摘要
拟通过酵母系统表达家蝇抗菌肽Defensin基因,并初步鉴定其抗菌活性。采用限制性内切酶SacⅠ将分泌型重组表达质粒pPIC9K/Defensin线性化后,运用氯化锂转化法将其转入巴斯德毕赤酵母GS115中,行G418加压筛选和PCR鉴定;所得阳性转化子转至摇瓶,28℃条件下0.5%甲醇诱导表达,连续诱导培养4 d后,上清液进行Tricine-SDS-PAGE检测表达效果,并采用琼脂糖孔穴扩散法检测表达产物对大肠杆菌E.coliK12D31的抑制作用。结果显示,家蝇抗菌肽Defensin在毕赤酵母中得到了成功表达,表达产物对大肠杆菌E.coliK12D31的生长有抑制作用。说明酵母系统适合抗菌肽的表达。
The research was desighed to study the expression of antibacterial peptide defensin in Pichia pastoris yeast and analysis of its antibacterial activity.After linearized by Sac I,pPIC9k/ Defensin was transformed into Pichia pastoris GS115 strain by the lithium chloride transformation method.Through G418 screening and PCR identification,the positive clones were transferred into shake flasks at 28℃ with 0.5% methanol induction.The supernatant was collected and detected by Tricine-SDS-PAGE,and bactericidal activity was analyzed by agarose diffusion assay after 4 days.The results showed that Musca domestica defensin was successfully expressed in Pichia pastoris and could inhibit the growth of E.coli K12D31.This study would lay the foundation for further research.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第9期194-197,共4页
Biotechnology Bulletin
基金
2007年度广东省科技计划项目(2007B020708015)
关键词
家蝇
抗菌肽
DEFENSIN
毕赤酵母
表达
Musca domestica Antibacterial peptide Defensin Pichia pastoris Expression
