恙虫病东方体Sta56抗原的原核表达、纯化及其在间接ELISA中的应用

目的　构建含恙虫病东方体sta5 6基因的重组表达质粒 ,在E .coli中表达Sta5 6重组抗原 ,纯化后用于间接ELISA检测恙虫病。方法　从含有恙虫病东方体Karp株sta5 6基因的重组质粒TOPO sta5 6扩增出截短的sta5 6 ,定向插入pET30a载体 ,转化大肠杆菌BL2 1(DE3) ,IPTG诱导表达 ,用SDS PAGE及Westernblot进行分析 ;采用电洗脱方法纯化重组抗原并应用于间接ELISA检测恙虫病。结果　以截短的sta5 6基因片段构建了重组表达质粒pETOt95 7,重组的Sta5 6抗原可在E .coli中以融合蛋白的形式有效表达 ,SDS PAGE显示了一条相对分子质量 (Mr)为 4 0 .3× 10 3 的蛋白表达带 ,West ernblot证实该融合蛋白能被恙虫病患者阳性血清所识别。电洗脱纯化的重组抗原应用于间接法ELISA检测恙虫病 ,与间接免疫荧光法IFAT比较 ,其敏感性和特异性分别为 91.7%和 76 .5 %。结论　在大肠杆菌中表达的Sta5 6重组抗原具有免疫反应性 ,纯化后可用作免疫诊断试剂。

The expression and purification of recombinant Sta56 antigen from Orientia tsutsugamushi and its application in indirect ELISA to detect scrub typhus infection

Objective To construct a recombinant plasmid containing the truncated gene of the major surface antigen Sta56 from Orientia tsutsugamushi ( Ot ) Karp strain and to express the antigen in E.coli . The purified recombinant antigen is applied in indirect ELISA to detect scrub typhus infection. Methods A truncated 957bp gene of sta56 of Ot Karp strain was amplified from the recombinant plasmid TOPO sta56 and directly cloned into plasmid pET30a and expressed in E.coli BL21(DE3) when induced by IPTG. The expressed protein was analyzed by SDS PAGE and Western blot. The recombinant protein was purified by electroelution and applied in indirect ELISA to detect scrub typhus infection. Results A recombinant plasmid containing a truncated sta56 gene was constructed to express the Sta56 recombinant protein in E.coli . The fusion proteins were identified by SDS PAGE and recognized by the positive serum of patients infected with Ot . Electroelution could be used to purify the protein. An indirect ELISA using this recombinant protein as coating antigen showed a sensitivity of 91.7% and a specificity of 76.5%, compared to IFAT. Conclusion The recombinant Sta56 antigen with immunoreactivity is a candidate diagnostic reagent for Ot infection.