CAJ | 학술논문

目的：构建含有His标签的结核分枝杆菌fbpB基因原核表达质粒，获得结核分枝杆菌Ag85B的表达蛋白。方法：制备结核分枝杆菌基因组DNA，从结核分枝杆菌H37Rv基因组中PCR扩增fbpB基因；连接至表达载体pET28a上，经序列测定证实正确后转化大肠杆菌（ E．coil） BL21，再经IPTG诱导表达His-Ag85B融合蛋白；用聚丙烯酰胺凝胶电泳和免疫印迹分析重组蛋白，ELISA检测融合蛋白的抗原性。结果：扩增出了结核分枝杆菌fgbB基因，构建了具有正确基因序列的质粒载体pET28a-fgbB，转化E．coil BL21后经诱导产生了高水平的表达产物。蛋白纯化后可被结核病患者血清特异性识别。结论：构建了质粒载体，并诱导表达了His-Ag85 B融合蛋白，为进一步研究结核菌的免疫机制奠定了基础。
목적：구건함유His표첨적결핵분지간균fbpB기인원핵표체질립，획득결핵분지간균Ag85B적표체단백。방법：제비결핵분지간균기인조DNA，종결핵분지간균H37Rv기인조중PCR확증fbpB기인；련접지표체재체pET28a상，경서렬측정증실정학후전화대장간균（ E．coil） BL21，재경IPTG유도표체His-Ag85B융합단백；용취병희선알응효전영화면역인적분석중조단백，ELISA검측융합단백적항원성。결과：확증출료결핵분지간균fgbB기인，구건료구유정학기인서렬적질립재체pET28a-fgbB，전화E．coil BL21후경유도산생료고수평적표체산물。단백순화후가피결핵병환자혈청특이성식별。결론：구건료질립재체，병유도표체료His-Ag85 B융합단백，위진일보연구결핵균적면역궤제전정료기출。
Aim:To construct His-tag prokaryotic expression plasmid of the fbpB gene of Mycobacterium tuberculosis, and to express the fusion protein efficiently in E.coli BL21.Methods:The fbpB gene was amplified by PCR from genomic DNA of Mycobacterium tuberculosis H37Rv strain,and was cloned into pET28a expression vector.E.coli BL21 strain was transformed with the recombinant vector that conformed by sequencing and induced to express recombinant protein .The re-combinant protein was confirmed by SDS-PAGE and immunoblot , and its antigenicity was analyzed by ELISA assay .Re-sults:The fbpB gene was amplified by PCR and the recombinant expressive vector pET 28a-fgbB was constructed.The E.coli BL21 strains with recombinant vector showed high level expression of Ag 85B fusion protein after IPTG induction .Conclu-sion:The purified fusion protein could be specifically recognized by sera of patients with tuberculosis.The expression of re-combinant Ag85B protein lays a basis for further studies on immunological mechanism of Mycobacterium tuberculosis.