乳酸菌素Gassericin T基因的人工合成及其组成型表达载体的构建

Synthesis of gatA Gene of Bacteriocin Gassericin T and Construction of its Constitutive Expression Vector Using the GAP Promoter

目的:为了研究乳酸菌素Gassericin T的作用机制及应用价值,人工合成Gassericin T基因并构建能高效表达外源蛋白的毕赤酵母组成型表达载体。方法:应用PCR方法从毕赤酵母染色体中扩增GAP启动子,经测序正确后与已线性化的不含pAOX1启动子的毕赤酵母诱导型表达载体pPIC9K连接,转化大肠杆菌DH5α。根据Gassericin T的基因序列,把Gassericin T的结构基因gatA的密码子转换成毕赤酵母偏爱的形式,设计了6条59nt的寡聚核苷酸引物,通过3次连续PCR反应,人工合成gatA片段(简称gat基因),经测序正确后插入pGAP9K质粒的多克隆位点。结果:用GAP启动子(pGAP)取代了pPIC9K上的pAOX1,构建了毕赤酵母组成型表达载体pGAP9K;PCR拼接获得250bp的目的基因序列,将目的基因克隆于pGAP9K,获得组成型表达载体pGAP9K-gat。结论:为下一步在毕赤酵母中组成型表达外源蛋白,研究其作用机理和遗传机制奠定了基础。

Objective： To construct a recombinant expression vector of Pichia pastoris expressing bacteriocin Gassericin T for study its mode of action and its value in application. Methods： The GAP gene promoter was amplified from P.pastoris GSII5 and used to replace the AOX1 promoter（pAOX1） on pPIC9K resulting in plasmid pGAPgK. The gassericin T gene was generated by three rounds of PCR from a total of six 59 nt oligoes after modification of the original sequence to the optional codon usage of P.pastoris. The gene（called gat gene） was cloned into pGAP9K. Results： A 250 bp DNA fragment encoding the bacteriocin gassericin T was gained. The recombinant constitutive expression vector pGAP9K-gat was constructed by inserting the aim gene into pGAP9K. Conclusion： The GAP promoter can be used to express foreign protein effectively and is the basis for studying the physicochemical properties and genetic mechanism of Gassericin T.