人α防御素5在酿酒酵母中的分泌表达及其活性分析  

Activity Analysis and Secretory Expression of Human Alpha-defensin 5 in Saccharomyces cerevisiae

作　　者：宋南[1] 邵婕 刘子昊 徐子龙 许琪瑶 张学文[1] 陈金军[1] 

机构地区：[1]湖南农业大学生物科学技术学院,中国湖南长沙410128

出　　处：《生命科学研究》2017年第6期471-476,共6页Life Science Research

基　　金：湖南省教育厅重点项目(16A098);湖南省科技厅重点项目(2017NK2311);国家自然科学基金资助项目(31672457;31772865);湖南农业大学大学生创新项目(XCX17071)

摘　　要：人α-防御素5(humanα-defensin 5,HD5)是人防御素α家族中发现的抑菌活性最高的多肽。为了探究HD5在酿酒酵母中分泌表达的可行性,首先利用PCR扩增获得酵母菌偏好的HD5核酸序列,构建酿酒酵母表达载体pVT102U/α-HD5,并将该重组质粒转入酿酒酵母S78中,通过营养缺陷筛选获得阳性转化菌株。然后,对重组菌进行发酵培养,取上清液纯化后通过tricine-SDS-PAGE和质谱检测表达产物。最后,利用琼脂扩散法检测表达的HD5的抑菌活性以及其对温度的耐受性,同时通过二倍稀释法检测表达的HD5对大肠杆菌、金黄色葡萄球菌以及沙门氏杆菌的最小完全抑制浓度。结果显示:表达的HD5对大肠杆菌、金黄色葡萄球菌以及沙门氏杆菌均具有明显的抑菌活性。发酵上清冻干粉对金黄色葡萄球菌完全抑制的最小浓度为10 mg/mL,对大肠杆菌和沙门氏杆菌完全抑制的最小浓度为40 mg/mL;且在不同温度处理下,表达的HD5对3种细菌仍具有一定的抑菌作用。上述结果表明具有高抑菌活性的HD5防御素在该重组系统中成功表达。The feasibility of secretion expression of human α-defensin 5 （HD5）, which exhibits the highest antimicrobial activity in human α-defensin family, in Saccharomyces cerevisiae was explored. The DNA fragment containing HD5 coding sequence with biased codons of S. cerevisiae was amplified by PCR, and inserted into the shuttle plasmid pVT102U/α of S. cerevisiae. The recombinant vector named pVT102U/α-HD5 was transformed into S. cerevisiae S78. The positive recombinant clones were screened out under nutrient deficiency. After fermentation, the recombinant protein could be detected in the culture supernatant by tricine-SDS-PAGE, and the protein was further subjected to mass spectroscopy to identify the relative molecular mass. The antimicrobial activities of the supernatant against the growth of Escherichia coli, Staphylococcus aureus and Salmonella pullorum were investigated. The minimal concentrations of the lyophilized powder of fermentation supernatant were 10 mg/mL to S. aureus and 40 mg/mL to both E. coli and S. pullorum for complete inhibition. Furthermore, the antibacterial activity of supernatant did not attenuate after treatment with various temperatures up to 140 ℃ for 10 min. The results showed that the recombinant HD5 could be produced with high antibacterial activity.

关 键 词：防御素 酿酒酵母 抗菌活性 载体构建 表达纯化 

分 类 号：Q28[生物学—细胞生物学]