论文部分内容阅读
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．
The plasmid pXZ-α-ANP-590 cloned with the chemically synthesized rat atrial natriuretic peptide gene was transformed into plasmid pLH280 and transformed into E. coli to express the fusion protein LacZ’-280-α-ANP. When the optimal host strain JM101 with this plasmid was cultured at 37 ° C and 270 r / min for 14 h, the expression level of the fusion protein reached its highest level. The fusion protein was extracted by guanidine hydrochloride, the guanidine hydrochloride was removed by dialysis, and then the fusion protein was cleaved by cyanogen bromide. The samples were collected by SephadexG-25 column chromatography. The free radical activity of each peak sample was determined by freeze-drying. The results show that the first peak sample has radioimmunoactivity. Both HPLC analysis and bioassay showed that the isolated samples were basically the same as the standard atrial natriuretic peptide samples. Compared with the fermentation broth carrying plasmid pXZ-α-ANP-590 (D (600) = 1), 1.2 mg of active α-ANP was obtained when 1 L of fermentation broth carrying plasmid pLH280 (D 2.0) produced a 4-fold increase in alpha-ANP production.