鸡IL-18cDNA的克隆及在大肠杆菌中的高效表达

Cloning of Chicken Interleukin-18 Mature Protein Gene and Its High Expression in E.coli

运用RT PCR方法,从经脂多糖(LPS)诱导的鸡马立克氏病成淋巴细胞样细胞系 MDCC MSB1 总 RNA中扩增得到了鸡白细胞介素18(Chicken interleukin 18,ChIL 18)成熟蛋白基因的 cDNA,并将其克隆到 pMD18 T载体上。DNA序列测定表明,克隆得到的ChIL 18 cDNA与国外报道的完全一致,包括终止密码子在内其编码区的长度为510bp,编码169个氨基酸残基的蛋白质。将ChIL 18 成熟肽段编码区定向克隆到原核表达载体 pGEX 6P 1中谷胱甘肽转移酶(GST)基因的下游,构建成原核表达质粒 pGEX ChIL18。该质粒的 BL21(DE3)LysS转化菌在 IPTG的诱导下可高效表达GST ChIL18基因融合蛋白,表达量约占菌体总蛋白的32%。

Chicken interleukin-18 (ChIL-18) mature protein gene was amplified from LPS-stimulated MDCC-MSB1 cells by RT-PCR.PCR product was cloned into the pMD18-T vector. Sequencing analysis showed that the nucleotide sequence of this ChIL-18 mature protein gene was 5l0bp including the stop coden and the same as the published ChIL-18 cDNA sequence by Schneider K. A prokaryotic expression plasmid of ChIL-18, pGEX-ChIL18, was obtained by subcloning the encoding region of the ChIL-18 mature peptide into pGEX-6P-1. The recombinant ChIL-18 (rChIL-18) was expressed efficiently in pGEX-ChIL18-transformed BL21(DE3)LysS induced by IPTG, the yield was accounted for 32% of the total bacterial protein.