金黄色葡萄球菌肠毒素SEK的纯化及DAS-ELISA检测方法的建立与应用
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摘要
本研究目的为建立一种快速检测金黄色葡萄球菌新型肠毒素SEK的双抗夹心酶联免疫吸附方法。将构建的原核表达载体pET-28a(+)-ΔNspSEK转化入BL 21(DE3)pLysS细胞中诱导表达,经Ni~(2+)-NTA亲和层析柱纯化获得重组SEK蛋白作为抗原。利用双抗夹心酶联免疫方法检测程序,确定抗SEK单克隆抗体及抗SEK多克隆血清的最佳稀释度,并使用该方法应用于检测SEK人工污染样品的加标回收率和茶多酚及乳酸链球菌素(Nisin)对7株sek阳性菌株的SEK蛋白分泌影响。结果表明,原核表达载体得到可溶性表达,重组SEK蛋白分子量约为27.7ku;建立的双抗夹心酶联免疫检测方法中,单克隆抗体最佳包被浓度为2.89μg/mL、抗血清的最佳稀释度为1:500,回归方程为y=0.2165x+0.1627,相关系数R~2=0.9993,最低检测限为0.1μg/mL;检测SEK人工污染脱脂奶、LB肉汤和牛肉糜中的加标回收率高达97%以上;并且,采用建立的方法测得茶多酚和Nisin的添加浓度在其MIC及以下浓度时,茶多酚对7株sek阳性菌株蛋白分泌抑制效果更明显。
To measure a newly recognized staphylococcal enterotoxin K(SEK), a double-antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) was established in this study. The recombinant protein SEK was expressed in BL21(DE3) pLysS cells and purified by Ni~(2+)-NTA affinity chromatography. Using the protein as an antigen, the monoclonal antibody and polyclonal antibodies were prepared. They were employed for detection and capture of SEK in the DAS-ELISA system that was developed to be capable of detecting SEK in piked skimmed milk, LB medium and minced beef. Subsequently, the ELISA system was applied to determine SEK secretion for 7 sek positive S. aureus isolates with tea polyphenols and Nisin treatment. The results showed that the recombinant protein SEK was successfully expressed and purified with the expected molecular weight of 27.7 ku. The DAS-ELISA for SEK was developed with the optimal anti-SEK monoclonal antibody concentration 2.89 μg/m L, the serum dilution ratio 1:500, the regression equation y=0.2165x+0.1627(R~2=0.9993), and the sensitivity 0.1 μg/mL. Using the developed ELISA assay, the recovery rates of SEK in spiked skimmed milk, LB medium and minced beef were more than 97%. Furthermore, both tea polyphenols and Nisin, especially tea polyphenols, inhibited the secretion of SEK at the concentration of MIC.
To measure a newly recognized staphylococcal enterotoxin K(SEK), a double-antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) was established in this study. The recombinant protein SEK was expressed in BL21(DE3) pLysS cells and purified by Ni~(2+)-NTA affinity chromatography. Using the protein as an antigen, the monoclonal antibody and polyclonal antibodies were prepared. They were employed for detection and capture of SEK in the DAS-ELISA system that was developed to be capable of detecting SEK in piked skimmed milk, LB medium and minced beef. Subsequently, the ELISA system was applied to determine SEK secretion for 7 sek positive S. aureus isolates with tea polyphenols and Nisin treatment. The results showed that the recombinant protein SEK was successfully expressed and purified with the expected molecular weight of 27.7 ku. The DAS-ELISA for SEK was developed with the optimal anti-SEK monoclonal antibody concentration 2.89 μg/m L, the serum dilution ratio 1:500, the regression equation y=0.2165x+0.1627(R~2=0.9993), and the sensitivity 0.1 μg/mL. Using the developed ELISA assay, the recovery rates of SEK in spiked skimmed milk, LB medium and minced beef were more than 97%. Furthermore, both tea polyphenols and Nisin, especially tea polyphenols, inhibited the secretion of SEK at the concentration of MIC.
引文
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[13]Nagaraj S,Ramlal S,Kingston J,et al.Development of IgYbased sandwich ELISA for the detection of staphylococcal enterotoxin G(SEG),an egc toxin[J].International Journal of Food Microbiology,2016,237:136-141
[14]朱安妮,唐俊妮,赵燕英,等.金黄色葡萄球菌新型肠毒素Ⅰ双抗夹心-酶联免疫检测方法的建立[J].食品科学,2016,37(16):193-198ZHU An-ni,TANG Jun-ni,ZHAO Yan-ying,et al.Development of a double-antibody sandwich enzyme linked immunosorbent assay for detection of staphylococcal enterotoxin I(SEI)[J].Food Science,2016,37(16):193-198
[15]Zhao Y Y,Zhu A N,Tang J N,et al.Identification and measurement of staphylococcal enterotoxin M from Staphylococcus aureus isolate associated with staphylococcal food poisoning[J].Letters in Applied Microbiology,2017,65(1):27
[16]杜玄,赵燕英,刘骥,等.金黄色葡萄球菌肠毒素P双抗夹心酶联免疫检测方法的建立[J].现代食品科技,2018,34(4):264-269DU Xuan,ZHAO Yan-ying,LIU Ji,et al.Development of a double-antibody sandwich enzyme linked immunosorbent assay for detection of staphylococcal enterotoxin P[J].Modern Food Science and Technology,2018,34(4):264-269
[17]刘骥,潘洁,田万帆,等.新型肠毒素蛋白SEl K编码基因的克隆测序、生物信息学分析和表达载体构建[J].西南民族大学学报:自然科学版,2016,42(3):274-280LIU Ji,PAN Jie,TIAN Wan-fan,et al.Cloning,sequence bioinformatic analysis and construction of expression vector for the newly identified staphylococcal enterotoxins SEIK-encoding Gene[J].Journal of Southwest Minzu University(Natural Science Edition),2016,42(3):274-280
[18]田万帆,刘骥,赵燕英,等.金黄色葡萄球菌新型肠毒素SEK原核表达、纯化及溶液构象分析[J].食品科学2019,40(4):138-145TIAN Wan-fan,LIU Ji,ZHAO Yan-ying,et al.Prokaryotic expression,purification,and solution conformation of staphylococcal enterot eoxin K[J].Food Science,2019,40(4):138-145
[19]Sparding N,Slotved H C,Nicolaisen G M,et al.Development of an ELISA for evaluation of swab recovery efficiencies of bovine serum albumin[J].Plos One,2014,9(11):378-390
[20]马伊萨兰,张荣,王洪志,等.食品中金黄色葡萄球菌生物被膜形成基因分析及影响因素研究[J].食品工业科技,2017,38(15):129-133MA Yi-sa-lan,ZHANG Rong,WANG Hong-zhi,et al.Analysis of biofilm formation related genes and its influence factors for Staphylococcus aures food isolates[J].Science and Technology of Food Industry,2017,38(15):129-133
[21]Cheng J,Wang Y,Cao Y,et al.The distribution of 18enterotoxin and enterotoxin-like genes in Staphylococcus aureus strains from different sources in east China[J].Foodborne Pathogens&Disease,2016,13(4):171
[22]Varshney A K,Mediavilla J R,Robiou N,et al.Diverse enterotoxin gene profiles among clonal complexes of Staphylococcus aureus isolates from the Bronx,New York[J].Applied&Environmental Microbiology,2009,75(21):6839
[23]Aguilar J L,Varshney A K,Wang X,et al.Detection and measurement of staphylococcal enterotoxin-like K(SEl-K)secretion by Staphylococcus aureus clinical isolates[J].Journal of Clinical Microbiology,2014,52(7):2536-2543
[24]Wu D J,Shen X Z,Li X M,et al.OL-064 Superantigen gene profiles and presence of exfoliative toxin genes in community-acquired meticillin-resistant Staphylococcus aureus,isolated from Chinese children[J].International Journal of Infectious Diseases,2011,15(1):S36-S36
[25]Orwin P M,Leung D Y M,Donahue H L,et al.Biochemical and biological properties of staphylococcal enterotoxin K[J].Infection&Immunity,2001,69(1):360
[26]Omoe K,Hu D L,Ono H K,et al.Emetic potentials of newly identified staphylococcal enterotoxin-like toxins[J].Infection and Immunity,2013,81(10):3627-3631
[27]李柯欣.茶多酚的提取、抑菌作用与抑菌机理研究[D].成都:西华大学,2017LI Ke-xin.Study on extraction,bacteriostasis and bacteriostatic mechanism of tea polyphenols[D].Chengdu:Xihua University,2017
[28]王琼,唐俊妮,汤承,等.6种食品防腐剂对金黄色葡萄球菌抑菌效果及肠毒素基因表达的影响[J].食品科学,2016,37(21):151-156WANG Qiong,TANG Jun-ni,TANG Cheng,et al.Inhibitory effect of six food antiseptics on the growth and staphylococcal enterotoxin(SE)gene expression of Staphylococcus aureus[J].Food Science,2016,37(21):151-156
[29]胡艳军,夏素琴,陈雨欣.茶多酚联合万古霉素对耐甲氧西林金黄色葡萄球菌生物膜的影响[J].检验医学与临床,2017,14(20):2977-2979HU Yan-jun,XIA Su-qin,CHEN Yu-xin.Effects of tea polyphenols and vancomycin on biofilm of methicillin-resistant Staphylococcus aureus[J].Laboratory Medicine and Clinic,2017,14(20):2977-2979
[2]Atanassova V,Meindl A,Ring C.Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham-a comparison of classical culturing detection and RFLP-PCR[J].International Journal of Food Microbiology,2001,68(1):105-113
[3]Tirado C,Schmidt K.WHO surveillance programme for control of foodborne infections and intoxications:preliminary results and trends across greater Europe.world health organization[J].Journal of Infection,2001,43(1):80-84
[4]Kadariya J,Smith T C,Thapaliya D.Staphylococcus aureus and staphylococcal food-borne disease:an ongoing challenge in public health[J].Biomed Research International,2014,827965
[5]黄嘉慧,吴诗,张峰,等.我国市售食用菌中金黄色葡萄球菌污染调查、耐药性及其肠毒素基因检测[J].食品科学技术学报,2018,36(3),25-32HUANG Jia-hui,WU Shi,ZHANG Feng,et al.Prevalence,antibiotic resistance,and enterotoxin gene detection of Staphylococcus aureus isolated from retail edible mushrooms in China[J].Journal of Food Science and Technology,2018,36(3):25-32
[6]Marrack P,Kappler J.The staphylococcal enterotoxins and their relatives[J].Science,1990,248(4956):705-711
[7]Le L Y,Baron F,Gautier M.Staphylococcus aureus and food poisoning[J].Genetics&Molecular Research,2003,2(1):63-76
[8]Pinchuk I V,Beswick E J,Reyes V E.Staphylococcal enterotoxins[J].Voprosy Pitaniia,2010,2(8):2177
[9]李琼琼,范一灵,宋明辉,等.食源性金黄色葡萄球菌肠毒素及其检测方法[J].食品安全质量检测学报,2016,7(2):555-560LI Qiong-qiong,FAN Yi-ling,SONG Ming-hui,et al.Research advances on foodborne staphylococcal enterotoxins and its detection methods[J].Journal of Food Safety and Quality,2016,7(2):555-560
[10]Wu S,Duan N,Ma X,et al.A highly sensitive fluorescence resonance energy transfer aptasensor for staphylococcal enterotoxin B detection based on exonuclease-catalyzed target recycling strategy[J].Analytica Chimica Acta,2013,782(9):59-66
[11]王华,潘家荣,熊汉国.免疫分析技术在食品安全检测中的应用现状[J].食品科技,2007,6:34-37WANG Hua,PAN Jia-rong,XIONG Han-guo.Application of immunoassay methods in food safety[J].Food Science and Technology,2007,6:34-37
[12]Postmes T J,Hout J C V,Saat G,et al.A radioimmunoassay study and comparison of seasonal variation in plasma triiodothyronine and thyroxine concentrations in normal healthy persons[J].Clinica Chimica Acta,1974,50(2):189-195
[13]Nagaraj S,Ramlal S,Kingston J,et al.Development of IgYbased sandwich ELISA for the detection of staphylococcal enterotoxin G(SEG),an egc toxin[J].International Journal of Food Microbiology,2016,237:136-141
[14]朱安妮,唐俊妮,赵燕英,等.金黄色葡萄球菌新型肠毒素Ⅰ双抗夹心-酶联免疫检测方法的建立[J].食品科学,2016,37(16):193-198ZHU An-ni,TANG Jun-ni,ZHAO Yan-ying,et al.Development of a double-antibody sandwich enzyme linked immunosorbent assay for detection of staphylococcal enterotoxin I(SEI)[J].Food Science,2016,37(16):193-198
[15]Zhao Y Y,Zhu A N,Tang J N,et al.Identification and measurement of staphylococcal enterotoxin M from Staphylococcus aureus isolate associated with staphylococcal food poisoning[J].Letters in Applied Microbiology,2017,65(1):27
[16]杜玄,赵燕英,刘骥,等.金黄色葡萄球菌肠毒素P双抗夹心酶联免疫检测方法的建立[J].现代食品科技,2018,34(4):264-269DU Xuan,ZHAO Yan-ying,LIU Ji,et al.Development of a double-antibody sandwich enzyme linked immunosorbent assay for detection of staphylococcal enterotoxin P[J].Modern Food Science and Technology,2018,34(4):264-269
[17]刘骥,潘洁,田万帆,等.新型肠毒素蛋白SEl K编码基因的克隆测序、生物信息学分析和表达载体构建[J].西南民族大学学报:自然科学版,2016,42(3):274-280LIU Ji,PAN Jie,TIAN Wan-fan,et al.Cloning,sequence bioinformatic analysis and construction of expression vector for the newly identified staphylococcal enterotoxins SEIK-encoding Gene[J].Journal of Southwest Minzu University(Natural Science Edition),2016,42(3):274-280
[18]田万帆,刘骥,赵燕英,等.金黄色葡萄球菌新型肠毒素SEK原核表达、纯化及溶液构象分析[J].食品科学2019,40(4):138-145TIAN Wan-fan,LIU Ji,ZHAO Yan-ying,et al.Prokaryotic expression,purification,and solution conformation of staphylococcal enterot eoxin K[J].Food Science,2019,40(4):138-145
[19]Sparding N,Slotved H C,Nicolaisen G M,et al.Development of an ELISA for evaluation of swab recovery efficiencies of bovine serum albumin[J].Plos One,2014,9(11):378-390
[20]马伊萨兰,张荣,王洪志,等.食品中金黄色葡萄球菌生物被膜形成基因分析及影响因素研究[J].食品工业科技,2017,38(15):129-133MA Yi-sa-lan,ZHANG Rong,WANG Hong-zhi,et al.Analysis of biofilm formation related genes and its influence factors for Staphylococcus aures food isolates[J].Science and Technology of Food Industry,2017,38(15):129-133
[21]Cheng J,Wang Y,Cao Y,et al.The distribution of 18enterotoxin and enterotoxin-like genes in Staphylococcus aureus strains from different sources in east China[J].Foodborne Pathogens&Disease,2016,13(4):171
[22]Varshney A K,Mediavilla J R,Robiou N,et al.Diverse enterotoxin gene profiles among clonal complexes of Staphylococcus aureus isolates from the Bronx,New York[J].Applied&Environmental Microbiology,2009,75(21):6839
[23]Aguilar J L,Varshney A K,Wang X,et al.Detection and measurement of staphylococcal enterotoxin-like K(SEl-K)secretion by Staphylococcus aureus clinical isolates[J].Journal of Clinical Microbiology,2014,52(7):2536-2543
[24]Wu D J,Shen X Z,Li X M,et al.OL-064 Superantigen gene profiles and presence of exfoliative toxin genes in community-acquired meticillin-resistant Staphylococcus aureus,isolated from Chinese children[J].International Journal of Infectious Diseases,2011,15(1):S36-S36
[25]Orwin P M,Leung D Y M,Donahue H L,et al.Biochemical and biological properties of staphylococcal enterotoxin K[J].Infection&Immunity,2001,69(1):360
[26]Omoe K,Hu D L,Ono H K,et al.Emetic potentials of newly identified staphylococcal enterotoxin-like toxins[J].Infection and Immunity,2013,81(10):3627-3631
[27]李柯欣.茶多酚的提取、抑菌作用与抑菌机理研究[D].成都:西华大学,2017LI Ke-xin.Study on extraction,bacteriostasis and bacteriostatic mechanism of tea polyphenols[D].Chengdu:Xihua University,2017
[28]王琼,唐俊妮,汤承,等.6种食品防腐剂对金黄色葡萄球菌抑菌效果及肠毒素基因表达的影响[J].食品科学,2016,37(21):151-156WANG Qiong,TANG Jun-ni,TANG Cheng,et al.Inhibitory effect of six food antiseptics on the growth and staphylococcal enterotoxin(SE)gene expression of Staphylococcus aureus[J].Food Science,2016,37(21):151-156
[29]胡艳军,夏素琴,陈雨欣.茶多酚联合万古霉素对耐甲氧西林金黄色葡萄球菌生物膜的影响[J].检验医学与临床,2017,14(20):2977-2979HU Yan-jun,XIA Su-qin,CHEN Yu-xin.Effects of tea polyphenols and vancomycin on biofilm of methicillin-resistant Staphylococcus aureus[J].Laboratory Medicine and Clinic,2017,14(20):2977-2979