Abstract: Abstract: Objective: Construct the recombinant RIP3 overexpressed plasmids and transfect them in breast cancer MCF7 cells. Identify the expression and localization of fusion protein, as well as its effect on the death way of MCF7 cells. Methods: The expression of RIP3 mRNA in four breast cancer cell lines and one mammary epithelial cell was detected by reverse transcription polymerase chain reaction (RT-PCR). The RIP3 coding sequence was amplified by polymerase chain reaction and subcloned into mCherry vector to construct recombinant plasmids. The plasmids were transfected into MCF7 cells by lentivirus after DNA sequencing, then screened by basticidin (4 mg/L) for 1 week. The efficiencies of RIP3 expression were validated by Western blotting. The death way of mCherry-RIP3-MCF7 cells was observed under the treatment of TNF-αand zVAD. Results: The lowest expression of RIP3 mRNA was seen in MCF7 cells. The sequencing results validated the well recombinant plasmids. The expression of mCherry-RIP3 fusion protein with a molecular weight of 85 ku was detected by Western blot. mCherry-RIP3 expression enhance the sensitivity of MCF7 cells to TNF-α and z-VAD induced cell death. Conclusion：The recombinant RIP3 overexpressed plasmids were successfully constructed, and the stable MCF7 cells with ectopic RIP3 transfection were obtained. The mCherry-RIP3 fusion protein was expressed in the cytoplasm and was conformed to mediate TNF-α induced necroptosis.

Key words: RIPK3 protein, human, Breast neoplasms, Plasmids, Necroptosis, TNF-α