重组金黄葡萄球菌肠毒素B及热休克蛋白65融合蛋白的原核表达及其在小鼠体内的体液免疫效果
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摘要
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。
Objective To express recombinant Staphylococcus aureus enteroxtoxin B(r SEB) and heat shock protein 65(HSP65)fusion protein in prokaryotic cells and investigate its humoral immune effect in mice. Methods Recombinant plasmid p ET-r SEB-HSP65 was constructed by fusion of a SEB gene in which TCR Vβ binding region was modified with HSP65 gene by molecular biological technique, and transformed to E. coli BL21(DE3) for expression under induction of IPTG. The expressed product was purified by affinity chromatography, with which mice were injected s.c. on days 0,14 and 28. Serum samples were collected on days 14, 28 and 42, and determined for anti-SEB Ig G by indirect ELISA.Results Restriction analysis and PCR proved that recombinant plasmid p ET-28a-r SEB-HSP65 was constructed correctly.The expressed r SEB-HSP65, with a relative molecular mass of about 85 000, mainly existed in a form of inclusion body,contained about 40. 81% of total somatic protein, and reached a purity of about 90% after purification. High antibody titers were induced in mice in various groups. Most of the GMTs of mice on days 14, 28 and 42 after immunization with aluminum adjuvant-containing vaccine were significantly higher than those with aluminum adjuvant-free vaccine at the same dosages(each P < 0. 05). However, the GMTs of mice 42 d after immunization with aluminum adjuvant-containing vaccine at a low dosage showed no significant difference with those with aluminum adjuvant-free vaccine at the samedosage(P > 0. 01). Conclusion Recombinant vaccine r SEB-HSP65 was successfully expressed in E. coli BL21(DE3),which induced high antibody level in mice.
Objective To express recombinant Staphylococcus aureus enteroxtoxin B(r SEB) and heat shock protein 65(HSP65)fusion protein in prokaryotic cells and investigate its humoral immune effect in mice. Methods Recombinant plasmid p ET-r SEB-HSP65 was constructed by fusion of a SEB gene in which TCR Vβ binding region was modified with HSP65 gene by molecular biological technique, and transformed to E. coli BL21(DE3) for expression under induction of IPTG. The expressed product was purified by affinity chromatography, with which mice were injected s.c. on days 0,14 and 28. Serum samples were collected on days 14, 28 and 42, and determined for anti-SEB Ig G by indirect ELISA.Results Restriction analysis and PCR proved that recombinant plasmid p ET-28a-r SEB-HSP65 was constructed correctly.The expressed r SEB-HSP65, with a relative molecular mass of about 85 000, mainly existed in a form of inclusion body,contained about 40. 81% of total somatic protein, and reached a purity of about 90% after purification. High antibody titers were induced in mice in various groups. Most of the GMTs of mice on days 14, 28 and 42 after immunization with aluminum adjuvant-containing vaccine were significantly higher than those with aluminum adjuvant-free vaccine at the same dosages(each P < 0. 05). However, the GMTs of mice 42 d after immunization with aluminum adjuvant-containing vaccine at a low dosage showed no significant difference with those with aluminum adjuvant-free vaccine at the samedosage(P > 0. 01). Conclusion Recombinant vaccine r SEB-HSP65 was successfully expressed in E. coli BL21(DE3),which induced high antibody level in mice.
引文
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[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.
[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.