侯天全;张国利;滕利荣;岳玉环;吴广谋;田园;付玉禾;赵鑫;张培培;

• 1:吉林大学生命科学学院
• 2:军事医学科学院军事兽医研究所
• 3:吉林省人兽共患病预防与控制重点实验室省部共建重点实验室

摘要(Abstract)：

目的原核表达重组金黄葡萄球菌肠毒素B(recombinant Staphylococcus aureus enterotoxin B,r SEB)及热休克蛋白65(heat shock protein 65,HSP65)融合蛋白r SEB-HSP65,并探讨其在小鼠体内的体液免疫效果。方法采用分子生物学方法将修改过TCR Vβ结合区的r SEB基因与HSP65基因融合,构建重组表达质粒p ET-28a-r SEB-HSP65,转化E.coli BL21(DE3),IPTG诱导表达,经铜柱亲和层析纯化。将纯化蛋白经小鼠背部皮下注射,分别于第0、14、28天各免疫1次,分别于第14、28、42 d经小鼠尾静脉采血,分离血清,间接ELISA法检测小鼠血清中抗-SEB的Ig G水平。结果重组表达质粒p ET-28a-r SEB-HSP65经双酶切及PCR鉴定,证明构建正确。表达的重组蛋白r SEB-HSP65的相对分子质量约85 000,主要以包涵体形式表达,表达量约占全菌总蛋白的40.81%,纯化蛋白纯度约为90%。各组免疫小鼠均可产生较高滴度抗体,且在第14、28及42天大部分含铝佐剂疫苗组的效价显著高于相同剂量的无铝佐剂疫苗组(P<0.05),第42天时无铝佐剂低剂量疫苗组与含铝佐剂低剂疫苗量组间差异无统计学意义(P>0.05)。结论成功在E.coli BL21(DE3)中表达了重组蛋白r SEB-HSP65,且可诱导小鼠产生较高的抗体水平。

关键词(KeyWords)： 金黄葡萄球菌肠毒素B;热休克蛋白65;原核细胞;基因表达;体液免疫

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 侯天全;张国利;滕利荣;岳玉环;吴广谋;田园;付玉禾;赵鑫;张培培;

Email:

DOI: 10.13200/j.cnki.cjb.001649

参考文献(References)：

• [1]ARCHER N K,MAZAITIS M J,COSTERTON J W,et al.Staphylococcus aureus biofilms:properties,regulation,and roles in human disease[J].Virulence,2011,2(5):445-459.
• [2]KRAKAUER T.Update on staphylococcal superantigen-induced signaling pathways and therapeutic interventions[J].Toxins(Basel),2013,5(9):1629-1654.
• [3]FAULKNER L,COOPER A,FANTINO C,et al.The mechanism of superantigen-mediated toxic shock:not a simple Th1cytokine storm[J].J Immunol,2005,175(10):6870-6877.
• [4]RAJAGOPALAN G,SEN M M,SINGH M,et al.Intranasal exposure to staphylococcal enterotoxin B elicits an acute systemic inflammatory response[J].Shock,2006,25(6):647-656.
• [5]MURSHID A,GONG J,STEVENSON M A,et al.Heat shock proteins and cancer vaccines:developments in the past decade and chaperoning in the decade to come[J].Expert Rev Vaccines,2011,10(11):1553-1568.
• [6]GBAGUIDI-HAORE H,THOUVEREZ M,COUETDIC G,et al.Usefulness of antimicrobial resistance pattern for detecting PVL-or TSST-1-producing meticillin-resistant Staphylococcus aureus in a French University Hospital[J].J Med Microbiol,2009,58(Pt 10):1337-1342.
• [7]LARKIN E A,CARMAN R J,KRAKAUER T,et al.Staphylococcus aureus:the toxic presence of a pathogen extraordinaire[J].Curr Med Chem,2009,16(30):4003-4019.
• [8]YANAGISAWA C,HANAKI H,NATAE T,et al.Neutralization of staphylococcal exotoxins in vitro by human-origin intravenous immunoglobulin[J].J Infect Chemother,2007,13(6):368-372.
• [9]YANG X,BUONPANE R A,MOZA B,et al.Neutralization of multiple staphylococcal superantigens by a single-chain protein consisting of affinity-matured,variable domain repeats[J].JInfect Dis,2008,198(3):344-348.
• [10]TILAHUN A Y,THEUER J E,PATEL R,et al.Detrimental effect of the proteasome inhibitor,Bortezomib in bacterial superantigen-and lipopolysaccharide-induced systemic inflammation[J].Mol Ther,2010,18(6):1143-1154.
• [11]PINCHUK I V,BESWICK E J,SAADA J I,et al.Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a:relevance to staphylococcal enterotoxigenic disease[J].J Immunol,2007,178(12):8097-8106.
• [12]VARSHNEY A K,WANG X,COOK E,et al.Generation,characterization,and epitope mapping of neutralizing and protective monoclonal antibodies against staphylococcal enterotoxin B-induced lethal shock[J].J Biol Chem,2011,286(11):9737-9747.
• [13]INSKEEP T K,STAHL C,ODLE J,et al.Oral vaccine formulations stimulate mucosal and systemic antibody responses against staphylococcal enterotoxin B in a piglet model[J].Clin Vaccine Immunol,2010,17(8):1163-1169.
• [14]LIANG J,AIHUA Z,YU W,et al.HSP65 serves as an immunogenic carrier for a diabetogenic peptide P277 inducing anti-inflammatory immune response in NOD mice by nasal administration[J].Vaccine,2010,28(19):3312-3317.