Construction of Mammalian Cell Expression Vector for pAcGFP-bFLIP(L) Fusion Protein and Its Expression in Follicular Granulosa Cells

Yang, Run Jun; Li, Wu Feng; Li, Jun Ya; Zhang, Lu Pei; Gao, Xue; Chen, Jin Bao; Xu, Shang Zhong

doi:2010.23.3.401

Article

Animal Biotechnology

Asian-Australasian Journal of Animal Sciences 2010;23(3): 401-409.
https://doi.org/10.5713/ajas.2010.90187 Published online February 22, 2010.

Construction of Mammalian Cell Expression Vector for pAcGFP-bFLIP(L) Fusion Protein and Its Expression in Follicular Granulosa Cells

Run Jun Yang, Wu Feng Li, Jun Ya Li, Lu Pei Zhang, Xue Gao, Jin Bao Chen, Shang Zhong Xu

Abstract

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFP-bFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) 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 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

Keywords: c-FLIP(L); pAcGFP-N l; Recombinant Plasmid; Follicular Granulosa Cell