【摘要】 目的构建梅毒硫氧还原蛋白TP0100基因的原核表达载体,在大肠埃希菌中表达重组蛋白,并评价其在梅毒血清学诊断中的价值。方法构建pET-28b-TP0100重组表达质粒,转化大肠埃希菌BL21(DE3)后诱导表达。经镍柱纯化,重组蛋白通过质谱和Western blot进行鉴定。以重组蛋白为抗原建立酶联免疫吸附试验(ELISA)间接法,并检测临床收集的梅毒血清。结果成功构建表达载体pET-28b-TP0100,测序结果表明质粒中的插入序列与GenBank中TP0100的基因序列相同。重组蛋白表达约占菌体总蛋白的40%,相对分子质量约为23×103。质谱和Western blot分别证实重组蛋白的酶解片段来自于TP0100蛋白,重组蛋白能与梅毒TPPA阳性血清发生特异性反应。ELISA检测20份梅毒螺旋体明胶凝集试验(TPPA)阳性血清和20份TPPA阴性血清,与TPPA方法的符合率分别为95%(19/20)和100%(20/20)。结论重组硫氧还原蛋白TP0100能够与梅毒患者阳性血清发生特异性反应,是一个潜在的梅毒血清学诊断抗原。更多还原

【Abstract】 Objective To construct the prokaryotic expression plasmid containing thioredoxin TP0100 gene of Treponema pallidum,express recombinant TP0100 protein in E.coli and to investigate the application in serodiagnosis of syphilis. Methods TP0100 gene was amplified by PCR and cloned into plasmid vector pET-28b.The recombinant plasmid pET-28b-TP0100 was transformed into E.coli BL21(DE3)for protein expression on IPTG induction.The expressed protein was purified using Ni2+ affinity chromatography and then identified by mass spectrometry and Western blotting.The indirect ELISA method was developed by using TP0100 protein as antigen.Twenty TPPA positive sera and twenty TPPA negative sera were tested by TP0100-ELISA. Results The recombinant plasmid pET-28b-TP0100 was constructed correctly.The sequencing result confirmed that the sequence of the fragment inserted in the plasmid was the same as the coding sequence of TP0100 protein recorded in GenBank.Protein with relative molecular mass of 23×103 was expressed in about 40% of total bacterial proteins.The result of mass spectrometric analysis demonstrated that the fragment was from TP0100 protein which could react with syphilitic sera.The coincidence rate was 95% in TPPA positive sera and 100% in TPPA negative sera with the result of TP0100-ELISA. Conclusion The recombinant thioredoxin TP0100 has specific reactivity with TPPA positive sera and could be an alternative antigen for serological diagnosis of syphilis.更多还原