基于pyrF的乳酸乳球菌食品级表达载体的构建

Construction of a Food-grade Expression Vector Based on pyrF Gene in Lactococcus lactis

基于pyrF筛选标记和来源于乳酸乳球菌(Lactococcus lactis,L.lactis)的基因组DNA为表达元件,构建L.lactis食品级表达载体,用于食品和药用多肽的表达和生产。首先,利用NZ3900 pyrF基因列构建同源重组突变盒,构建NZ3900 ΔpyrF突变株;然后,分别以来源于L.lactis的repA和repC基因为复制元件、pyrF基因为筛选标记、P_(32)和P_(8)为启动子、以及Tusp_(45)和TpepN为终止子,构建食品级表达质粒pLD;最后以绿色荧光蛋白ZsGreen为报告基因,验证ZsGreen在NZ3900 ΔpyrF突变株的表达及pLD-ZsG的遗传稳定性。实验结果表明,原养型ZsGreen阳性转化子可在普通Elliker培养基中正常生长,在荧光显微镜下观察到明显的绿色荧光信号;此外,PCR和Western blotting也证实ZsGreen能在NZ3900中表达且能稳定传代至30代,说明pLD食品级表达载体构建成功且使得外源蛋白在L.lactis中稳定表达。综上所述,基于pyrF营养缺陷型标记构建的L.lactis食品级表达载体的方法切实可行,可为推动L.lactis在食品和药用多肽生产中的应用提供研究基础。

In this study,the pyrF screening marker and the genomic DNA fragments were used to construct the expression vectors in food-grade Lactococcus lactis(L.lactis).Such expression system could potentially be used to express and produce food-grade and medicinal polypeptides.Firstly,the NZ3900ΔpyrF auxotrophic strain was created from the homologous recombination mutant cassette.Secondly,the repA and repC genes were used as the replication elements,the pyrF gene as the screening marker,the P_(32) and P_(8) elements from L.lactis as the promoters,and the Tusp_(45) and TpepN from L.lactis as the terminators,all of which were constructed in the expression plasmid pLD.Finally,the reporter gene ZsGreen(a fluorescent protein)was used to verify the expression of recombinant protein in the NZ3900 ΔpyrF mutant strain and the genetic stability of pLD-ZsG plasmid.The result showed that the prototrophic ZsGreen positive transformants could grow normally in common Elliker culture medium,and the green fluorescent signal was observed under a fluorescence microscope.In addition,ZsGreen protein could be highly expressed in NZ3900 ΔpyrF and the expression plasmid could be stably transmitted through at least 30 generations,according to the results of the PCR and Western blotting,indicating that the recombinant protein was expressed in L.lactis in a stable manner.Based on the above results,the approach for creating an L.lactis expression vector(without antibiotic resistance gene)based on the pyrF auxotrophic marker is feasible and offers a basis for further investigation into the use of L.lactis to manufacture food-and pharmaceutical-grade polypeptides.