells) by activating related genes. At present, it has been demonstrated that mammal iPS cells can be obtained safely by directly delivering the proteins of reprogramming factors to somatic cells. This research is the preliminary stage of inducing Takifugu rubripes testicular cells to iPS cells, which will further lay foundation for cell engineering and breeding. Transcription factors Oct4 and Sox2 were connected with cell penetrating peptide sequence to get the recombinant transcription factor proteins, which were then transfered into in vitro cultured Takifugu rubripes testicular cells. The intracellular location of the recombinant protein was determined.The primers of Oct4 and Sox2 were designed by the published genome database of Takifugu rubripes. The two genes, cloned from the liver of Takifugu rubripes, were introduced restriction enzyme sites BamH I and Kpn I at both ends. Similarly, the both ends of the synthesized cell penetrating peptide sequence (11R), composed of 11 arginines transduction domain, were introduced restriction enzyme sites Kpn I and EcoR I. The 11R were connected to the 3'terminal of reprogramming factors after both digestion by the restriction enzyme Kpn I. The product was then ligased to the prokaryotic expression vector pET32a after both digestion by BamH I and EcoR I to construct the expression plasmids pET32a-oct4-11R and pET32a-sox2-11R. The two recombinant plasmids were transformed into E.coli BL21(DE3) and the expression were induced by Isopropylβ-D-1-Thiogalactopyranoside (IPTG) for 5h at 30℃. Then E.coli cells were broken by freeze-thaw and ultrasonic. The recombinant proteins Oct4, which present in the cytoplasm, were purified by Ni-NTA resin. The Sox2, in the form of inclusion bodies, were denatured by urea firstly, purified using Ni-NTA resin, and renaturation by dialysis. After sterilization by filtration, the two transcription factor proteins were added to the culture medium of Takifugu rubripes testicular cells. Through observation of cell morphology under light microscope and Western blot detection of recombinant proteins, the optimal concentrations of the recombinant proteins were determined at 8μg/ml. The proteins could be observed distributing throughout the cell by immunofluorescence labeling, especially focus on the nucleus. It indicates that the two recombinant proteins can enter the nucleus mediated by the cell penetrating peptide.The above results confirmed that the constructed expression vectors of Oct4 and Sox2 can be effectively expressed in E. coli. The recombinant proteins of Oct4 and Sox2 were introduced effectively into the nucleus of Takifugu rubripes testicular cells mediated by the penetrating peptide. Cells have survived for 10 days now with recombinant proteins in them, which lay early foundation for further inducing Takifugu rubripes testicular cells to iPS cells.