人β-防御素3基因的克隆及其在酿酒酵母中的表达

Secreted Expression of Human Beta-defensin-3 in Saccharomyces cerevisiae

摘要:

以质粒pPIC9K-hBD3为模板，扩增得到一段上游带信号肽因子（α-factor）的人β-防御素-3（hBD-3）基因，克隆至酿酒酵母穿梭质粒pYES2中，构建了酿酒酵母表达载体pYES2-α-factor-hBD3 (pYα-hBD3)。将重组质粒转化至Saccharomyces cerevisiae INVSC1中，鉴定阳性重组子，经2%半乳糖诱导表达。实验发现表达所得hBD-3对金黄色葡萄球菌（ATCC6538）和大肠杆菌（ATCC10231）具有明显的抑菌活性。hBD-3基因片段在酿酒酵母中的表达，为进一步探讨hBD-3的生物活性及防御素应用安全性打下基础。

Abstract:

DNA fragment containing hBD-3 with α-factor in the upstream sequence was amplified and inserted into the shuttle plasmid-pYES2 of Saccharomyces cerevisiae after digestion. The recombinant vector called pYES2-α-factor-hBD3 was transformated into Saccharomyces cerevisiae INVSC1. The recombinant positive clones, which were screening by PCR reduced with 2% galactose. The recombinant protein could be detected in the cultured supernatant by protein assay reagents. Plate test indicated that the supematant could inhibit the growth of Escherichia coli ATCC6538 and Staphylococcus aureus ATCC10231. The strategy of secretion expression of Human beta-defensin-3 gene in Saccharomyces cerevisiae may lay the foundation for further exploration of the biological activity and security application of the Human beta-defensin-3.