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
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摘要
金黄色葡萄球菌K型肠毒素(Staphylococcus aureus enterotoxin K,SEK)由金黄色葡萄球菌肠毒素基因簇编码,流行病学显示,编码SEK蛋白的sek基因在食品源金黄色葡萄球菌菌株中具有较高的检出率,说明SEK也可能是一种重要的食品中毒潜在致病因子。本研究将截去N端信号肽的SEK蛋白编码基因与原核表达载体pET-28a(+)连接,构建重组表达质粒pET-28a(+)-ΔNspsek;通过对sek基因在不同原核表达宿主菌中的表达及表达条件优化分析,确立SEK蛋白的最优表达条件;利用镍亲合层析纯化含组氨酸(His)标签的SEK融合蛋白,将凝血酶切除His标签后的SEK蛋白进行聚合状态及热稳定分析、荧光发射谱和圆二色谱分析。结果表明,重组蛋白SEK表达成功,热处理导致蛋白部分降解;荧光光谱揭示SEK蛋白在278 nm和295 nm波长处具有相同的最大色氨酸发射峰;344 nm波长处表明SEK蛋白处于紧密折叠的天然状态;圆二色谱分析表明,ΔNspSEK重组蛋白富含β-折叠(23.6%)、β-转角(28%)以及α-螺旋(29.1%)等二级结构。为深入研究SEK蛋白的结构与功能提供基础,也对改进食品加工工艺和提高食品安全具有指导意义。
Staphylococcus aureus enterotoxin K(SEK) is encoded by the enterotoxin gene cluster(egc) of S. aureus.Epidemiologic survey showed that the sek gene had a relatively high prevalence in foodborne isolates of S. aureus, which indicates that SEK protein may be an important virulence factor causing staphylococcal food poisoning. In this study, the sek gene from S. aureus without N-terminal signal peptide was firstly ligated into plasmid pET-28 a(+). Then, the recombinant expression plasmid pET-28 a(+)-ΔNspsek was transformed into competent cells of Escherichia coli. The expression conditions were optimized and the His-ΔNspSEK fusion protein was purified to homogeneity by Ni-Sepharose affinity chromatography.The purified protein without His tag was used to assay polymerization status, thermal stability, fluorescence emission and circular dichroism spectra. The results showed that the recombinant protein ΔNspSEK was successfully expressed and purified. It could be partially degraded by heat treatment. Fluorescence emission spectra of ΔNspSEK exhibited identical tryptophan emission peaks at 278 and 295 nm, as well as excitation at 344 nm indicating that ΔNspSEK is tightly folded in nature. Circular dichroism spectra revealed that His-ΔNspSEK tagged with 6 × His sequence was rich in β-sheet(23.6%),β-turn(28%), and α-helix(29.1%). The results of this investigation will facilitate further study of the structure and function of SEK, and also provide a useful guideline to improve food processing technology and food safety.
Staphylococcus aureus enterotoxin K(SEK) is encoded by the enterotoxin gene cluster(egc) of S. aureus.Epidemiologic survey showed that the sek gene had a relatively high prevalence in foodborne isolates of S. aureus, which indicates that SEK protein may be an important virulence factor causing staphylococcal food poisoning. In this study, the sek gene from S. aureus without N-terminal signal peptide was firstly ligated into plasmid pET-28 a(+). Then, the recombinant expression plasmid pET-28 a(+)-ΔNspsek was transformed into competent cells of Escherichia coli. The expression conditions were optimized and the His-ΔNspSEK fusion protein was purified to homogeneity by Ni-Sepharose affinity chromatography.The purified protein without His tag was used to assay polymerization status, thermal stability, fluorescence emission and circular dichroism spectra. The results showed that the recombinant protein ΔNspSEK was successfully expressed and purified. It could be partially degraded by heat treatment. Fluorescence emission spectra of ΔNspSEK exhibited identical tryptophan emission peaks at 278 and 295 nm, as well as excitation at 344 nm indicating that ΔNspSEK is tightly folded in nature. Circular dichroism spectra revealed that His-ΔNspSEK tagged with 6 × His sequence was rich in β-sheet(23.6%),β-turn(28%), and α-helix(29.1%). The results of this investigation will facilitate further study of the structure and function of SEK, and also provide a useful guideline to improve food processing technology and food safety.
引文
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[2]NEMA V,AGRAWAL R,KAMBOJ D V,et al.Isolation and characterization of heat resistant enterotoxigenic Staphylococcus aureus from a food poisoning outbreak in Indian subcontinent[J].International Journal of Food Microbiology,2007,117(1):29-35.DOI:10.1016/j.ijfoodmicro.2007.01.015.
[3]LI S J,HU D L,MAINA E K,et al.Superantigenic activity of toxic shock syndrome toxin-1 is resistant to heating and digestive enzymes[J].Applied Microbiology,2011,110(3):729-736.DOI:10.1111/j.1365-2672.2010.04927.x.
[4]AKHTAR M,PARK C E,RAYMAN K.Effect of urea treatment on recovery of staphylococcal enterotoxin A from heat-processed foods[J].Applied and Environmental Microbiology,1996,62(9):3274-3276.
[5]FUNG D Y,STEINBERG D H,MILLER R D,et al.Thermal inactivation of staphylococcal enterotoxins B and C[J].Applied Microbiology,1973,26(6):938-942.
[6]HUMBER J Y,DENNY C B,BOHRER C W.Influence of pH on the heat inactivation of staphylococcal enterotoxin A as determined by monkey feeding and serological assay[J].Applied Microbiology,1975,30(5):755-758.
[7]SPERO L,GRIFFIN B Y,MIDDLEBROOK J L,et al.Effect of single and double peptide bond scission by trypsin on the structure and activity of staphylococcal enterotoxin C[J].Journal of Biological Chemistry,1976,251(18):5580-5588.
[8]REGENTHAL P,HANSEN JS,ANDRéI,et al.Thermal stability and structural changes in bacterial toxins responsible for food poisoning[J].PLoS ONE,2017,12(4):e0175989.DOI:10.1371/journal.pone.0175989.
[9]KLEIN E Y,SUN L,SMITH D L,et al.The changing epidemiology of methicillin-resistant Staphylococcus aureus in the United States:a national observational study[J].American Journal of Epidemiology,2013,177(7):666-674.DOI:10.1093/aje/kws273.
[10]ARGUDíN Má,MENDOZA M C,RODICIO M R.Food poisoning and Staphylococcus aureus enterotoxins[J].Toxins,2010,2(7):1751-1773.DOI:10.3390/toxins2071751.
[11]PETERSSON K,FORSBERG G,WALSE B.Interplay between superantigens and immunoreceptors[J].Scandinavian Journal of Immunology,2004,59(4):345-355.DOI:10.1111/j.0300-9475.2004.01404.x.
[12]ORWIN P M,LEUNG D Y,DONAHUE H L,et al.Biochemical and biological properties of staphylococcal enterotoxin K[J].Infection and Immunity,2001,69(1):360-366.DOI:10.1128/IAI.69.1.360-366.2001.
[13]JARRAUD S,PEYRAT M A,LIM A,et al.egc,a highly prevalent operon of enterotoxin gene,forms a putative nursery of superantigens in Staphylococcus aureus[J].Journal of Immunology,2001,166(1):669-677.DOI:10.4049/jimmunol.166.1.669.
[14]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 and Environmental Microbiology,2009,75(21):6839-6849.DOI:10.1128/AEM.00272-09.
[15]WU D,LI X,YANG Y,et al.Superantigen gene profiles and presence of exfoliative toxin genes in community-acquired meticillin-resistant Staphylococcus aureus isolated from Chinese children[J].Journal of Medical Microbiology,2011,60(Pt1):35-45.DOI:10.1099/jmm.0.023465-0.
[16]ORWIN P M,LEUNG D Y,TRIPP T J,et al.Characterization of a novel staphylococcal enterotoxin-like superantigen,a member of the group V subfamily of pyrogenic toxins[J].Biochemistry,2002,41(47):14033-14040.
[17]GüNTHER S,VARMA A K,MOZA B,et al.A novel loop domain in superantigens extends their T cell receptor recognition site[J].Journal of Molecular Biology,2007,371(1):210-221.DOI:10.1016/j.jmb.2007.05.038.
[18]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.DOI:10.1128/IAI.00550-13.
[19]ONO H K,HIROSE S,NAITO I,et al.The emetic activity of staphylococcal enterotoxins,SEK,SEL,SEM,SEN and SEO in a small emetic animal model,the house musk shrew[J].Microbiology and Immunology,2017,61(1):12-16.DOI:10.1111/1348-0421.12460.
[20]王琼,唐俊妮,汤承,等.金黄色葡萄球菌新型肠毒素sek基因在3株食品分离菌株中的表达[J].食品科学,2016,37(13):140-147.DOI:10.7506/spkx1002-6630-201613025.
[21]王琼,张荣,陈娟,等.金黄色葡萄球菌在猪肉上的生长以及新型肠毒素基因sek的表达[J].食品工业科技,2016,37(12):190-194.DOI:10.13386/j.issn1002-0306.2016.12.028.
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[24]刘骥,潘洁,田万帆,等.新型肠毒素蛋白SElK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):274-280.DOI:10.11920/xnmdzk.2016.03.006.
[25]王琼,唐俊妮,汤承,等.一种采用微波炉加热快速提取细菌DNA用于PCR扩增的方法[J].西南民族大学学报(自然科学版),2015,41(2):150-155.DOI:10.11920/xnmdzk.2015.02.004.
[26]刘骥,杨帆,田万帆,等.金黄色葡萄球菌M型肠毒素原核表达、纯化、鉴定及溶液构象研究[J].食品科学,2017,38(24):40-46.DOI:10.7506/spkx1002-6630-201724007.
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