在乳酸乳球菌中表达乳源血管紧张素转移酶抑制肽。选取了4种不同的来源于牛酪蛋白的血管紧张素转移酶(ACE)抑制肽,为了确保能够在人体消化液作用下正常发挥它们的ACE抑制活性,4种短肽以串联多肽(TP)的形式进行表达,并在各短肽单体间引入了人体内主要消化酶的酶切位点。根据TP的氨基酸序列和乳酸乳球菌的偏爱密码子,人工合成TP基因。然后将TP基因和绿色荧光蛋白(GFP)基因串联于载体pSEC-E7,从而构建了pSEC-TP:GFP质粒,实现了2种蛋白在乳酸乳球菌中的共表达。经电击转化,将该重组质粒转入乳酸乳球菌NZ9000中,获得重组菌株NZ9000(pSEC-TP:GFP)。用Nisin诱导TP:GFP蛋白表达。RT-PCR、激光共聚焦扫描显微镜和SDS-PAGE鉴定表达产物。RT-PCR结果表明,TP:GFP蛋白在RNA水平表达成功;SDS-PAGE表明目标产物是35 ku的条带。在乳酸乳球菌中实现了乳源血管紧张素转移酶抑制肽的表达。

Objective:To express Milk-derived angiotensin-I converting enzyme(ACE) inhibitory peptides in Lactococcus lactis(L.lactis).Methods:The four different Bovine casein-derived peptides were chosen and designed as tandem peptides(TP) with digestive enzyme restriction site among them to ensure the potent ACE inhibitory activity of TP could be activated by pepsin and trypsin in human digestive juice.The TP gene was synthesized artificially based on its amino acid sequence and bias codon of L.lactis.Then,the TP gene was co-expressed with green fluorescent protein(GFP),which was cloned in expression vector pSEC-E7 resulting in the pSEC-TP:GFP plasmid.This recombinant plasmid was transformed into L.lactis strain NZ9000 carrying regulatory genes nisR and nisK to obtain the strain NZ9000(pSEC-TP:GFP) by electroporation.The expression of TP:GFP gene induced by Nisin was identified by RT-PCR,SDS-PAGE and Confocal scanning laser micrographs.Results:RT-PCR showed that the TP:GFP was expressed at the RNA level.SDS-PAGE analysis showed target proteins product of 35 ku.Conclusion:The expression of Milk-derived ACE inhibitory peptide has been detected in Lactococcus lactis.