恙虫病东方体Gilliam株56kDa蛋白部分基因的原核表达及初步鉴定

目的　对恙虫病东方体Gilliam株 5 6kDa蛋白部分基因进行原核表达 ,以获得高效表达、有生物活性目的蛋白 ,为恙虫病诊断试剂的研制打下基础。方法　根据Gilliam株东方体 5 6kDa蛋白基因序列设计特定引物 ,用PCR法扩增出编码5 6kDa抗原富含亲水区及同源性高的C端一段长约 70 0bp的DNA ,插入原核载体PGEX - 4T - 2 ,转化大肠杆菌TGI ,抽提质粒 ,酶切鉴定 ,含插入片段的重组质粒进行序列分析 ,再转化表达宿主菌大肠杆菌BL2 1(DE3) ,IPTG诱导表达 ,用SDS -PAGE及Western -blot进行分析鉴定。结果　SDS -PAGE检测表明该截短片段以融合蛋白的方式得到成功表达 ,在相对分子量 5 8kDa处有表达条带 ,经薄层扫描分析 ,目的蛋白条带占全菌蛋白的 30 5 %。Western -blot证实该融合蛋白能被恙虫病患者阳性血清识别。结论　表达产物初步鉴定具有生物活性 ,有望应用于恙虫病诊断试剂的研制

Expression and identification of Truncated 56kDa protein of Orientia tsutsugamushi Gilliam Strain in prokaryotic cells

Aim The purpose of this studies is to express the truncated 56kDa gene of Orientia tsutsugamushi (Ot)Gilliam strain in Escherichia coli to obtain the abundant recombinant protein with high activity and to play a foundation for preparing the diagnosis kit of scrub typhus Methods The truncated gene(about 700bp)encoding for the C terminus amino acids of 56kDa protein of Ot with high hydrophilicity and hemologization was amplified by using PCR technique PCR product was cloned initially into the PGEX 4T 2 expression vector,then transformed into E coli TGI,the plasmid DNA was extracted and digested with enzymes Plasmids containing the right insertion were sequenced to confirm its identity and retransformed the combinants into E coli BL21(DE3) After IPTG induction,the bacterial lysates were analyzed by SDS PAGE and western blot Results The recombinant plasmid containing the aim gene has been expressed successfully,the expressed fusion protein was visualized on gel at molecular mass about 58kDa and could be detected by the positive serum from patients of scrub typhus by western blot Conclusions The expression product of the truncated 56kDa gene of Gilliam strain has immunoreactivity and may be a promising diagnostic antigen for further preparing the diagnosis kit of scrub typhus