摘要
目的构建红色荧光蛋白(dsRed)与卵白蛋白(Ovalbumin,OVA)表位融合蛋白的高效表达质粒,并表达与纯化此红荧光蛋白标记的OVA模式抗原。方法合成的寡聚核甘酸变性退火成双链DNA接头,此接头含有OVA257-264表位的编码序列和一个AgeⅠ酶切位点,然后将其与dsRed蛋白编码序列克隆连接进pET16b,此表达质粒(pET-16bOR)转化大肠杆菌BL21,异丙基硫代半糖苷诱导表达后,流式细胞术和激光共聚焦显微镜检测融合蛋白的表达,并进一步经Ni2+-NTA琼脂糖柱纯化,SDS-PAGE和Westernblot分析纯化的融合蛋白。结果成功构建dsRed与OVA表位融合蛋白的高效表达质粒pET-16bOR,此融合蛋白能被488nm和568nm激发荧光,此蛋白被纯化并经SDS-PAGE和Westernblot分析证实。结论红荧光蛋白与OVA表位的融合蛋白具有荧光特性,并在大肠杆菌表达、纯化,可作为进一步分析抗原递呈的模式抗原。
Objective To express and purify the fusion protein dsRed tagged OVA epitope antigen via construction an expression vector pET16bOR. Methods The overlapping oligonucleotides that contain an Age I site and OVA257-264 encoding sequence were hybridized to a double strand adaptor. The adaptor and dsRed fragment from plasmid pdsRed-C1 were cloned into pET-16b, and then E. coli BL21 cells were transformed with the correct insertion （pET-16bOR） and induced with isopropyl beta-D-thiogalactoside. The expression of recombinant protein was analyzed by confocal microscopy and flow cytometry. The dsRed tagged OVA epitope fusion protein was purified by Ni-NTA agaroses and then identified by SDS-PAGE and Western blotting. Results The dsRed tagged OVA epitope fusion protein expression vector was successfully coustmcted and the fusion protein was expressed and purified with red fluorescent trader 488 and 568 nm excitation. Conclusion The dsRed tagged OVA epitope fusion protein is obtained by genetic engineering methods, which keep the fluorescent characters of dsRed and can be used for further functional analysis for antigen presentation.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第5期382-385,共4页
Immunological Journal
基金
国家自然科学基金资助项目(30400392)
