一种新型融合蛋白RANKL-Fc的表达和纯化  

Expression and purification of a novel fusion protein,RANKL-Fc

作　　者：王永林[1,2] 刘亭[1,2] 王红勋 王静娴[1,4] 王爱民[1,4] 何彬[1,4] 李勇军[1,4] 

机构地区：[1]贵阳医学院药学院,贵阳550004 [2]贵阳医学院贵州省药物制剂重点实验室,贵阳550004 [3]上海科新生物技术有限公司,上海201203 [4]贵阳医学院民族药与中药开发应用教育部工程研究中心,贵阳550004

出　　处：《中国新药杂志》2015年第4期439-442,共4页Chinese Journal of New Drugs

基　　金：贵州省中药现代化科技产业研究开发专项(黔科合重G字[2013]4001);贵州省科技支撑计划社会发展攻关([2012]3079);贵州省高等学校创新能力提升计划(黔教合协同创新字[2013]04)

摘　　要：目的：研究一种新型融合蛋白核转录因子NF．KB受体激动剂配体一免疫球蛋白Fc（RANKL-Fc）的表达和纯化。方法：通过聚合酶链反应（PCR）将RANKL（140317）蛋白片段的DNA片段和人源免疫球蛋白IgGFc（1232）片段的DNA片段放大，并把它们同时亚克隆到哺乳动物细胞表达载体（PCDNA3．1）。将构建好的表达载体在哺乳动物CHO细胞进行表达、纯化。通过细胞染色和流式细胞仪分析融合蛋白RANKL-Fc分别与RANKL，FcRII相互作用。结果和结论：在CHO．K1细胞中成功表达融合蛋白RANKL-Fc，该蛋白纯化纯度超过90％。融合蛋白RANKL—Fc能分别与RANKL，FcRII相互作用，结果表明该融合蛋白是以RANKL／RANK通路为靶向的潜在的活性蛋白。Objective ： To investigate the expression and purification of a novel fusion protein, RANKL-Fc. Methods： The carboxyl-terminal 177 amino acids of RANKL （140 -317） and the 232 amino acids of human IgG Fc （1 -232） were amplified by PCR. The amplified DNA fragment was subcloned into a mammalian cell expres- sion vector （PCDNA 3. 1 ） to generate a fusion protein of RANKL-Fc for protein expression in mammalian CHO cells. The interactions of RANKL-Fc with RANKL and Fc RII were investigated by cell staining and flow cytometric analysis, respectively. Results and Conclusion： RANKL-Fc was successfully expressed and purified in the CHO- K1 cells with a purity of greater than 90%. Preliminary studies demonstrated that this fusion protein could interact with RANK and Fc RII. This result indicates that this fusion protein might work as an alternative agent to treat cancer metastasis by targeting RANKL/RANK pathway.

关 键 词：转录因子NF—κB受体激动剂配体 人源免疫球蛋白IgG FC 融合蛋白RANKL-Fc 

分 类 号：R914.5[医药卫生—药物化学]