含2A片段的重组黄热病毒17D疫苗表达载体的构建

Construction of Recombinant Yellow Fever Virus 17D Containing 2A Fragment as a Vaccine Vector

黄热疫苗是一种减毒的黄热病毒17D(YF-17D)活疫苗,是现有疫苗中最安全、最有效的疫苗之一,适于发展为疫苗载体。用RT-PCR法扩增出覆盖YF-17D全长基因组的3个cDNA片段:5′cDNA(A)、3′cDNA(B)和中间cDNA(C),同时引入SP6增强子序列、酶切位点和重复序列。顺序将A和B同E.coli-yeast穿梭质粒pRS424连接,再与C共转染酵母菌,利用缺少色氨酸和尿嘧啶的选择性固体培养基筛选出含YF-17D全长基因组的cDNA质粒。以该质粒为模板,经过DNA重组和酵母同源重组,获得含有口蹄疫病毒蛋白水解酶2A片段的重组YF-17D表达载体。将该表达载体体外转录后,电击转染BHK-21细胞。间接免疫荧光检测结果表明,RNA转录体在BHK-21细胞中进行了稳定的表达;滴度测定与形态学观察结果表明,重组病毒在细胞中的生长曲线等特征同母本YF-17D十分相似。结果提示,利用酵母菌同源重组在2A部位引入异种抗原基因,重组YF-17D表达载体pRS-YF-2A1具有成为高效活疫苗表达载体的潜力。

The Yellow Fever （YF） vaccine, an attenuated yellow fever 17D （YF-17D） live vaccine, is one of the most effective and safest vaccines in the world and is regarded as one of the best candidates for viral expression vector. We here first reported in China the construction and characterization of the recombinant expression vector of yellow fever 17D which contained the proteinase 2A fragment of foot-and-mouth disease virus （FMDV）. Three cDNA fragments representing the full-length YF-17D genome, named 5＇-end cDNA （A）, 3＇-end cDNA （B） and middle cDNA （C）, were obtained by reverse transcription polymerase chain reaction （ RT-PCR）, together with the introduction of SP6 enhancer, necessary restriction sites and overlaps for homologous recombination in yeast. Fragment A and B were then introduced into pRS424 in turn by DNA recombination, followed by transfection of fragment C and the recombinant pRS424 containing A and B （pRS-A-B） into yeast. A recombinant vector containing full length cDNA of YF-17D （pRS-YF） was obtained by screening on medium lack of tryptophan and uracil. A recombinant YF-17D expression vector containing FMDV-2A gene fragment （pRS-YF-2A1） was then constructed by methods of DNA recombination and homologous recombination in yeast described above. In vitro transcription of the recombinant vector pRS- YF-2A1 was then carried out and introduced into BHK-21 cells by electroporation. Results of indirect immunofluorescence assay （IFA） and titer determination showed a stable infectious recombinant virus was gotten, whose features such as growth curve were similar to those of the parental YF-17D. The results suggest that the recombinant vector pRS-YF-2A1, by introduction of heterogenous genes via 2A region, is potential to be an effective live vaccine expression vector.