【摘要】 采用乳酸乳球菌表达载体对PlnF抗菌肽基因进行克隆表达,旨在使重组乳酸菌可以直接应用于食品的发酵和防腐之中。在乳酸乳球菌pNZ8149/NZ3900表达载体中,构建含有胞外表达信号肽的PlnF抗菌肽重组质粒,进一步在电阻200Ω、电容25μF条件下电转入NZ3900感受态内。经溴甲酚紫培养基筛选、菌落聚合酶链式反应验证、测序等鉴定正确的重组菌株,以1 ng/mL Nisin诱导6 h后离心获得的上清液对金黄色葡萄球菌具有(14.03±0.23)mm的抑菌圈,经Tricine-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测到在5.8～7.8 kDa间出现一条与预期大小相近的条带,进一步通过纳升液相色谱-电喷雾-串联质谱验证表明PlnF蛋白在pNZ8149/NZ3900乳酸乳球菌表达载体中正确地进行了胞外表达。更多还原

【Abstract】 This study aimed to clone and express the gene encoding antimicrobial peptide PlnF from Lactobacillus plantarum in Lactococcus lactis for the purpose of obtaining a recombinant strain which can be applied directly in in food preservation and preservation. The pln F gene was cloned and inserted into plasmid vector pNZ8149. The resulting recombinant plasmid was transformed by electroporation into competent cells of L. lactis NZ3900 under the conditions: resistance 200 Ω and capacitance 25 μF. The recombinant strain was screened out using a bromocresol purple-supplemented medium, confirmed by PCR and sequenced. The expression was induced by 1 ng/m L nisin for 6 h. The culture supernatant was found to be active against Staphylococcus aureus with an inhibition diameter of (14.03 ± 0.23) mm and it displayed a protein band of 5.8–7.8 kDa as determined by Tricine-SDS-PAGE, which was consistent with the predicted value. Furthermore, the extracellular expression of recombinant PlnF was confirmed by nano LC-ESI-MS/MS.更多还原