Construction of Mammalian Cell Expression Vector for pAcGFP-bFADD Fusion Protein and Its Expression in CHO-K1 Cell

Fas-associated death domain (FADD) is a signal connection protein in the Fas/FasL apoptotic path, which may play a key role in apoptosis, by transferring the apoptotic signal. To reveal the intracellular signal transduction molecules involved in the procedure of follicular development in the bovine ovary, the authors cloned the FADD gene in the bovine ovary tissue with reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified FADD gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme Bgl II/EcoR I and sequencing, the transfected pAcGFP-bFADD into CHO-K1 cell, mediated by Lipofectamine 2000, observed the expression of AcGFP and detected the transcription and expression of FADD by RT-PCR and Western blotting. The results showed that the cattle FADD was successfully cloned, the pAcGFP-bFADD fusion protein recombinant plasmid was successfully constructed by introducing Bgl II, EcoR I cloning site at the two ends of the FADD open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 654 bp transcription was amplified by RT-PCR, and 51.4 kD target protein was detected by Western blot. Construction of the pAcGFP-bFADD recombinant plasmid should be helpful for further understanding the mechanism and regulation of FADD on bovine oocyte formation and development.