Gassericin T基因的合成及其组成型表达载体的构建

目的:构建Gassericin T基因毕赤酵母(Pichia pastoris)组成型表达载体。方法:根据乳酸菌素Gassericin T的基因序列,把Gassericin T的结构基因gatA编码的氨基酸的密码子转换成P.pastoris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,获得了250bp左右的gat A片段(简称gat基因)。应用PCR方法从P.pastoris染色体中扩增了GAP启动子,大小为500bp左右,以其取代诱导型表达载体pPIC9K上的pAOX1,构建了组成型表达载体pGAP9K。将合成的gat基因克隆到pGAP9K质粒的多克隆位点中。结果:获得的gat及gap基因与预期结果一致,序列无碱基突变,构建的表达载体pGAP9K-gat经PCR、酶切鉴定完全正确。结论:成功构建了Gassericin T基因P.pastoris组成型表达载体,为下一步高效表达Gassericin T蛋白,进一步研究其作用机理及应用价值打下基础。

Synthesis of Gassericin T Gene and Construction of Its Constitutive Expression Vector

Objective:To construct a constitutive expression vector of the gassericin T gene using the GAP promoter.Methods: A 250bp DNA fragment encoding the bacteriocin Gassericin T called gat gene was generated by three rounds of PCR from a total of six 59nt oligoes after modification of the original sequence to the optional codon usage of Pichia pastoris.The GAP gene promoter which is about 500 bp was amplified from P.pastoris GS115 and used to replace the AOX1 promoter(pAOX1) on pPIC9K resulting in plasmid pGAP9K.The recombinant expression vector pGAP9K-gat was constructed by inserting the gat gene into pGAP9K.Results: The obtained sequences of gat and gap gene were detected and aligned to the expected sequences.Also,the integrity constitutive expression vector pGAP9K-gat were verified by PCR and restriction enzyme digestion.Conclusion: Gat gene was cloned into pGAP9K and its constitutive expression vector pGAP9K-gat was successfully constructed,which facilitates further functional study of the Gassericin T.