关键词: 性别决定区Y盒17, 慢病毒载体, PC12细胞, 实时荧光定量PCR, 感染复数

Abstract:

Objective To construct the sex-determined region Y-box 17 （SOX17） over-expression lentiviral vector， and to construct the cell line stably over-expressing SOX17 by using the PC12 cells infected with SOX17 over-expression lentivirus. Methods The over-expression sequence of SOX17 was designed and synthesized based on the National Center for Biotechnology Information （NCBI） Database， and was connected to the GV492 lentiviral vector after being doubly digested with BamHⅠ and AgeⅠ enzymes to construct the GV492-SOX17 over-expression recombinant plasmid. The agarose gel electrophoresis was used to identify the PCR products， and the positive bacteria carrying the GV492-SOX17 over-expression recombinant plasmid were selected， cloned and sequenced. The GV492 empty plasmid and GV492-SOX17 over-expression recombinant plasmid were transfected into the human embryonic kidney HEK 293T cells， respectively. After transfected for 48 h， the GV492 control lentivirus and GV492-SOX17 over-expression lentivirus were packaged and the viral titer was detected. The PC12 cells were divided into blank group， GV492 control group， and GV492-SOX17 group. The cells in blank group was not treated， and the cells in GV492 control and GV492-SOX17 groups were infected with the corresponding lentivirus （multiplicity of infection = 100）， and the PC12 cells successfully infected with lentivirus were selected with 10 mg·L^－1 puromycin. The growth state and green fluorescence expression of the PC12 cells were observed under fluorescence microscope；real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression level of SOX17 mRNA in the PC12 cells in various groups；Western blotting method was used to detect the expression level of SOX17 protein in the PC12 cells in various groups. Results The gene fragment length of GV492-SOX17 over-expression recombinant plasmid was about 744 bp， and the sequence of GV492-SOX17 over-expression recombinant plasmid gene was identical to the designed and synthesized SOX17 over-expression sequence. The titers of GV492 control lentivirus and GV492-SOX17 over-expression lentivirus were both 2.5×108 TU·mL^－1. The growth state of the PC12 cells in various groups was good and the green fluorescence was expressed. The RT-qPCR results showed that the expression level of SOX17 mRNA in the cells in GV492-SOX17 group was significantly higher than those in blank group and GV492 control group（P<0.01）. The Western blotting results showed that there was a specific band appeared at a relative molecular mass of 44 000， suggesting the SOX17 protein was successfully expressed in the PC12 cells； compared with blank group and GV492 control group， the expression level of SOX17 protein in the cells in GV492-SOX17 group was increased （P<0.05）. Conclusion The GV492-SOX17 lentiviral expression vector is successfully constructed and the PC12 cell line stably over-expressing GV492-SOX17 is established.

Key words: Sex-determined region Y box 17, Lentiviral vector, PC12 cells, Real-time fluorescence quantitative PCR, Multiplicity of infection