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

文献类型： 外文期刊

第一作者： Yang, Run Jun

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

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关键词： Molecular Genetics: Biochemistry and Molecular Biophysics;Enzymology: Biochemistry and Molecular Biophysics;Endocrine System: Chemical Coordination and Homeostasis;Reproductive System: Reproduction

期刊名称：ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES （ 影响因子：2.509； 五年影响因子：2.604 ）

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收录情况： SCI

摘要： 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-N1, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme Bg/IIEcoRI 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 Bg/IIEcoRI 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.

分类号： Q95

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