作　　者： ; ; ; ; ; ; ; ;

机构地区： 海南大学海洋学院

出　　处： 《中山大学学报（自然科学版）》 2012年第2期86-90,96,共6页

摘　　要： 采用聚合酶链反应从花鳗鲡垂体cDNA中分别扩增出GTHα、LHβ基因,再利用搭桥技术,中间以lin-ker将两个基因连接成单个基因LHβα。然后利用双酶切、连接,将LHβα基因连接到载体pPICZαA中,构建出促黄体生成素的酵母共表达载体pPICZαA-LHβα。再利用电转化法将其转入到酵母野生型菌株X-33中,在含不同浓度的zeocinYPDS平板上筛选高拷贝转化子,阳性克隆经φ=0.7%甲醇诱导表达,表达产物采用SDS-PAGE和western blot进行分析,得到所表达的产物相对分子质量约为45 000。 The GTHa, LHβ gene fragments were amplified by PCR, which the template was pituitary cDNA of marble eels. In the way of bridging, the two fragments were connected to a single gene LHflct, a linker inserted between them. The LHβa gene fragment was subcloned to pPICZctA with double enzymedigestion and connection, constructing eukaryotic co-expression plasmid pPICZaA-LHβa. The recombi- nant plasmid was transfected into a native yeast strain X-33 by electrotransformation. The high-copy clones were selected by the YPDS plates containing different concentrations of Zeocin. The positive clones were induced by 0. 7% methanol and expression products were tested by SDS-PAGE and western blot, and its molecular weight was determined as about 45 000.

关 键 词： 花鳗鲡 促黄体生成激素 共表达载体 毕赤酵母

领　　域： [生物学]