恙虫病东方体sta56部分基因的克隆和表达

CLONING AND EXPRESSION OF TRUNCATED sta56 GENE OF ORIENTIA TSUTSUGAMUSHI IN ESCHERICHIA COLI

目的 构建恙虫病东方体sta5 6基因的重组表达质粒 ,并在E .coli中表达Sta5 6重组抗原。方法 从恙虫病东方体Karp株基因组中扩增出sta5 6基因的ORF及其中长 10 5 3bp的大片段 ,用TA克隆技术将此大片段克隆 ,并以重组质粒为模板 ,扩增编码Sta5 6抗原亲水氨基酸区的一段长 3 4 2bp的DNA ,插入 pProEXHTb载体 ,转化大肠杆菌DH5α ,IPTG诱导表达 ,用SDS -PAGE及Westernblot进行分析。结果 从恙虫病东方体基因组中扩增出sta5 6基因的ORF ,以截短片段构建了重组表达质粒 pHTbOt3 4 2 ,SDS -PAGE显示蛋白表达带的分子量约为 15 .4kDa ,Westernblot证实该融合蛋白能被恙虫病患者阳性血清所识别。结论 在大肠杆菌中表达的Sta5

Aim To construct a recombinant plasmid containing the truncated gene of the major surface antigen Sta56 of Orientia tsutsugamushi(Ot.)Karp strain,and express the protein antigen in E.coli.Methods The ORF of sta56 and a 1053bp fragment of the ORF were amplified from the genomic DNA of Karp strain by PCR technique,then the 1053bp fragment was cloned into T-vector.A 342bp truncated gene of sta56 encoding a hydrophilic amino acid region was amplified from the recombinant plasmid and subcloned into the expression vector pProEX HTb and expressed in E.coli DH5α.The expression was induced by IPTG and the expressed protein was analyzed by SDS-PAGE and Western blot.Results A recombinant plasmid was constructed to express the Sta56 recombinant protein in E.coli.The fusion protein was 15.4 kDa in SDS-PAGE and could be recognized by the positive serum of patients infected by Ot.Conclusion A truncated sta56 gene of Karp strain was expressed in E.coli as a fusion protein with immunoreactivity.