【摘要】 为在杆状病毒表达系统中表达口蹄疫病毒(FM D V)结构蛋白VP1,利用限制性酶切位点将VP1基因插入到杆状病毒表达载体pFastBacHTB,得到重组转移载体p FastBacHTB-VP1,并将其转化至DH10Bac感受态细胞进行蓝白斑筛选。将鉴定正确的重组杆状病毒质粒转染Sf9细胞后收获重组杆状病毒rBacHTB-VP1,通过间接免疫荧光(IFA)和蛋白质印迹(Western-blot)鉴定结构蛋白VP1的表达情况。IFA结果表明结构蛋白VP1能够在昆虫细胞中表达;Western-blot检测到32 ku左右的表达产物,与预期结果相符,且能够被A型FMDV豚鼠阳性超免疫血清所识别。上述结果表明,经杆状病毒真核表达系统表达的可溶性蛋白VP1具有良好的反应原性,为进一步研究A型口蹄疫病毒结构蛋白的免疫原性以及研制新型口蹄疫亚单位疫苗提供了实验依据。更多还原

【Abstract】 In order to express the structural protein VP1 of foot-and-mouth disease virus in the baculovirus expression system, VP1 gene was cloned into the baculovirus vector p Fast Bac HTB using restriction enzyme cutting sites and then the recombinant plasmid p Fast Bac HTB-VP1 was constructed. Subsequently the recombinant plasmid was transformed into DH10 Bac competent cells for blue-white selection, and then the identified recombinant baculovirus plasmid was transfected into Sf9 cells to obtain the recombinant baculovirus r Bac-HTB-VP1. The indirect immunofluorescent assay(IFA) and Western-blot were performed to identify the expression of VP1 protein. The IFA result indicated that VP1 protein was successfully expressed in Sf9 cells.Western-blot analysis showed that the size of product is about 32 ku,which correspond to the anticipated size, and the products could combine with the positive guinea pig antiserum against FMDV type A.This study indicated the soluble VP1 protein expressed in eukaryotic baculovirus expression system could keep the effective reactionogenicity. Besides, our study also provides experimental bases for analyzing the immunogenicity of structural protein of FMDV type A and developing new subunit vaccines against FMD.更多还原