恙虫病东方体Karp株56kD表膜蛋白基因的表达及免疫诊断应用研究

目的实现恙虫病东方体56kD表膜蛋白的原核表达并评价重组蛋白作为抗原的免疫诊断价值。方法亚克隆技术构建原核表达质粒载体pGEX-Sta56,转化宿主菌BL21(DE3),IPTG诱导表达GST-Sta56融合蛋白,亲和层析纯化融合蛋白,应用ELISA以评价其免疫诊断价值。结果pGEX-Sta56经测序鉴定Sta56基因以正确的表达框架连入载体,经过诱导表达得到正确大小的融合蛋白,应用纯化的GST-Sta56的ELISA检测结果与全细胞抗原有很高线性相关性,在小鼠血清检测中敏感性,特异性和准确性分别大于90%,70%和80%。结论成功表达重组的56kD表膜蛋白并可能替代传统的全细胞纯化蛋白,为建立快速、敏感和特异的免疫学诊断奠定了基础。

Expression of gene encoding for the 56 kDa surface membrane protein of Orientia tsutsugamushi strain Karp and its application in immunological diagnosis

To investigate the prokaryotis expression of the 56 kDa surface membrane protein of Orientia tsutsugamushi strain Karp and to evaluate the value of the recombinant protein as antigen in the immunological diagnosis,the prokaryotic expression plasmid pGEX Sta56 was constructed by cub cloning technique, and then transformed to host bacteria BL21 (DE3). The expression of GST Sta56 fusion protein was induced by IPTG and purified by GST purification module. The recombinant protein was used to cover the ELISA plate and the ELISA results were compared with those by using the indirect immunofluorescence assay.It was found that the Sta56 gene was linked to vector pGEX Sta56 with correct open reading frame by sequencing analysis, and the correct fusion protein was obtained by induced expression As demonstrated by ELISA assay a high liner correlation was obtained with that of the whole cell antigen. The sensitivity, specificity and accuracy were 90%, 70% and 80% respectively as demonstrated by using the mouse sera as samples.It is concluded that the recombinant 56kDa surface membrane protein is expressed successfully. This protein can be used to substitute the traditional antigen purified from the whole cell as to establish a rapid, sensitive and specific method for immunological diagnosis.