乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。
Lactococcus lactis is a good carrier for the delivery of therapeutic agents in vivo. To investigate its actual delivery in vivo, L. lactis needs to be labeled. In this study, L. lactis NZ9000 was labeled with an enhanced green fluorescent protein (eGFP) using the CRISPR/Cas9 system to study its transport in vivo and evaluate its function as a probiotic. Firstly, a recombinant plasmid pYSH carrying eGFP and homologous arms was constructed based on the existing L. lactis CRISPR/Cas9 editing plasmid pLL25 in our laboratory. Secondly, pYSH was electro transferred into L. lactis NZ9000 competent cells, replacing the lactate dehydrogenase (ldh) gene in the genome with the enhanced green fluorescent protein gene, thus enabling L. lactis NZ9000 to express a green fluorescent protein. Finally, we measured the fluorescence intensity of the green fluorescence-labeled L. lactis NZ9000 mutant. The quantitative results of fluorescence intensity showed that eGFP could be stably expressed in different growth stages of L. lactis NZ9000.
[1] 陈俊亮, 田芬, 霍贵成, 等.乳酸乳球菌对切达干酪成熟过程中质构和风味的影响[J].食品科学, 2013, 34(21):163-167.
CHEN J L, TIAN F, HUO G C, et al.Effect of Lactococcus lactis on texture and flavor of cheddar cheese during ripening[J].Food Science, 2013, 34(21):163-167.
[2] 赵贵丽, 罗爱平, 黄名正, 等.乳酸乳球菌豆腐生物凝固剂性能评价研究[J].食品工业, 2017, 38(10):146-149.
ZHAO G L, LUO A P, HUANG M Z, et al.Research on the performance evaluation of biological coagulant fermented by Lactococcus lacis[J].The Food Industry, 2017, 38(10):146-149.
[3] 司克辉. 优良乳酸菌菌株在凝固型酸奶中的生产应用[J].农业科技与信息, 2011(10):57-58.
SI K H.Application of good Lactobacillus strains in the production of solidified yoghurt[J].Information of Agricultural Science and Technology, 2011(10):57-58.
[4] YU J, SONG Y Q, REN Y, et al.Genome-level comparisons provide insight into the phylogeny and metabolic diversity of species within the genus Lactococcus[J].BMC Microbiology, 2017, 17(1):213.
[5] SONG A A L, IN L L A, LIM S H E, et al.A review on Lactococcus lactis:From food to factory[J].Microbial Cell Factories, 2017, 16(1):55.
[6] GARCIA-FRUITORS E.Lactic acid bacteria:A promising alternative for recombinant protein production[J].Microbial Cell Factories, 2012, 11(1):157.
[7] LINARES D M, KOK J, POOLMAN B.Genome sequences of Lactococcus lactis MG1363 (revised) and NZ9000 and comparative physiological studies[J].Journal of Bacteriology, 2010, 192(21):5 806-5 812.
[8] MORELLO E, BERMU'DEZ-HUMARÁN L G, LLULL D, et al. Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion[J].Journal of Molecular Microbiology & Biotechnology, 2008, 14:48-58.
[9] GU Q, SONG D F, ZHU M Y.Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B[J].FEMS Immunology & Medical Microbiology, 2009, 56(3):197-203.
[10] MISHRA V, PRASAD D N.Application of in vitro methods for selection of Lactobacillus casei strain as potential probiotics[J].International Journal of Food Microbiology, 2005, 103(1):109-115.
[11] SILVIA B, VALENTINA T, GRETA R, et al.The vaginal isolate Lactobacillus paracasei LPC-S01 (DSM26760) is suitable for oral administration[J].Frontiers in Microbiology, 2015, 6:952.
[12] CHAMPS C D, MARONCLE N, BALESTRINO D, et al.Persistence of colonization of intestinal mucosa by a probiotic strain, Lactobacillus casei subsp. rhamnosus Lcr35, after oral consumption[J].Journal of Clinical Microbiology, 2003, 41(3):1 270-1 273.
[13] 陈佩, 党辉, 贺国旗, 等.一株干酪乳杆菌生物学特性及其发酵豆乳的研究[J].食品研究与开发, 2017, 38(17):17-21.
CHEN P, DANG H, HE G Q, et al.Study on the biological characteristics of Lactobacillus casei and application in the fermented soymilk produce[J].Food Research and Development, 2017, 38(17):17-21.
[14] KONG L H, XIONG Z Q, SONG X, et al.Characterization of a panel of strong constitutive promoters from Streptococcus thermophilus for fine-tuning gene expression[J].Acs Synthetic Biology, 2019, 8(6):1 469-1 472.
[15] MIERAU I, KLEEREBEZEM M.10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis[J].Applied Microbiology and Biotechnology, 2005, 68(6):705-717.
[16] ZHU D L, LIU F L, XU H J, et al.Isolation of strong constitutive promoters from Lactococcus lactis subsp.lactis N8[J].FEMS Microbiology Letters, 2015, 362(16):fnv107.
[17] SONG X, ZHANG X Y, XIONG Z Q, et al.CRISPR-Cas-mediated gene editing in lactic acid bacteria[J].Molecular Biology Reports, 2020, 47(10):8 133-8 144.
[18] SONG X, LIU L, LIU X X, et al.Single plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis[J].Journal of Dairy Science, 2021, 104(10):10 576-10 585.
[19] 韦云莹, 王立峰, 熊智强, 等.响应面法优化乳酸乳球菌电转化效率研究[J].上海理工大学学报, 2018, 40(6):566-571.
WEI Y Y, WANG L F, XIONG Z Q, et al.Optimization of electroporation efficiency of Lactococcus lactis using response surface methodology[J].Journal of University of Shanghai for Science and Technology, 2018, 40(6):566-571.
[20] 刘璐, 艾连中, 夏永军, 等.乳酸乳球菌NZ9000中增强型绿色荧光蛋白报告系统的构建[J].食品与发酵工业, 2020, 46(11):46-51.
LIU L, AI L Z, XIA Y J, et al.A reporter system with the enhanced green fluorescent protein in Lactococcus lactis NZ9000[J].Food and Fermentation Industries, 2020, 46(11):46-51.
[21] 刘璇. 细胞荧光探针的合成与应用[D].长沙:湖南师范大学, 2012.
LIU X.Synthesis and application of cellular fluorescent probes[D].Changsha:Hunan Normal University, 2012.
