论文部分内容阅读
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。
Yellow fever vaccine is an attenuated yellow fever virus 17D (YF-17D) live vaccine that is one of the safest and most effective vaccines available in existing vaccines and is suitable for development as a vaccine vector. Three cDNA fragments covering the full-length genome of YF-17D were amplified by RT-PCR: 5'cDNA (A), 3'cDNA (B) and middle cDNA (C). At the same time, SP6 enhancer was introduced and digested Site and repeat. Sequences A and B were ligated with the E.coli-yeast shuttle plasmid pRS424 and cotransfected with C into yeast, and the cDNA containing the full-length genome of YF-17D was screened using a selective solid medium lacking tryptophan and uracil Plasmid. Using the plasmid as a template, a recombinant YF-17D expression vector containing the 2A fragment of foot-and-mouth disease virus proteolytic enzyme was obtained after DNA recombination and yeast homologous recombination. The expression vector was transcribed in vitro and then electroporated into BHK-21 cells. The results of indirect immunofluorescence assay showed that the RNA transcripts were stably expressed in BHK-21 cells. The titer and morphological observation showed that the growth curves of the recombinant virus in the cells were similar to those of female parent YF-17D . The results suggested that heterologous antigen gene was introduced into 2A by homologous recombination of yeast, and the recombinant YF-17D expression vector pRS-YF-2A1 has the potential to become a highly efficient live vaccine expression vector.