Construction Of Human Recombinant RIZ1Eukaryotic Expression Vectors And Its Expression In Human Esophageal Squamous Cell Carcinoma TE-13Cells

Objective:To construct human RAZ1eukaryotic expression vectors and transfect human esophageal squamous cell carcinoma TE-13cells, and observe the expression of RIZ1in TE-13cells.Methods:1. Extract tRNA from the human esophageal carcinoma tissues using RT-PCR method.2.Reverse transcriptase tRNA extracted from the esophageal carcinoma tissue to cDNA.3. According to the RIZ1sequence released by NCBI design5primer.4. PCR amplification from the cDNA of esophageal carcinoma.5.Connect PCR product to T carrier, and sequencing.6. Connected plasmid which digested by restriction enzyme to pcDNA3.1(+) eukaryotic expression vector.7. RIZ1which connected to pcDNA3.1(+) was identified by restriction enzyme digestion and sequencing.8. Recover and cultivate human esophageal squamous cell carcinoma TE-13cells.9. Extract pcDNA3.1(+)/RIZ1ultra pure plasmid.10. PcDNA3.1(+)/RIZ1ultra pure plasmid was transfected into TE-13.11. Evaluate the protein expression of pcDNA3.1(+)/RIZ1in human esophageal squamous cell carcinoma TE-13cells using western-blotting.Result:1.Destination gene amplified by PCR was about5157bp in length analysed by12g/L agarose gel electrophoresis, which met the expectancy. Human recombinant RIZ1eukaryotic expression plasmid vectors pcDNA3.1(+)/RIZ1were constructed, and the result of DNA sequencing compared by BLAST was consistent with that of NCBI released.2.Western blotting analysis revealed that the relative expression of RIZ1protein was stable in pcDNA3.1(+)/RIZ1transfected cells.Conclusion:Human recombinant RIZ1eukaryotic expression vectors were successfully constructed, TE-13was transfected, and RIZ1protein was expressed in transfected cells.