Lesseva Milena, Christova Iva, Miloshev George
Laboratory of Yeast Molecular Genetics, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Folia Med (Plovdiv). 2007;49(3-4):58-62.
Lyme borreliosis is an arthropod transmitted infection caused by some species of the Borrelia genus. Current diagnosis employs serological testing and detection of Borrelia-specific antibodies. Using recombinant Borrelia burgdorferi antigens may improve assay specificity and sensitivity. One of the immunodominant Borrelia antigens that elicit a strong and early immune response is FlaB, making it appropriate for recombinant protein based serological diagnostic tests.
Borrelia burgdorferi genomic DNA was isolated and used as a template for the amplification of the flaB gene. The gene was cloned in the expression vector pGEX-2T.
The amplified flaB gene was cloned in the expression vector yielding a GST-FlaB fusion gene. The gene ligated in-frame was expressed as the recombinant GST-FlaB protein. After visualization by polyacrylamide gel electrophoresis the successful expression of the FlaB protein was confirmed by immunoblotting.
The expression and purification of the recombinant FlaB protein is a prerequisite for obtaining large amounts of the product through a simple and labour-free procedure, which will facilitate the diagnosis of Lyme disease.
莱姆病是一种由某些疏螺旋体属物种引起的节肢动物传播感染。目前的诊断采用血清学检测和检测疏螺旋体特异性抗体。使用重组伯氏疏螺旋体抗原可能会提高检测的特异性和敏感性。引发强烈且早期免疫反应的免疫显性疏螺旋体抗原之一是FlaB，这使其适用于基于重组蛋白的血清学诊断测试。
分离伯氏疏螺旋体基因组DNA，并用作扩增flaB基因的模板。该基因被克隆到表达载体pGEX - 2T中。
扩增的flaB基因被克隆到表达载体中，产生了一个GST - FlaB融合基因。框内连接的基因表达为重组GST - FlaB蛋白。通过聚丙烯酰胺凝胶电泳可视化后，通过免疫印迹确认了FlaB蛋白的成功表达。
重组FlaB蛋白的表达和纯化是通过简单且无需费力的程序获得大量产物的先决条件，这将有助于莱姆病的诊断。
