PlnF抗菌肽在乳酸乳球菌中的分泌表达及抑菌活性鉴定
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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。
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.
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.
引文
[1]邓泽静,李毅,洪程基,等.食品中金黄色葡萄球菌污染状况及其毒素检测[J].浙江预防医学,2011,23(2):92-93.DOI:10.3969/j.issn.1007-0931.2011.02.043.
[2]冯疆蓉,李春杰.生鲜乳有害微生物污染与危害分析[J].草业科学,2016,33(9):1875-1892.DOI:10.11829/j.issn.1001-0629.2015-0705.
[3]赵凤亭,程振云,徐莉娟,等.耐甲氧西林金黄色葡萄球菌的毒力基因检测及研究[J].中国病原生物学杂志,2015,10(10):931-935.DOI:10.13350/j.cjpb.151016.
[4]孙强正,熊衍文,叶长芸,等.食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达[J].微生物学报,2008(3):293-298.DOI:10.13343/j.cnki.wsxb.2008.03.003.
[5]任大勇,荣凤君,宫圣洁,等.发酵食品乳酸菌分离鉴定及功能特性研究[J].食品研究与开发,2016,37(19):194-199.DOI:10.3969/j.issn.1005-6521.2016.19.044.
[6]任大勇,荣凤君,宫圣洁,等.不同来源乳酸菌对2种致病菌的抑菌活性比较及抑菌性质研究[J].食品工程,2015(4):41-44.DOI:10.3969/j.issn.1673-6044.2015.04.012.
[7]REN D Y,GONG S J,SHU J Y,et al.Mixed Lactobacillus plantarum strains inhibit Staphylococcus aureus induced inflammation and ameliorate intestinal microflora in mice[J].BioMed Research International,2017,2017:7476467.DOI:10.1155/2017/7476467.
[8]王翠艳,王玉华,朴春红,等.绿色木霉纤维二糖水解酶基因的克隆及其在乳酸菌中的表达与鉴定[J].食品科学,2016,37(19):141-146.DOI:10.7506/spkx1002-6630-201619024.
[9]WANG Z H,CAO X H,DU X G,et al.Mucosal and systemic immunity in mice after intranasal immunization with recombinant Lactococcus lactis expressing ORF6 of PRRSV[J].Cellular Immunology,2014,287(2):69-73.DOI:10.1016/j.cellimm.2013.12.004.
[10]马超,王丕武,付永平,等.乳酸乳球菌食品级表达载体的改造(英文)[J].Agricultural Science&Technology,2010,11(4):59-63.DOI:10.3969/j.issn.1009-4229-B.2010.04.018.
[11]HOLO H,NES I F.High-frequency transformation by electroporation of Lactococcus lactis subsp.cremoris grown with glycine in osmotically stabilized media[J].Applied and Environmental Mircobiology,989,55(12):3119-3123.
[12]潘荣荣,王茂鹏,李昌,等.乳酸菌破壁方法的比较研究[J].食品工业科技,2017,38(12):3 4-3 8.D O I:1 0.1 3 3 8 6/j.issn1002-0306.2017.12.007.
[13]张波,李晨,董慧,等.1种快速高效提取乳酸菌质粒的方法[J].中国食品学报,2014,14(9):7-13.DOI:10.16429/j.1009-7848.2014.09.001.
[14]任大勇,李昌,秦艳青,等.乳酸乳球菌高效电转化方法的建立[J].中国兽药杂志,2012,46(1):5-9.DOI:10.3969/j.issn.1002-1280.2012.01.002.
[15]孟庆森,姜艳平,崔文,等.两段牛乳铁蛋白肽基因在乳酸乳球菌中的共表达[J].畜牧与兽医,2013,45(4):1-4.
[16]张小娟,张荣光,段广才,等.乳酸乳球菌NZ3900电转化条件的优化[J].山东医药,2010,50(11):53-55.DOI:10.3969/j.issn.1002-266X.2010.11.019.
[17]张艳丽,聂春明,周利伟,等.牛凝乳酶原基因在食品级乳酸乳球菌中的重组表达[J].食品科学,2014,35(7):123-127.DOI:10.7506/spkx1002-6630-201407025.
[18]KUDA T,SARENGAOLE,TAKAHASHI H,et al.Alcohol-brewing properties of acid-and bile-tolerant yeasts co-cultured with lactic acid bacteria isolated from traditional handmade domestic dairy products from Inner Mongolia[J].LWT-Food Science and Technology,2016,65:62-69.DOI:10.1016/j.lwt.2015.07.065.
[19]张小娟,张荣光,段广才.幽门螺杆菌尿素酶B在乳酸乳球菌中表达与优化[J].中国公共卫生,2011,27(9):1134-1136.DOI:10.11847/zgggws2011-27-09-27.
[20]任大勇,李昌,秦艳青,等.植物乳杆菌melA基因的克隆及其作为食品级筛选标记的初步研究[J].食品科学,2011,32(9):135-139.
[21]ZHANG W J,WANG X M,DAI L,et al.Novel conjugative plasmid from Escherichia coli of swine origin that coharbors the multiresistance gene cfr and the extended-spectrum-β-lactamase gene bla CTX-M-14b[J].Antimicrobial Agents&Chemotherapy,2015,59(2):1337-1340.DOI:10.1128/AAC.04631-14.
[22]钟平.对临床微生物检验及细菌耐药性的监测分析[J].中国社区医师,2016,32(31):17-18.
[23]任璐雅,杨艳艳,章检明,等.乳酸菌抗菌肽(细菌素)抗菌机理的研究进展[J].中国食品添加剂,2015(1):143-149.DOI:10.3969/j.issn.1006-2513.2015.01.018.
[24]李冠楠,夏雪娟,隆耀航,等.抗菌肽的研究进展及其应用[J].动物营养学报,2014,26(1):17-25.DOI:10.3969/j.issn.1006-267x.2014.01.003.
[25]MENOUSEK J,MISHRA B,HANKE M L,et al.Database screening and in vivo efficacy of antimicrobial peptides against methicillinresistant Staphylococcus aureus USA300[J].International Journal of Antimicrobial Agents,2012,39(5):402-406.DOI:10.1016/j.ijantimicag.2012.02.003.
[26]MENG F,ZHAO H,ZHANG C,et al.Expression of a novel bacteriocin-the plantaricin Pln1-in Escherichia coli and its functional analysis[J].Protein Expression&Purification,2015,119:85-93.DOI:10.1016/j.pep.2015.11.008.
[27]JIANG H,LI P,GU Q.Heterologous expression and purification of plantaricin NC8,a two-peptide bacteriocin against Salmonella spp.from Lactobacillus plantarum ZJ316[J].Protein Expression&Purification,2016,127:28.DOI:10.1016/j.pep.2016.06.013.
[28]MENG D M,DAI H X,GAO X F,et al.Expression,purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris[J].Protein Expression&Purification,2016,127:35-43.DOI:10.1016/j.pep.2016.07.001.
[29]谭嘉圣.人源及杂合抗菌肽在乳酸乳球菌中重组分泌表达[D].广州:南方医科大学,2015.
[30]李朴,文阳安,刘靳波,等.抗菌肽Bactenecin7重组质粒构建及其在乳酸菌的分泌表达和活性鉴定[J].中国生物工程杂志,2009,29(1):70-74.DOI:10.13523/j.cb.20090114.
[2]冯疆蓉,李春杰.生鲜乳有害微生物污染与危害分析[J].草业科学,2016,33(9):1875-1892.DOI:10.11829/j.issn.1001-0629.2015-0705.
[3]赵凤亭,程振云,徐莉娟,等.耐甲氧西林金黄色葡萄球菌的毒力基因检测及研究[J].中国病原生物学杂志,2015,10(10):931-935.DOI:10.13350/j.cjpb.151016.
[4]孙强正,熊衍文,叶长芸,等.食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达[J].微生物学报,2008(3):293-298.DOI:10.13343/j.cnki.wsxb.2008.03.003.
[5]任大勇,荣凤君,宫圣洁,等.发酵食品乳酸菌分离鉴定及功能特性研究[J].食品研究与开发,2016,37(19):194-199.DOI:10.3969/j.issn.1005-6521.2016.19.044.
[6]任大勇,荣凤君,宫圣洁,等.不同来源乳酸菌对2种致病菌的抑菌活性比较及抑菌性质研究[J].食品工程,2015(4):41-44.DOI:10.3969/j.issn.1673-6044.2015.04.012.
[7]REN D Y,GONG S J,SHU J Y,et al.Mixed Lactobacillus plantarum strains inhibit Staphylococcus aureus induced inflammation and ameliorate intestinal microflora in mice[J].BioMed Research International,2017,2017:7476467.DOI:10.1155/2017/7476467.
[8]王翠艳,王玉华,朴春红,等.绿色木霉纤维二糖水解酶基因的克隆及其在乳酸菌中的表达与鉴定[J].食品科学,2016,37(19):141-146.DOI:10.7506/spkx1002-6630-201619024.
[9]WANG Z H,CAO X H,DU X G,et al.Mucosal and systemic immunity in mice after intranasal immunization with recombinant Lactococcus lactis expressing ORF6 of PRRSV[J].Cellular Immunology,2014,287(2):69-73.DOI:10.1016/j.cellimm.2013.12.004.
[10]马超,王丕武,付永平,等.乳酸乳球菌食品级表达载体的改造(英文)[J].Agricultural Science&Technology,2010,11(4):59-63.DOI:10.3969/j.issn.1009-4229-B.2010.04.018.
[11]HOLO H,NES I F.High-frequency transformation by electroporation of Lactococcus lactis subsp.cremoris grown with glycine in osmotically stabilized media[J].Applied and Environmental Mircobiology,989,55(12):3119-3123.
[12]潘荣荣,王茂鹏,李昌,等.乳酸菌破壁方法的比较研究[J].食品工业科技,2017,38(12):3 4-3 8.D O I:1 0.1 3 3 8 6/j.issn1002-0306.2017.12.007.
[13]张波,李晨,董慧,等.1种快速高效提取乳酸菌质粒的方法[J].中国食品学报,2014,14(9):7-13.DOI:10.16429/j.1009-7848.2014.09.001.
[14]任大勇,李昌,秦艳青,等.乳酸乳球菌高效电转化方法的建立[J].中国兽药杂志,2012,46(1):5-9.DOI:10.3969/j.issn.1002-1280.2012.01.002.
[15]孟庆森,姜艳平,崔文,等.两段牛乳铁蛋白肽基因在乳酸乳球菌中的共表达[J].畜牧与兽医,2013,45(4):1-4.
[16]张小娟,张荣光,段广才,等.乳酸乳球菌NZ3900电转化条件的优化[J].山东医药,2010,50(11):53-55.DOI:10.3969/j.issn.1002-266X.2010.11.019.
[17]张艳丽,聂春明,周利伟,等.牛凝乳酶原基因在食品级乳酸乳球菌中的重组表达[J].食品科学,2014,35(7):123-127.DOI:10.7506/spkx1002-6630-201407025.
[18]KUDA T,SARENGAOLE,TAKAHASHI H,et al.Alcohol-brewing properties of acid-and bile-tolerant yeasts co-cultured with lactic acid bacteria isolated from traditional handmade domestic dairy products from Inner Mongolia[J].LWT-Food Science and Technology,2016,65:62-69.DOI:10.1016/j.lwt.2015.07.065.
[19]张小娟,张荣光,段广才.幽门螺杆菌尿素酶B在乳酸乳球菌中表达与优化[J].中国公共卫生,2011,27(9):1134-1136.DOI:10.11847/zgggws2011-27-09-27.
[20]任大勇,李昌,秦艳青,等.植物乳杆菌melA基因的克隆及其作为食品级筛选标记的初步研究[J].食品科学,2011,32(9):135-139.
[21]ZHANG W J,WANG X M,DAI L,et al.Novel conjugative plasmid from Escherichia coli of swine origin that coharbors the multiresistance gene cfr and the extended-spectrum-β-lactamase gene bla CTX-M-14b[J].Antimicrobial Agents&Chemotherapy,2015,59(2):1337-1340.DOI:10.1128/AAC.04631-14.
[22]钟平.对临床微生物检验及细菌耐药性的监测分析[J].中国社区医师,2016,32(31):17-18.
[23]任璐雅,杨艳艳,章检明,等.乳酸菌抗菌肽(细菌素)抗菌机理的研究进展[J].中国食品添加剂,2015(1):143-149.DOI:10.3969/j.issn.1006-2513.2015.01.018.
[24]李冠楠,夏雪娟,隆耀航,等.抗菌肽的研究进展及其应用[J].动物营养学报,2014,26(1):17-25.DOI:10.3969/j.issn.1006-267x.2014.01.003.
[25]MENOUSEK J,MISHRA B,HANKE M L,et al.Database screening and in vivo efficacy of antimicrobial peptides against methicillinresistant Staphylococcus aureus USA300[J].International Journal of Antimicrobial Agents,2012,39(5):402-406.DOI:10.1016/j.ijantimicag.2012.02.003.
[26]MENG F,ZHAO H,ZHANG C,et al.Expression of a novel bacteriocin-the plantaricin Pln1-in Escherichia coli and its functional analysis[J].Protein Expression&Purification,2015,119:85-93.DOI:10.1016/j.pep.2015.11.008.
[27]JIANG H,LI P,GU Q.Heterologous expression and purification of plantaricin NC8,a two-peptide bacteriocin against Salmonella spp.from Lactobacillus plantarum ZJ316[J].Protein Expression&Purification,2016,127:28.DOI:10.1016/j.pep.2016.06.013.
[28]MENG D M,DAI H X,GAO X F,et al.Expression,purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris[J].Protein Expression&Purification,2016,127:35-43.DOI:10.1016/j.pep.2016.07.001.
[29]谭嘉圣.人源及杂合抗菌肽在乳酸乳球菌中重组分泌表达[D].广州:南方医科大学,2015.
[30]李朴,文阳安,刘靳波,等.抗菌肽Bactenecin7重组质粒构建及其在乳酸菌的分泌表达和活性鉴定[J].中国生物工程杂志,2009,29(1):70-74.DOI:10.13523/j.cb.20090114.
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