摘要
将克隆有化学合成的鼠心钠素基因的质粒pXZ－α－ANP－590改建成质粒pLH280，转化大肠杆菌表达融合蛋白LacZ＇－280－α－ANP．当带有此质粒的最适宿主菌JM101在37℃，270r／min摇床培养14h，融合蛋白表达量可达最高值．利用超声波破碎菌体；通过盐酸胍抽提融合蛋白；透析除去盐酸胍后用溴化氰裂解融合蛋白；SephadexG－25柱层析后收集样品；各峰样品冷冻干燥后进行放免活性测定．结果说明第一峰样品具有放免活性．HPLC分析和生物活性测定均证明此分离样品与标准心钠素样品基本一致．处理1L携带质粒pLH280的发酵液（D（600）＝2．0）可得1．2mg有活性的α－ANP，较之携带质粒pXZ－α－ANP－590的发酵液（D（600）＝2．0）的α－ANP产量提高了4倍．
A plasmid pLH280 was cortstructed from pXZ-α-ANP-590 which contained a chemical synthesized α-ANP gene. This plasmid was transferred into E. coli by transformation,the expression of fusion protein LacZ'-280-α-ANP showed the highest level in host cell of JM101, when it grew at 37℃ for 14 h. The fusion protein of the inclusing body was resolved in 6 mol/L Gucl, and cleaved by CNBr at the site of Met which link LacZ' and α-ANP, then the released α-ANP was purified by Sephadex G-25 chromatography. Purified α-ANP was collected after RIA activity test. From HPLC analysis and biological assay, it is proved the sample is identical with authentic α-ANP. About 1. 2 mg α-ANP was obtained from 1 L fermentation LB broth [D(600)=2. 0]. The yield of α-ANP using pLH280 plasmid is five times as high as the yield of α-ANP of pXZ-α-ANP-590.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
1998年第4期428-432,共5页
Journal of Fudan University：Natural Science
