摘要
目的 在大肠埃希菌中表达梅毒密螺旋体 (Treponemapallidum ,Tp)Tp47重组蛋白 ,用其建立诊断梅毒的酶联免疫法 (enzymeimmunoassay ,EIA)。 方法 应用聚合酶链反应法 (PCR)从Tp全基因组中扩增目的片段Tp47,并将其克隆到表达载体 pET - 3 0a中 ,构建重组质粒pET - 3 0a -Tp47。在大肠埃希菌中表达重组蛋白Tp47,亲和层析柱纯化 ,建立EIA法 ,检测血清中抗 -Tp抗体。 结果 获得了Tp47基因重组蛋白的高效表达 ,该重组蛋白存在于细菌上清中 ,占全菌体蛋白 12 %左右 ,分子量约 3 9kDa。蛋白印迹法 (westernblot,WB)显示 ,Tp47重组蛋白具有良好的抗原性。用Tp47重组蛋白建立EIA法 ,检测 11份梅毒血凝试验阳性和 2 8份阴性血清 ,灵敏度为 10 0 % (11/ 11) ,特异性为 96 4% (2 7/ 2 8)。
Objective To express the recombinant Tp47 protein of Treponema pallidum(TP)in E.Coli,and to develop enzyme immunoassay(EIA) for serological diagnosis of syphilis.Methods The target gene Tp47 was amplified from the Tp complete genome by polymerase chain reaction(PCR).The PCR products were cloned into the expression vector pET-30a to generate recombinant plasmid pET-30a-Tp47.The recombinant Tp47 protein was expressed in E.Coli,and purified with affinity chromatography columns.Then it was used for the development of EIA.Results The recombinant Tp47 protein with the molecular weight of 39kDa was expressed in the supernatant of E.coli and contained about 12% of the total cellular protein.The antigenicity of the precombinant Tp47 protein was confirmed by both western immunoblot and EIA.11 THHA(Treponema pallidum hemagglutination assay)positive and 28 negative sera were tested by the EIA,with the sensitivity of 100%(11/11) and the specificity of 96.4%(27/28).Conclusion The recombinant Tp47 protein can be used for development of EIA for serological diagnosis of syphilis.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2003年第2期145-146,共2页
Chinese Journal of Public Health
