人γ心钠素在大肠杆菌中的高效表达

利用聚合酶链式反应（ＰＣＲ）技术扩增人γ心钠素（γ－ｈＡＮＰ）ｃＤＮＡ编码序列，在其５′和３′端分别引入ＥｃｏＲⅠ和ＢａｍＨⅠ限制性内切酶位点，定向克隆到表达质粒载体ｐＭＳ－３１ｂ，在大肠杆菌ｐｏｐ２１３６中高效表达出人γ心钠素融合蛋白，表达产物占菌体总蛋白的３０～４０％。双向免疫扩散法测定证明表达产物与人α心钠素（α－ｈＡＮＰ）抗血清呈阳性反应，纯化的表达产物经复性处理后能引起大鼠胸主动脉条舒张．

High-level Expression of Human-Atrial Natriuretic Peptide in E. coli

Through the polymerase chain reaction (PCR) technique,the whole coding sequence of γ-hANP was amplified while both EcoR Ⅰ and BamH Ⅰ recognition sites had been introduced to their 5′adn 3′ends respectively. The amplified fragments were cloned downstream to Bacteriophage MSZ replicase gene in plasmid pMS-31b in result to form high-expression plasmid pMS31b/γ-hANP. E. coli pop2136 strain transformed by pMS-31b/γ-hAVP grew at 30℃ to the cell density of A = 0. 6 ,and then was induced at 42℃. As assessed by SDSPAGE analysis,a novel protein band with Mr of 25 kD appears in the lysate, which accounts for 30-40% of the total bacterial protein. Double-direction immunodiffusion test has revealed that the expressed products reacted positively with standard anti-α-hANP antisera. Purified and folded products can cause relaxation of rat aorta strips.