Neurturin基因克隆及在大肠杆菌中表达

Ｎｅｕｒｔｕｒｉｎ（ Ｎ Ｔ Ｎ）是最近发现的一种与胶质细胞源性神经营养因子（ Ｇ Ｄ Ｎ Ｆ）相关的神经营养因子．利用 Ｐ Ｃ Ｒ 方法以染色体 Ｄ Ｎ Ａ 为模板，扩增获得了编码人 Ｎ Ｔ Ｎ 成熟蛋白的基因，将其克隆于 ｐ Ｕ Ｃ１９ 质粒，进行序列分析，结果与文献报道一致．将基因重组于硫氧还蛋白融合表达载体ｐ Ｔｈｉｏ Ｈｉｓ 系统，在宿主菌 Ｔｏｐ１０ 中获得了高效、稳定表达，表达的ｈ Ｎ Ｔ Ｎ 占菌体总蛋白 ２０％ 左右．这为进一步的基础研究与临床应用奠定了基础．

Cloning of Human Neurturin Gene and Its Expression in E.coli

Neurturin (NTN) is a novel neurotrophic factor related to glial cell derived neurotrophic factor.The cDNA fragment encoding mature hNTN was amplified by PCR from human chromosome DNA extracted from liver tissue,and ligated into cloning vector pUC19.Sequence analysis verified that the fragment cloned in pUC19 was hNTN cDNA.The gene was inserted into thioredoxin fusion expression vector pThioHisA between Eco R Ⅰ and Pst Ⅰ sites,and the recombinant plasmid was transformed into E.coli Top10.Higher levels of expression of hNTN fusion protein was obtained 8 or 10 hours after induction with 1 mmol/L IPTG.The expressed fusion protein,with molecular weight of about 28 kD,was about 20% of the total bacteria protein as detected by SDS PAGE and densitometry analysis.