牛FADD基因的克隆分析及其真核表达载体的构建

Cloning and analysis of the cattle FADD gene and construcion of eukaryotic expression vector

【目的】克隆牛FADD基因并构建真核表达载体,以探讨其在牛卵泡发育过程中的调控作用。【方法】采用RT-PCR技术,从牛卵巢组织总RNA中反转录FADD cDNA,对编码区核苷酸序列分析后,将其全长cDNA删除终止密码子,采用定向克隆技术连接克隆到含有水母绿色荧光蛋白(AcGFP)报告基因的真核表达载体pAcGFP-Nl中,用BglⅡ、EcoRⅠ双酶切、测序进行鉴定,并进行了牛FADD mRNA的组织表达谱分析。【结果】克隆的牛FADD基因编码区全长序列与NCBI公布的序列完全一致,核苷酸和氨基酸序列与猪的同源性最高,与鸡的同源性最低,仅为42.5%和44%;通过PCR方法在FADD阅读框两端引入了BglⅡ和EcoRⅠ克隆位点,并于起始位点前加入Kozak序列,成功构建pAcGFP-N1-bFADD融合蛋白表达载体。组织表达谱分析表明,牛FADD mRNA高表达于肝脏、淋巴组织及睾丸、卵巢组织,在小肠、心、胰腺、肺、肾及肌肉中则弱表达或无表达。【结论】成功克隆了牛FADD基因,并构建pAcGFP-N1-bFADD融合蛋白表达载体,为FADD基因在牛卵母细胞发育调控中的基础研究提供了一种简便可靠的方法。

[Objective] Cattle FADD gene was cloned and its eukaryotic expression vector was constructed to exploit the gene＇s regulation role in the procedure of follicular development in bovine ovary. [Method] The FADD gene was amplified in cattle ovary tissue by RT-PCR,the nucleotide sequence in the coding region was analyzed and the termination codon in its cDNA was deleted. The amplified FADD gene was directionally cloned into eukaryotic expression vector pAcGFP-N1 including AcGFP, then identifed by restrictive enzyme Bgl Ⅱ/EcoR Ⅰ and sequencing. [Result] The sequence was consistent with that of NC- BI,the homology with pig was the highest in nucleotide and amino acid sequence, while that with poultry was the lowest,only 42.5% and 44%. The pAcGFP-NI-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. [Conclusion] Bovine FADD gene was cloned successfully and the pAcGFP-NI-bFADD recombinant plasmid was constructed,it should be helpful for further understanding the mechanism of regulation of FADD on bovine oocyte formation and development.