牛pAcGFP-FADD融合蛋白真核表达载体构建及在CHO-K1细胞中的表达

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

摘要:

FADD是Fas/FasL系统的一个信号连接蛋白, 通过传递凋亡信号, 介导细胞凋亡。为了揭示FADD在牛卵泡发育过程中的调控作用, 采用RT-PCR从牛卵巢组织中扩增FADD基因, 将其cDNA终止密码子删除, 采用定向克隆技术连接到带有水母绿色荧光蛋白(AcGFP)报告基因的真核表达载体pAcGFP-Nl中, 构建融合蛋白重组质粒, 经BglⅡ/EcoRⅠ酶切、测序鉴定后, 用脂质体介导质粒转染CHO-K1细胞, 观察有无荧光的表达及用RT-PCR和Western blotting方法检测基因转录、表达情况。结果表明, 成功克隆牛FADD基因, 通过PCR方法在FADD阅读框两端引入了Bgl Ⅱ和EcoRⅠ克隆位点, 并于起始位点前加入Kozak序列, 成功构建pAcGFP- bFADD融合蛋白真核表达载体, 重组质粒转染CHO-K1 24 h后在荧光显微镜下观察到绿色荧光, 转染效率可达65%, 通过RT-PCR扩增出654 bp的转录产物, 并用Western blotting检测到51.4 kD目的蛋白的表达。

Abstract:

Fas-associated death domain (FADD) is a signal connection protein in Fas/FasL apoptotic path which might play a key role on apoptosis by transferring apoptotic signal. To reveal the intracellular signal transduction molecules involved in the procedure of follicular development in bovine ovary, we cloned FADD gene in bovine ovary tissue with RT-PCR, deleted the termination codon in its cDNA and directionally cloned the amplified FADD gene into eukaryotic expression vector pAcGFP-Nl including AcGFP, successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme Bgl II/EcoR I and sequencing, 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 successfuly constructed by introducing Bgl II, EcoR I cloning site at two ends of FADD open reading frame and inserting a Kozak sequence before 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 blotting. Construction of pAcGFP-bFADD recombinant plasmid should be helpful for further understanding the mechanism of regulation of FADD on bovine oocytes formation and development.