梅毒螺旋体特异性抗原TpN47基因片段的克隆与表达Cloning and Expression of Gene Fragment Encoding TpN47 Lipoprotein of Treponema pallidum
王宪灵;张卫军;邹全明;曾明;罗萍;郭刚;
摘要(Abstract)：
目的在大肠埃希菌中表达梅毒螺旋体(Tp)TpN47(68410 aa)重组蛋白。方法采用PCR法从Tp全基因组中扩增TpN47(202410 aa)重组蛋白。方法采用PCR法从Tp全基因组中扩增TpN47(2021230 bp)片段,T-A克隆后构建原核表达质粒pET-22 b(+)-TpN47,经酶切鉴定后转化EcoliBL21(DE3),IPTG诱导表达,表达产物经Ni2+亲和层析柱纯化,Western blot鉴定其免疫反应性。结果PCR法扩增出约1 000 bp的目的片段,原核表达质粒pET-22 b(+)-TpN47经酶切鉴定正确,表达的蛋白约占菌体总蛋白的43%,相对分子质量约为39 000,以包涵体形式存在。经纯化后蛋白纯度达95%以上,Western blot证实该蛋白能与梅毒患者阳性血清发生特异性反应。结论所表达的TpN47重组蛋白具有良好的免疫反应性,为开发临床检测效果更好的梅毒诊断试剂盒奠定了实验基础。
关键词(KeyWords)： 梅毒密螺旋体;特异性抗原;基因克隆;表达
基金项目(Foundation):
作者(Authors): 王宪灵;张卫军;邹全明;曾明;罗萍;郭刚;
DOI: 10.13200/j.cjb.2008.04.30.wangxl.004
参考文献(References)：
- [1]Fujimura K,Ise N,Ueno E,et al.Reactivity of recombinant Treponemapallidum(r-Tp)antigens with anti-Tp antibodies in human syphilitic seraevaluated by ELISA.J Clin Lab Anal,1997,11(6):315-322.
- [2]Brinkman MB,McKevitt M,McLoughlin M,et al.Reactivity of antibodiesfrom syphilis patients to a protein array representing theTreponema pal-lidumproteome.J Clin Microbiol,2006,44(3):888-891.
- [3]Baughn RE,Jiang A,Abraham R,et al.Molecular mimicry between animmunodominant amino acid motif on the 47-kDa lipoprotein ofTrepone-ma pallidum(Tpp47)and multiple repeats of analogous sequences in fi-bronectin.J Immunol,1996,157(2):720-731.
- [4]Fraser CM,Norris SJ,Weinstock GM,et al.Complete genome sequence ofTreponema pallidum,the syphilis spirochete.Science,1998,281(5375):375-388.
- [5]McKevitt M,Patel K,Smajs D,et al.Systematic cloning ofTreponemapallidumopen reading frames for protein expression and antigen discov-ery.Genome Res,2003,13(7):1665-1674.
- [6]Castro R,Prieto ES,Santo I,et al.Evaluation of an enzyme immunoassaytechnique for detection of antibodies againstTreponema pallidum.J Clin-Microbiol,2003,41(1):250-253.
- [7]Sambri V,Marangoni A,Eyer C,et al.Western immunoblotting with fiveTreponema pallidumrecombinant antigens for serologic diagnosis ofsyphilis.Clin Diagn Lab Immunol,2001,8(3):534-539.
- [8]Sato NS,Suzuki T,Ueda T,et al.Recombinant antigen-based immuno-slotblot method for serodiagnosis of syphilis.Braz J Med Biol Res,2004,37(7):949-955.
- [9]党倩丽,陆学东,张小艳,等.梅毒螺旋体感染兔模型中特异性抗体分析.中国皮肤性病学杂志,2004,18(1):38~40.
- [10]De Lemos EA,Belem ZR,Santos A,et al.Characterization of the West-ern blotting IgG reactivity patterns in the clinical phases of acquiredsyphilis.Diagn Microbiol Infect Dis,2007,58(2):177-183.