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

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。

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

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 GS115 and used to replace the AOX1 promoter(pAOX1)on pPIC9K resulting in plasmid pGAP9K.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.