Ⅰ型鸭甲型肝炎病毒VP1基因在昆虫细胞中的表达及鉴定

摘要

为在昆虫细胞中表达Ⅰ型鸭甲型肝炎病毒(DHAV-Ⅰ)的主要结构蛋白VP1,根据GenBank已发表的DHAV-Ⅰ SH株VP1基因序列,设计1对特异性引物,通过RT-PCR扩增VP1基因,将其克隆至杆状病毒转移载体pFastBac1,获得重组杆状病毒转移载体pFB-VP1,将其转化E.coliDH10Bac感受态细胞,经抗性、蓝白斑筛选及PCR鉴定后,构建重组穿梭质粒rBacmid-VP1.在脂质体介导下将重组质粒rBacmid-VP1转染昆虫细胞Sf 9,制备含有DHAV-ⅠVP1基因的重组杆状病毒rBac-VP1.Western blot结果显示,表达的重组蛋白VP1大小约27 ku,能与DHAV-ⅠVP1多克隆抗体发生特异性反应;间接免疫荧光检测结果显示,重组蛋白VP1能与DHAV-Ⅰ全病毒阳性血清发生特异性反应,细胞内出现较强的荧光.以上结果表明,在昆虫细胞中成功表达了具有免疫原性的DHAV-Ⅰ主要结构蛋白VP1.%In order to express the main structural protein VP1 of duck hepatitis A virus type Ⅰ (DHAV-Ⅰ)in insect cells,one pair of specific primers were designed according to the published genome sequences of DHAV-Ⅰ to amplify VP1 gene by RT-PCR,the amplified fragment was cloned into baculovirus expression vector pFastBac1.The recombinant vector pFB-VP1 was transformed into E.coli DH10Bac competent cells,and the positive recombinant bacmid rBacmid-VP1 was screened according to the resistant and the blue-white plague screening,confirmed by PCR.The recombinant bacmid rBacmid-VP1 was transfected into the Sf 9 insect cells by liposome to obtain the recombinant baculovirus,and was named as rBac-VP1.The result of Western blot showed that the molecular weight of the recombinant proteins was about 27 ku,and could be recognized by the antiserum of DHAV-Ⅰ VP1.Indirect immunofluorescence analysis proved that the recombinant proteins could be recognized by the positive anti-virus serum.These results suggested that the main structural protein VP1 of DHAV-Ⅰ with immunogenicity was expressed successfully in insect cells.