采用定点基因缺失突变技术在大肠杆菌中分泌鲑鱼生长激素

Site-Directed Mutagenesis of Cloned sGH Gene and Its Secretive Expression in Escherichia Coli

目的：为获得与天然鲑鱼生长激素一致的基因工程产物，对已构建的鲑鱼生长激素基因分泌型表达质粒ｐＯｓＧＨ１５３进行改造，删除所编码表达产物氨基端多余的９个碱基。方法：采用ＰＡＬＴＥＲ系统进行寡聚核苷酸指导下的基因定点缺失突变。突变质粒ｐＯｓＧＨ１５８经限制性酶切图谱及Ｓｏｕｔｈｅｒｎ印迹杂交分析加以证实。结果：含有表达质粒ｐＯｓＧＨ１５８的大肠杆菌株经ＩＰＴＧ诱导后提取周质蛋白。经ＳＤＳ－ＰＡＧＥ及免疫印迹分析表明：周质表达产物具有与天然ｓＧＨ免疫活性和分子量一致的特性，其分子质量约为２３ｋｕ。结论：应用ＰＩＮ－Ⅲ－ｏｍｐＡ ２分泌型载体系统和ＰＡＬＴＥＲ突变系统构建鲑鱼生长激素基因在大肠杆菌表达载体的方法，可作为原核系统分泌型高效表达的模型。

Purpose and methods: A recombinant secretion vector pOsGH 153 containing genes ceding for the E. coli ompA signal peptide and mature sGN was constructed by inserting mature sGH coding sequence into the EcoR1 site and BamH1 site of the secretion pIN-Ⅲ-ompA2. To remove the extra 9 bp between the ompA signal peptide and the amino terminus of the mature sGH,a mutant vector pOsGH 158 was constructed by using a modified mutagenesis system containing a phagemid PALTER 1 vector and 1 mutagenetic. one for deletion of the 9 bp,another for selection of the oligonucleotide-directed mu- tant. The mutant was confirmed by southern blot analysis and DNA sequencing. Upon induction by IPTG, sGH with an authentic amino termius was expressed and secreted into E. coli periplasma. The expression product was identical by SDS- PAGE and western blot with rabbit anti-salmon GH antibodies. sGH products were estimated as 90 ng/ml medium.