人表皮生长因子基因的化学合成和克隆及其在酵母中的表达

Chemical Synthesis, Cloning and Expression of Human Epidermal Growth Factor Gene in Yeast Saccharomyces cerevisiae

用固相亚磷酰胺法合成了人表皮生长因子基因,全长为173个核苷酸,它被分成8个寡核苷酸,分别在DNA合成仪上合成。经分离纯化后的寡聚核苷酸进行酶促连接,然后被克隆到噬菌体M13mp18中,克隆经分子杂交、限制酶酶切以及DNA序列分析检测,证明合成的人表皮生长因子基因和设计的完全一致。将人表皮生长因子基因的DNA片段插入酵母分泌型表达载体YFD59 HindⅢ位点中,建成表达人表皮生长因子基因的质粒YFD104,再将此质粒转化酿酒酵母,所得转化子经摇瓶发酵、发酵上清液用受体结合试验表明有人表皮生长因子表达。

A gene coding for human epidermal growth factor (hEGF) has beenchemically synthesized by solid-phase phosphoramidite method. The 173base-pair synthetic DNA duplex consists of structural gene encodinghEGF, a stop codon TGA at 3′end and some convenient restrictionsites at both ends of the gene. The synthesis of the gene involved en-zymatic joining of 8 oligonucleotides to form DNA duplex which wascloned into vector M13mp18. The recombinant colonies were identifiedby dot hybridization and restriction enzyme digestion. Its accuracy wasconfilmed by DNA sequence analysis.The hEGF DNA was inserted inyeast secretion vector YFD59. The resulted expression plasmid YFD104was introduced into yeast Saccharomyces cerevisiae. The binding assaysbowed that the yeast transformants could express and secret hECF.