呼吸道合胞病毒分泌型融合蛋白的真核表达和纯化

目的 研究人呼吸道合胞病毒(human respiratory syneytial viruse,RSV)分泌型融合蛋白(secreted fusion protein,sF)基因在杆状病毒中的表达及纯化.方法 根据编码F蛋白的基因序列设计引物,以PCR方法将F蛋白基因的跨膜区和胞质区核酸序列替换为6×His核酸序列.获得COOH'端带有His标签的重组sF-His蛋白编码基因,利用Bac-to-Bac杆状病毒表达系统,构建可表达sF蛋白的重组杆状病毒,用脂质体Cellfectine reagent转染sf9细胞,实现sF在sf9细胞中的表达,Ni柱亲和层析纯化sF蛋白.结果 成功获得了sF-His编码基因,构建的重组杆状病毒可高效表达sF,Ni-柱纯化后sF的浓度为1.084 mg/ml,纯度达90%以上.结论 杆状病毒系统是制备8F的有效方法,纯化的sF蛋白为RSV新型疫苗及单克隆抗体和诊断试剂等研究奠定了基础.

Expression and purification of a secreted form of fusion glycoprotein of human respiratory syncytial virus encoded by recombinant baculovirus

Objective To study the expression and purification of a secreted form of fusion glycoprotein (sF)of human respiratory syneytial virus(RSV)encoded by recombinant baculovirus.Methods According the ORF of F protein,a pair of specific primers was designed and PCR technique was exploited to amplify the gene of sF in which the gene sequence of the transmembrane and cytoplasmic tail domains were replaced by a C-terminal six-histidine tag.Then,a recombinant baculovirus encoding sF-His was constructed,and transfected into sf9 insect cells by Lipofectamine cellfectine reagent.Finally,the expressed sF was purified by Ni2+-affinity chromatograph.Results The gene encoding sF-His was obtained.The resulting construct of recombinant baculovirus is capable of expressing sF protein.The concentration of Ni2+-affinity chromatograph purified sF is 1.084 mg/ml with the purity of no less than 90%.Conclusion Baeulovirus expression system is a good method for large scale of preparation of sF.The purified F paves the way for the development of potential RSV vaccine and diagnostic kit,ere.