乳酸乳球菌食品级表达载体的改造

Reconstruction of A food-grade Expression Vector in Lactococcus lactis

[目的]改造乳酸乳球菌食品级表达载体,拓宽原系统的应用范围。[方法]以pNZ8149为基础,在pNZ8149的启动子PnisA下游插入乳酸乳球菌MG1363未知分泌蛋白(Usp45)的胞外分泌信号肽基因序列SPusp45,利用特异性引物通过PCR方法删除启动子序列和信号肽基因序列间的酶切位点,确保SD序列和起始密码子间的距离相对最佳,构建分泌表达载体pNZS。将报告基因gus重组到pNZS的多克隆位点中,构建pNZS-gus,电击转化L.lactisNZ3900。以10ng/ml的nisin诱导培养,取培养液进行GUS染色试验。[结果]新构建的L.lactisN3900/pNZS-gus系统可以正确表达有活性的GUS蛋白,并可以将GUS蛋白分泌到细胞外。[结论]该系统的构建成功,为目的蛋白的分泌性表达研究和口服级疫苗的研制奠定了基础。

[Objective] In order to improve expression vector of L.lactis at food grade and widen application range of orginal system.[Method] Taking pNZ8149 as basis,the promoter PnisA of pNZ8149 was inserted L.lactis MG1363 and SPusp45 of unknown secretory protein in downstream.Through PCR technology,specific primers were used to delete restriction sites between promoter sequence and signal peptide gene sequence and ensure better distance between SD sequence and start codon to construct secreting expression vector pNZS.The reporter gene gus was recombined into multiple cloning site of pNZS to construct pNZS-gus and L.lactis was transformed by electroporation.10 ng/ml nisin was used for induction culture,then culture solution was conducted GUS staining test.[Result] The new constructed L.lactis N3900/pNZS-gus system could express active GUS protein and GUS protein could be secreted out of cell.[Conclusion] The successful construction of this system lays foundation for secretion expression study of protein and oral vaccine research.