梅毒螺旋体优势表位抗原嵌合表达构建双抗原夹心法的研究

目的用基因工程方法表达嵌合的梅毒螺旋体优势表位抗原,建立检测血清梅毒抗体的双抗原夹心酶联免疫方法(double antigen sandwich enzyme-linked immunosorbent assay,DAS-EIA)。方法通过计算机软件分析选择梅毒螺旋体优势抗原表位,用聚合酶链式反应(PCR)扩增优势表位基因,构建了梅毒螺旋体多优势表位嵌合抗原(rTpN15-TpN17-TpN47)表达载体,转化宿主菌BL21(DE3)进行表达,亲和层析柱法纯化获得高纯度融合抗原,并用其建立检测梅毒抗体的DAS-EIA。结果表达的优势表位嵌合抗原具有很好的抗原性。用其建立的嵌合抗原DAS-EIA检测确诊的50份阳性和30份阴性血,阳性检出率和阴性检出率都是100%。结论嵌合抗原DAS-EIA法具有比间接EIA和重组单抗原DAS-EIA更高的灵敏度和检出正确率,其检测水平已经达到国外TPHA的水平。该方法的建立为临床检测梅毒开辟了新的方法。

Study of the recombinant treponema pallidum multi-epitope chimeric antigen-based double antigen sandwiched enzyme-linked immunosorbent assay

Objective To establish a new method by using the gene engineering which is named double antigen sandwiched enzyme-linked immunosorbent assay(DAS-EIA)to detect serum syphilis antibody.Methods The dominant epitopes of Treponema pallidum were analyzed and picked out by using computer software.The recombinant multi-epitope chimeric antigen(rTpN15-TpN17-TpN47)expression vector of Treponema Pallidum was constructed,and transformed into E.coli BL21(DE3).Then,the antigen was highly expressed,purified and used for the development of DAS-EIA consequently.Results Fifty positive sera and 30 negative sera which had been diagnosed were detected.The sensitivity and specificity of EIA were 100%(50/50)and 100%(30/30),respectively.Conclusions The result has shown that the DSA-ELISA is a sensitive method which has reached the same level as TPHA and can be used as an potential tool for detecting serum syphilis antibody.