CAJ | 학술논문

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.
목적 연구인호흡도합포병독(human respiratory syneytial viruse,RSV)분비형융합단백(secreted fusion protein,sF)기인재간상병독중적표체급순화.방법 근거편마F단백적기인서렬설계인물,이PCR방법장F단백기인적과막구화포질구핵산서렬체환위6×His핵산서렬.획득COOH'단대유His표첨적중조sF-His단백편마기인,이용Bac-to-Bac간상병독표체계통,구건가표체sF단백적중조간상병독,용지질체Cellfectine reagent전염sf9세포,실현sF재sf9세포중적표체,Ni주친화층석순화sF단백.결과 성공획득료sF-His편마기인,구건적중조간상병독가고효표체sF,Ni-주순화후sF적농도위1.084 mg/ml,순도체90%이상.결론 간상병독계통시제비8F적유효방법,순화적sF단백위RSV신형역묘급단극륭항체화진단시제등연구전정료기출.
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.