【摘要】 为构建A型口蹄疫病毒(FMDV)多抗原表位杆状病毒表达载体,将A型FMDV上5个B细胞表位(VP1140-160aa、VP270-80aa、VP352-67aa、3B29-42aa和3D16-30aa)和4个辅助性T细胞表位(VP1200-213aa、VP420-34aa、3A21-35aa和3D346-370aa)通过人工合成的方法串联起来构建复合多表位基因B。然后将其克隆至杆状病毒表达载体pFastBac HTB。将酶切和测序鉴定正确的阳性重组质粒pFastBac HTB-B转化至大肠杆菌DH10Bac感受态细胞进行蓝白斑筛选。将PCR鉴定正确的重组杆状病毒质粒利用CellfectinⅡReagent转染至Sf9细胞。通过间接免疫荧光试验和Western-blot检测表达情况。结果显示,能够得到与A型FMDV猪抗阳性血清结合的蛋白,大小约为22.3ku,与预期结果相符。结果表明,成功构建了A型FMDV多抗原表位杆状病毒表达载体,并在昆虫细胞中正确表达,为下一步蛋白纯化奠定了基础。更多还原

【Abstract】 To construct the baculovirus expression vector for multi-epitopes of foot-and-mouth disease virus(FMDV)type A,a nucleotide sequence B which is containing five B cell epitopes(VP1140-160 aa,VP270-80 aa,VP352-67 aa,3B29-42 aaand 3D16-30aa)and four helper T cell epitopes(VP1200-213 aa,VP420-34 aa,3A21-35aaand3D346-370aa)of FMDV was synthesized and cloned into the baculovirus expression vector pFastBacTMHTB.With the identification of sequencing and enzyme digestion,the positive recombinant plasmids pFastBacTM HTB-B were selected and transformed into Escherichia coli DH10 Bac competent cells for white and blue clones screening.After the identification with PCR assay,the positive recombinant bacmid was transfected into Sf9 cells by using CellfectinⅡ Reagent.Then the protein was expressed and confirmed by indirect immunofluorescence assay and Western-blot.As expected,the expressed protein was 22.3ku in size and could be recognized by the positive porcine antiserum against FMDV type A.In the present study,the baculovirus expression vector for multi-epitopes of FMDV type A was successfully constructed and expressed in insect cells,which laid the foundation for the protein purification in the further works.更多还原