食品级乳酸乳球菌pNZ8149-luc质粒的构建及表达
|
摘要
目的:构建能够稳定表达萤火虫荧光素酶报告基因(luc)的乳酸乳球菌(Lactococcus lactis, L.lactis)食品级表达系统,以便后续研究对目的基因进行示踪。方法:从pGL4.10质粒中PCR扩增萤火虫荧光素酶报告基因,测序,克隆至载体pNZ8149,构建pNZ8149-luc表达质粒;电击转化宿主乳酸乳球菌NZ3900,采用乳糖筛选法获得重组的乳酸乳球菌,Nisin诱导,采用微孔板发光检测仪检测荧光素酶的存在,Western Blot检测目标蛋白luc的表达。结果:PCR扩增的荧光素酶报告基因成功克隆至pNZ8149质粒,并电击转化宿主乳酸乳球菌NZ3900,得到乳酸乳球菌表达系统NZ3900/pNZ8149-luc。Nisin诱导后,检测到荧光素酶随诱导时间的延长活性逐渐增强,时间超过24 h之后荧光素酶活性逐渐下降。Western Blot检测到目标蛋白luc在胞内表达。结论:成功构建了p NZ8149-luc表达载体,并能够在乳酸乳球菌体内稳定表达。
Objective: A food-grade expression system for Lactococcus lactis was constructed to stably express the firefly luciferase reporter gene for subsequent study of the target gene. Methods: The luciferase reporter gene was amplified by PCR from pGL4.10 plasmid, sequenced and cloned into vector pNZ8149. The expression plasmid pNZ8149-luc was constructed, which was transformed into the host L.lactis NZ3900 by electric shock. Recombinant L.lactis was obtained by lactose screening and induced through using Nisin.The presence of luciferase was detected by microplate fluorescence detector and the expression of Luc was detected by Western Blot.Results: The luciferase reporter gene was successfully cloned into the pNZ8149 plasmid that was transformed into the host L.lactis NZ3900 to obtain the L.lactis expression system NZ3900/pNZ8149-luc. After induction by Nisin, the activity of luciferase increased gradually with the prolongation of induction time, and decreased gradually after 24 hours. Western Blot detected intracellular expression of the target protein luc. Conclusion: The pNZ8149-luc expression vector has been successfully constructed and can be expressed stably in L. lactis.
Objective: A food-grade expression system for Lactococcus lactis was constructed to stably express the firefly luciferase reporter gene for subsequent study of the target gene. Methods: The luciferase reporter gene was amplified by PCR from pGL4.10 plasmid, sequenced and cloned into vector pNZ8149. The expression plasmid pNZ8149-luc was constructed, which was transformed into the host L.lactis NZ3900 by electric shock. Recombinant L.lactis was obtained by lactose screening and induced through using Nisin.The presence of luciferase was detected by microplate fluorescence detector and the expression of Luc was detected by Western Blot.Results: The luciferase reporter gene was successfully cloned into the pNZ8149 plasmid that was transformed into the host L.lactis NZ3900 to obtain the L.lactis expression system NZ3900/pNZ8149-luc. After induction by Nisin, the activity of luciferase increased gradually with the prolongation of induction time, and decreased gradually after 24 hours. Western Blot detected intracellular expression of the target protein luc. Conclusion: The pNZ8149-luc expression vector has been successfully constructed and can be expressed stably in L. lactis.
引文
[1]Castro CPD,Drumond MM,Batista VL,et al.Vector development timeline for mucosal vaccination and treatment of disease using Lactococcus lactis and design approaches of next generation food grade plasmids[J].Frontiers in Microbiology,2018,9:1805
[2]Kobierecka PA,Olech B,Ksi諭ek M,et al.Cell wall anchoring of the campylobacter Antigens to Lactococcus lactis[J].Frontiers in Microbiology,2016,7:165
[3]Song AA,In LL,Lim SH,et al.A review on Lactococcus lactis:from food to factory[J].Microbial Cell Factories,2017,16(1):55-69
[4]Wyszyńska A,Kobierecka P,Bardowski J,et al.Lactic acid bacteria--20 years exploring their potential as live vectors for mucosal vaccination[J].Appl Microbiol Biotechnol,2015,99(7):2967-2977
[5]Loh JMS,Lorenz N,Tsai CJ,et al.Mucosal vaccination with pili from Group A.Streptococcus expressed on Lactococcus lactis generates protective immune responses[J].Sci Rep,2017,7(1):7174
[6]Yagnik B,Padh H,Desai P.Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis[J].Microbes&Infection,2016,18(4):237-244
[7]Sun Xiao-hui,Tang Xu,Wang Qing-hua,et al.Fusion expression of bifunctional enzyme complex for luciferin-recycling to enhance the luminescence imaging[J].J Photochem Photobiol B,2018,185:66-72
[8]Li Jing,Chen Lai-zhong,Du Lu-pei,et al.Cage the firefly luciferin!-a strategy for developing bioluminescent probes[J].Chemical Society Reviews,2012,42(2):662-676
[9]Dagil YA,Sharova VS,Pinegin BV,et al.A cell-based test system for the assessment of pharmacokinetics of NOD1 and NOD2 receptor agonists[J].International Immunopharmacology,2018,63:94-100
[10]Saiki P,Nakajima Y,Van Griensven LJLD,et al.Real-time monitoring of IL-6 and IL-10 reporter expression for anti-inflammation activity in live RAW 264.7 cells[J].Biochemical and Biophysical Research Communications,2018,505:885-890
[11]范园园,李铁,陈爽,等.用于活体成像裸鼠肺癌肿瘤模型的建立[J].中国兽医学报,2018,38(06):1177-1184
[12]韩笑笑,刘丽雅,谭超月,等.稳定表达荧光素酶基因的TC-1细胞系的构建[J].郑州大学学报(医学版),2018,53(04):429-432
[13]Holo H,Nes IF.High-frequency transformation,by electroporation,of Lactococcus lactis subsp.cremoris grown with glycine in osmotically stabilized media[J].Applied and Environmental Microbiology,1989,55(12):3119-3123
[14]Azizpour M,Hosseini SD,Jafari P,et al.Lac tococcus lactis:a new strategy for vaccination[J].Avicenna J Med Biotechnol,2017,9(4):163-168
[15]Vassaux G,Nitcheu J,Jezzard S,et al.Bacterial gene therapy strategies[J].J Pathol,2006,208(2):290-298
[16]Pellissery AJ,Nair UR.Lactic acid bacteria as mucosal delivery vaccine[J].Adv Anim Vet Sci,2013,1(6):183-187
[17]Azevedo MD,Meijerink M,Taverne N,et al.Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer[J].Vaccine,2015,33(38):4807-4812
[18]Bermúdez Humarán LG.Lactococcus lactis as a live vector for mucosal delivery of therapeutic proteins[J].Hum Vaccin,2009,5(4):264-267
[19]Sáez D,Fernández P,Rivera A,et al.Oral immunization of mice with recombinant Lactococcus lactis expressing Cu,Zn superoxide dismutase of Brucella abortus triggers protective immunity[J].Vaccine,2012,30(7):1283-1290
[20]彭晓燕,张荣光,段广才,等.幽门螺杆菌中性粒细胞激活蛋白在乳酸菌中的表达及免疫活性[J].中国卫生检验杂志,2017,27(10):1372-1374
[21]王莉平,韩先杰,王孝彬,等.表达猪流行性腹泻病毒S1蛋白的重组乳酸乳球菌的构建及其免疫原性的研究[J].中国兽医科学,2017,47(09):1118-1123
[22]Boucher I,Parrot M,Gaudreau H,et al.Novel food-grade plasmid vector based on melibiose fermentation for the genetic engineering of Lactococcus lactis[J].Applied and Environmental Microbiology,2002,68(12):6152-6161
[23]Loera-Arias MJ,Villatoro-Hernández J,Parga-Castillo MA,et al.Secretion of biologically active human interleukin 22(IL-22)by Lactococcus lactis[J].Biotechnology Letters,2014,36(12):2489-2494
[24]Wang Bo-Hua,Xu Yang,Li Yan-Ping.Use of the pyr G gene as a food-grade selection marker in Monascus[J].Biotechnology Letters,2010,32(11):1631-1635
[25]Ng DTW,Sarkar CA.Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000[J]Biotechnology&Bioengineering,2011,108(8):1987-1996
[26]王传文,周宏专,郭芳芳,等.表达空肠弯曲菌Cfr A蛋白重组乳酸乳球菌的构建及其免疫效果[J].微生物学报,2016,56(8):1326-1334
[27]Lehmann C,Sieg H,Lampen A,et al.Disturbance of firefly luciferase-based bioassays by different aluminum species[J].Analytical Biochemistry,2016,504:27-29
[28]程佳,庞田田,缑晶.利用荧光素酶报告基因检测维生素D生物活性[J].药物分析杂志,2017,37(07):1275-1279
[29]Zhang Fu-quan,Perez-Martin E,Juleff N,et al.A replication-competent foot-and-mouth disease virus expressing a luciferase reporter[J]Journal of Virological Methods,2017,247:38-44
[30]Becker JP,Clemens JR,Theile D,et al.Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays[J].Analytical Biochemistry,2016,509:124-129
[2]Kobierecka PA,Olech B,Ksi諭ek M,et al.Cell wall anchoring of the campylobacter Antigens to Lactococcus lactis[J].Frontiers in Microbiology,2016,7:165
[3]Song AA,In LL,Lim SH,et al.A review on Lactococcus lactis:from food to factory[J].Microbial Cell Factories,2017,16(1):55-69
[4]Wyszyńska A,Kobierecka P,Bardowski J,et al.Lactic acid bacteria--20 years exploring their potential as live vectors for mucosal vaccination[J].Appl Microbiol Biotechnol,2015,99(7):2967-2977
[5]Loh JMS,Lorenz N,Tsai CJ,et al.Mucosal vaccination with pili from Group A.Streptococcus expressed on Lactococcus lactis generates protective immune responses[J].Sci Rep,2017,7(1):7174
[6]Yagnik B,Padh H,Desai P.Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis[J].Microbes&Infection,2016,18(4):237-244
[7]Sun Xiao-hui,Tang Xu,Wang Qing-hua,et al.Fusion expression of bifunctional enzyme complex for luciferin-recycling to enhance the luminescence imaging[J].J Photochem Photobiol B,2018,185:66-72
[8]Li Jing,Chen Lai-zhong,Du Lu-pei,et al.Cage the firefly luciferin!-a strategy for developing bioluminescent probes[J].Chemical Society Reviews,2012,42(2):662-676
[9]Dagil YA,Sharova VS,Pinegin BV,et al.A cell-based test system for the assessment of pharmacokinetics of NOD1 and NOD2 receptor agonists[J].International Immunopharmacology,2018,63:94-100
[10]Saiki P,Nakajima Y,Van Griensven LJLD,et al.Real-time monitoring of IL-6 and IL-10 reporter expression for anti-inflammation activity in live RAW 264.7 cells[J].Biochemical and Biophysical Research Communications,2018,505:885-890
[11]范园园,李铁,陈爽,等.用于活体成像裸鼠肺癌肿瘤模型的建立[J].中国兽医学报,2018,38(06):1177-1184
[12]韩笑笑,刘丽雅,谭超月,等.稳定表达荧光素酶基因的TC-1细胞系的构建[J].郑州大学学报(医学版),2018,53(04):429-432
[13]Holo H,Nes IF.High-frequency transformation,by electroporation,of Lactococcus lactis subsp.cremoris grown with glycine in osmotically stabilized media[J].Applied and Environmental Microbiology,1989,55(12):3119-3123
[14]Azizpour M,Hosseini SD,Jafari P,et al.Lac tococcus lactis:a new strategy for vaccination[J].Avicenna J Med Biotechnol,2017,9(4):163-168
[15]Vassaux G,Nitcheu J,Jezzard S,et al.Bacterial gene therapy strategies[J].J Pathol,2006,208(2):290-298
[16]Pellissery AJ,Nair UR.Lactic acid bacteria as mucosal delivery vaccine[J].Adv Anim Vet Sci,2013,1(6):183-187
[17]Azevedo MD,Meijerink M,Taverne N,et al.Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer[J].Vaccine,2015,33(38):4807-4812
[18]Bermúdez Humarán LG.Lactococcus lactis as a live vector for mucosal delivery of therapeutic proteins[J].Hum Vaccin,2009,5(4):264-267
[19]Sáez D,Fernández P,Rivera A,et al.Oral immunization of mice with recombinant Lactococcus lactis expressing Cu,Zn superoxide dismutase of Brucella abortus triggers protective immunity[J].Vaccine,2012,30(7):1283-1290
[20]彭晓燕,张荣光,段广才,等.幽门螺杆菌中性粒细胞激活蛋白在乳酸菌中的表达及免疫活性[J].中国卫生检验杂志,2017,27(10):1372-1374
[21]王莉平,韩先杰,王孝彬,等.表达猪流行性腹泻病毒S1蛋白的重组乳酸乳球菌的构建及其免疫原性的研究[J].中国兽医科学,2017,47(09):1118-1123
[22]Boucher I,Parrot M,Gaudreau H,et al.Novel food-grade plasmid vector based on melibiose fermentation for the genetic engineering of Lactococcus lactis[J].Applied and Environmental Microbiology,2002,68(12):6152-6161
[23]Loera-Arias MJ,Villatoro-Hernández J,Parga-Castillo MA,et al.Secretion of biologically active human interleukin 22(IL-22)by Lactococcus lactis[J].Biotechnology Letters,2014,36(12):2489-2494
[24]Wang Bo-Hua,Xu Yang,Li Yan-Ping.Use of the pyr G gene as a food-grade selection marker in Monascus[J].Biotechnology Letters,2010,32(11):1631-1635
[25]Ng DTW,Sarkar CA.Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000[J]Biotechnology&Bioengineering,2011,108(8):1987-1996
[26]王传文,周宏专,郭芳芳,等.表达空肠弯曲菌Cfr A蛋白重组乳酸乳球菌的构建及其免疫效果[J].微生物学报,2016,56(8):1326-1334
[27]Lehmann C,Sieg H,Lampen A,et al.Disturbance of firefly luciferase-based bioassays by different aluminum species[J].Analytical Biochemistry,2016,504:27-29
[28]程佳,庞田田,缑晶.利用荧光素酶报告基因检测维生素D生物活性[J].药物分析杂志,2017,37(07):1275-1279
[29]Zhang Fu-quan,Perez-Martin E,Juleff N,et al.A replication-competent foot-and-mouth disease virus expressing a luciferase reporter[J]Journal of Virological Methods,2017,247:38-44
[30]Becker JP,Clemens JR,Theile D,et al.Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays[J].Analytical Biochemistry,2016,509:124-129