恙虫病东方体Gilliam株56kDa外膜蛋白基因的克隆与表达

目的　表达恙虫病东方体 (Orientiatsutsugamushi)Gilliam株 5 6kDa外膜蛋白 ,探索其作为诊断抗原的可能性。方法　采用PCR方法 ,从Ot Gilliam株菌种基因组DNA中扩增出 5 6kDa外膜蛋白基因片段 ,将目的基因片段定向插入原核表达载体pQE30 ,再用重组质粒转化大肠杆菌 ,最后用IPTG诱导转化菌 ,SDS -PAGE和Western -blotting检测重组质粒目的基因的表达。结果　 (1)获得长约 15 0 0bp的Ot Gilliam株 5 6kDa外膜蛋白基因 ,SDS -PAGE检测表达产物 ,在相对分子量5 6× 10 4处有表达带 ;(2 )DNA测序证明重组质粒 pQE30 / 5 6中插入的基因片段的序列与已报告的Ot Gilliam株 5 6kDa外膜蛋白基因序列基本一致。 (3)诱导表达菌体经超声破碎后 ,显示目的蛋白主要以包涵体形式存在 ;(4 )免疫印迹显示该重组质粒转化的大肠杆菌有一相对分子量约为 5 6× 10 4的独特蛋白带 ,该蛋白约占细菌蛋白总量的 2 9 4 %。结论　Ot Gilliam株5 6kDa外膜蛋白基因在大肠杆菌中获得了高效表达 ,表达的重组蛋白具有免疫反应性 ,与Ot Gilliam株感染的小鼠血清在免疫印迹中呈阳性反应。

Cloning and expression of the 56 kDa outer membrane protein gene of Orientia tsutsugamushi Gilliam strain

To express 56 kDa outer membrane protein gene of Orienia tsutsugamushi for the exploration of possiblity of use as a diagnostic antigen,this gene was amplified,inserted into prokaryotic expression vector pQE30,transduced to E.coli with the recombinant plasmid and finally the transfered bacteria was induced with IPTG.The expression product was assayed with SDS-PAGE and Western blotting.A 56 kDa outer membrane protein gene with approximate 1500 bp in length was obtained.And a specific protein band of 5.6×10 4 of the relative molecular weight was found in the immunoblotting assay.This protein accounting to 29.4% of total amount of bacterial proteins was verified to react with the polyclonal antisera,to the Gilliam strtain.It cocludes that high efficiency expression of the 56 kDa outer membrane protein gene of the Galliam strain of Orientia tsutsugamushi in E.coli is obtained,and the expressed protein shows immunogenicity,revealing a positive reaction with the sera of mice infected with the Galliam strain of Orientia tsutsugamushi in immunoblotting.