构象稳定的破伤风毒素Hc片段突变体(HcM)的制备及其免疫原性分析
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摘要
破伤风是由破伤风杆菌侵入人体伤口、生长繁殖、产生毒素而引起的一种急性特异性感染,其死亡率高,严重危害人民生命健康。研究证实破伤风毒素重链C端(Hc)具有与毒素受体结合的活性,完全保留了全分子的免疫原性,有望开发成为新的基因工程破伤风亚单位疫苗以替换传统的甲醛灭活类毒素疫苗。由于野生型Hc蛋白(HcW)易形成分子间及分子内二硫键,且各构象分子之间易发生不稳定的转换,为疫苗的生产工艺带来困难,因此,通过将破伤风HcW蛋白的869位半胱氨酸突变为丙氨酸,构建构象稳定的破伤风亚单位疫苗突变体HcM,对HcM进行表达、发酵和纯化,研究其在不同条件下的稳定性,比较其与受体GT1b亲和力的变化及免疫原性。实验结果表明HcM在大肠杆菌中获得了高效可溶性表达,通过QXL柱、phenyl-Hs柱和source30Q柱三步纯化,可获得纯度95%以上的HcM蛋白。纯化后的HcM蛋白在不同保存条件下构象稳定且保持了与破伤风毒素受体GT1b结合的活性。免疫小鼠后,HcM能够诱导小鼠产生高滴度抗体并能够保护小鼠抵御1×103 LD50剂量的破伤风毒素攻击。以上结果表明构象稳定的HcM有望成为新的破伤风重组亚单位疫苗候选者,其生产工艺简单、稳定且保持了与野生型相似的免疫原性,不论是针对日常破伤风治疗还是对于国家应对突发事件(战争、地震等灾害)的药品储备均具有重大意义。
Tetanus is caused by tetanus toxin synthesized by Clostridium tetani.Fragment C(Hc),the 50 kDa carboxy-terminal portion of tetanus toxin,is nontoxic but has receptor protein binding activities,which has been evaluated as a potential new recombinant subunit vaccine to replace the traditional formaldehyde inactivated toxoid vaccine.It is easy for wild Hc(HcW) to form inter-and intra-molecular disulfide bonds and the different conformations changes unstably,which brings difficulties for vaccine production technology.In our study,the Cys 869 of HcW was mutated to Ala and the conformation-stable fragment-C mutant of tetanus toxin(HcM) was constructed.The HcM was expressed,fermented and purified and its stability,receptor binding and immunogenicity were evaluated.The result showed that the HcM got high-level expression and was purified to >95% of purity.The purified HcM was conformation-stable at different temperature for different time and kept the binding activities with one of its receptor GT1b.Mice given three vaccinations by HcM developed a protective immune response and were 100% protected against an intraperitoneal administration of 1×103 50% lethal doses(LD50s) of tetanus neurotoxin.All the results showed that the conformation-stable HcM had potent immunogenicity as a recombinant tetanus vaccine candidate with simple production process and similar immunogenicity with HcW.Whether for routine tetanus therapy or for countries to respond to unexpected events(war,earthquake or other disaster),it is of great significance.
Tetanus is caused by tetanus toxin synthesized by Clostridium tetani.Fragment C(Hc),the 50 kDa carboxy-terminal portion of tetanus toxin,is nontoxic but has receptor protein binding activities,which has been evaluated as a potential new recombinant subunit vaccine to replace the traditional formaldehyde inactivated toxoid vaccine.It is easy for wild Hc(HcW) to form inter-and intra-molecular disulfide bonds and the different conformations changes unstably,which brings difficulties for vaccine production technology.In our study,the Cys 869 of HcW was mutated to Ala and the conformation-stable fragment-C mutant of tetanus toxin(HcM) was constructed.The HcM was expressed,fermented and purified and its stability,receptor binding and immunogenicity were evaluated.The result showed that the HcM got high-level expression and was purified to >95% of purity.The purified HcM was conformation-stable at different temperature for different time and kept the binding activities with one of its receptor GT1b.Mice given three vaccinations by HcM developed a protective immune response and were 100% protected against an intraperitoneal administration of 1×103 50% lethal doses(LD50s) of tetanus neurotoxin.All the results showed that the conformation-stable HcM had potent immunogenicity as a recombinant tetanus vaccine candidate with simple production process and similar immunogenicity with HcW.Whether for routine tetanus therapy or for countries to respond to unexpected events(war,earthquake or other disaster),it is of great significance.
引文
[1]Mallick IH,Winslet MC.A review of the epidemiology,pathogenesis and management of tetanus.Int J surg,2004,2(2):109-112.
[2]Lacy DB,Tepp W,Cohen AC,et al.Crystal structure ofbotulinum neurotoxin type A and implications for toxicity.Nature Struct Biol,1998,5(10):898-902.
[3]Quinn HE,McIntyre PB.Tetanus in the elderly–Animportant preventable disease in Australia.Vaccine,2007,25(7):1304-1309.
[4]Bizzini B,Stoeckel K,SchwabM.An antigenicpolypeptide fragment isolated from tetanus toxin:chemical characterization,binding to gangliosides andretrograde axonal transport in various neuron systems.JNeurochem,1977,28(3):592-542.
[5]Matsuda M,Lei DL,Sugimoto N,et al.Isolation,purification,and characterization of fragment B,theNH22-terminal half of the heavy chain of tetanus toxin.Infect Immun,1989,57(11):3588-3593.
[6]Schiavo G,Poulain B,Rossetto O,et al.Tetanus toxin is azinc protein and its inhibition of neurotiansmitter releaseand protease activity depend on zinc.EMBO J,1992,11(10):3577-3583.
[7]Lafaye P,Nato F,MaziéJC,et al.Similar bindingproperties for a neutralizing anti-tetanus toxoid humanmonoclonal antibody and its bacterially expressed Fab.Res Immunol,1995,146(6):373-382.
[8]Rummel A,Bade S,Alves J,et al.Two carbohydratebinding sites in the Hcc-domain of tetanus neurotoxin arerequired for toxicity.J Mol Biol,2003,326(3):835-847.
[9]Halpern JL,Loftus A.Characterization of thereceptor-binding domain of tetanus toxin.J Biol Chem,1993,268(15):11188-11192.
[10]Herreros J,Lalli G,Schiavo G.C-terminal half of tetanustoxin fragment C is sufficient for neuronal binding andinteraction with a putative protein receptor.Biochem J,2000,347(Pt 1):199-204.
[11]Wells JM,Wilson PW,Norton PM,et al.Lactococcuslactis:high-level expression of tetanus toxin fragment Cand protection against lethal challenge.Mol Microbiol,1993,8(6):1155-1162.
[12]Qazi O,Bolgiano B,Crane D,et al.The HC fragment oftetanus toxin forms stable,concentration-dependentdimers via an intermolecular disulphide bond.J Mol Biol,2007,365(1):123-134.
[2]Lacy DB,Tepp W,Cohen AC,et al.Crystal structure ofbotulinum neurotoxin type A and implications for toxicity.Nature Struct Biol,1998,5(10):898-902.
[3]Quinn HE,McIntyre PB.Tetanus in the elderly–Animportant preventable disease in Australia.Vaccine,2007,25(7):1304-1309.
[4]Bizzini B,Stoeckel K,SchwabM.An antigenicpolypeptide fragment isolated from tetanus toxin:chemical characterization,binding to gangliosides andretrograde axonal transport in various neuron systems.JNeurochem,1977,28(3):592-542.
[5]Matsuda M,Lei DL,Sugimoto N,et al.Isolation,purification,and characterization of fragment B,theNH22-terminal half of the heavy chain of tetanus toxin.Infect Immun,1989,57(11):3588-3593.
[6]Schiavo G,Poulain B,Rossetto O,et al.Tetanus toxin is azinc protein and its inhibition of neurotiansmitter releaseand protease activity depend on zinc.EMBO J,1992,11(10):3577-3583.
[7]Lafaye P,Nato F,MaziéJC,et al.Similar bindingproperties for a neutralizing anti-tetanus toxoid humanmonoclonal antibody and its bacterially expressed Fab.Res Immunol,1995,146(6):373-382.
[8]Rummel A,Bade S,Alves J,et al.Two carbohydratebinding sites in the Hcc-domain of tetanus neurotoxin arerequired for toxicity.J Mol Biol,2003,326(3):835-847.
[9]Halpern JL,Loftus A.Characterization of thereceptor-binding domain of tetanus toxin.J Biol Chem,1993,268(15):11188-11192.
[10]Herreros J,Lalli G,Schiavo G.C-terminal half of tetanustoxin fragment C is sufficient for neuronal binding andinteraction with a putative protein receptor.Biochem J,2000,347(Pt 1):199-204.
[11]Wells JM,Wilson PW,Norton PM,et al.Lactococcuslactis:high-level expression of tetanus toxin fragment Cand protection against lethal challenge.Mol Microbiol,1993,8(6):1155-1162.
[12]Qazi O,Bolgiano B,Crane D,et al.The HC fragment oftetanus toxin forms stable,concentration-dependentdimers via an intermolecular disulphide bond.J Mol Biol,2007,365(1):123-134.
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