牙鲆干扰素调节因子核心区序列的克隆及初步表达

Cloning of cDNA for flounder Interferon Regulatory Factor (fIRF) and its primary expression in prokaryocyte

采用逆转录—聚合酶链式反应 (RT -PCR)方法 ,从牙鲆 (Paralichthysolivaceus)肝脏总RNA中扩增出干扰素调节因子 (fIRF)的核心区序列。定向插入质粒 pUC18,克隆的牙鲆IRFcDNA序列分析表明 ,与Yabu报道的牙鲆IRFcDNA相比有 1个碱基差异 ,但与推断的氨基酸序列完全一致。将牙鲆IRF的核心区序列cDNA定向插入原核表达载体 pBV2 2 0 ,构建成牙鲆IRF表达载体pBVfIRF2 2。SDS -PAGE分析表明 ,经 4 2℃诱导 ,含 pBVfIRF2 2质粒的大肠杆菌可表达一分子量约 12 0 0 0的特异蛋白。

The cDNA encoding core region of flounder Interferon Regulatory Factor (fIRF) mature peptide was amplified by reverse transcription PCR (RT PCR) method using total RNA isolated from flounder (Paralichthys olivaceus) liver as template. The amplified cDNA fragment was inserted in plasmid pUC18 and the cloned nucleotide sequence shows there is only one codon different from that reported by Yabu in 1998, and the amino acid sequence is the same as that inferred from the nucleotide sequences. Then by subcloning the cDNA into the downstream of the PRPL promoter of expression plasmid pBV220, the expression plasmid pBVfIRF22 was constructed. The SDS PAGE results show that after 4 hours of induction at 42℃, E. coli DH5α containing pBV220 fIRF22 can express a specific protein with 12 kD molecular weight.