Asia1型口蹄疫病毒含RSD受体识别位点感染性克隆的构建与病毒拯救

摘要

口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)具有高的突变率和很强的适应性,在环境条件变化时会迅速从准种种群中选择变异株来适应新的环境,这些变异株包括在RGD受体结合基序内部或附近氨基酸的替换.FMDV Asia1/JS/China/05株经不同宿主传代后产生含RSD和RDD受体识别基序的变异株,为了研究含RDD受体识别基序的FMDV在受体结合位点内1个氨基酸的变化是否影响感染性病毒子的产生,作者利用已构建的含RDD受体结合位点的FMDV Asia1/JS/China/05株的全长感染性cDNA克隆为骨架,采用融合PCR方法,构建了该毒株含RSD受体位点的全长cDNA克隆pFMDV-RSD.线性化的pFMDV-RSD重组质粒和表达T7RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,56 h后可观察到典型的FMDV致细胞病变效应.对收获的病毒分别进行序列测定、间接免疫荧光试验、电子显微镜观察和乳鼠致病性分析,结果证实该毒株细胞受体位点1个氨基酸的替换不影响活病毒的拯救.该试验为进一步研究含RSD受体结合位点FMDV的生物学特性奠定了基础.%Foot-and-Mouth Disease Virus (FMDV) has the high mutation rates and exhibits an important potential for adaptations. The new variants will be generated upon environment change to adapt new environmental conditions, these variants contained one or several amino acid replacements within the capsid protein, including the RGD cell receptor-binding motif. Following serial passages of FMDV Asia1/JS/China/05 isolates under different host condition, the viruses which contained an RSD and RDD sequence in the cell receptor-binding site were generated. To study the effect of single amino acid replacements within RDD receptor-binding motif on production of infectious virus particle, we constructed type Asia 1 FMDV full-length cDNA clone pFMDV-RSD by using over-lap PCR, based on FMDV Asia1/JS/China/05 full-length infectious cDNA clone. BHK-21 cells were cotransfected with linearized recombinant plasmids and plasmids expressing T7 RNA polymerase. Apparent CPE were observed after 56 h incubation. Furthermore, the rescued FMDV were verified by RT-PCR, IFA, electron microscope and sulk mice pathogenicity analysis. These results suggested that the infectious FMDV were acquired, and one amino acid replacement in the cell receptor-binding did not affect the virus viability. This study lays a foundation for further study of biology characteristic of rescued virus with RSD receptor recognition site.