口蹄疫病毒Asia 1/JSWX株基因组全长感染性克隆的体外拯救与序列分析

In vitro rescue and sequence analysis of an infectious full-length cDNA clone of foot-and-mouth disease virus Asia 1/JSWX strain

通过反转录聚合酶链反应,获得了口蹄疫病毒(FMDV)Asia1/JSWX株基因组3′端长片段(长约7.5kb)和5′UTR中ploy(C)前后的2个短片段。5′UTR的2个短片段经融合PCR扩增得到长约710bp的片段。用引物在基因组5′末端引入AflⅡ酶切位点和T7启动子,在5′UTR内引入SpeⅠ酶切鉴定位点,在3′末端引入NotⅠ酶切位点,将融合片段和3′端长片段顺次连接到载体pSL1180。经T7体外转录系统获取的RNA转录本与脂质体共转染BHK21细胞。测序结果表明,构建的病毒基因组全长cDNA为8 197nt,分别包括1个长为1 095nt的5′UTR[含有1个17nt的ploy(C)];1个长6 990nt的ORF;1个长为93nt的3′UTR;之后是18nt的poly(A)尾巴。该全长cDNA克隆与Asia 1/Jiangsu/China/2005株基因组序列的同源性为98.4%。测序和酶切鉴定结果均表明,该口蹄疫病毒株全长cDNA克隆已构建成功,该方法极大地简化了获得FMDV全长cDNA克隆的过程。通过反转录聚合酶链反应、间接免疫荧光试验和蚀斑试验等鉴定,本试验获得了感染性分子克隆;体外拯救获得的基因工程病毒连续传代培养后,可致BHK21细胞产生病变。上述结果表明,构建的cDNA克隆可以作为基因操作的载体,为深入研究安全性好、稳定性高和免疫原性强的基因工程疫苗奠定了基础。

A fragment covering foot-and-mouth disease virus(FMDV)Asia 1/JSWX strain genome 3′end full-length(about 7.5kb)and two short segments locating around the ploy(C)region in 5′UTR were amplified by RT-PCR,respectively.Subsequently the fragment about 710bp was obtained by over-lap PCR amplification of two short segments in the 5′UTR.Restriction enzyme site AflⅡ,a T7promoter at the 5′end,restriction enzyme site SpeⅠ within the 5′UTR and NotⅠat the 3′end were introduced into primers.Then the over-lap fragment and 3′end full-length were sequentially connected to the plasmid vector pSL1180.With the T7transcription system,the RNA transcripts in vitro with lipofection were transfected into BHK21cells.The sequencing result showed that the full-length cDNA was 8 197nt in length,which contains a 1 095nt 5′UTR with a 17nt ploy(C),a 6 990nt ORF,and a 93nt 3′UTR followed by a 18nt poly(A).Compared to the full-length cDNA clone sequence with Asia 1/JiangSu/China/2005strain genome,the homology was 98.4%.Sequencing and restriction analysis results showed that the FMDV full-length cDNA had been successfully constructed.The construction method greatly simplified the process of obtaining FMDV full-length cDNA clone.The cloned virus was identified by RT-PCR,indirect immunofluorescent assay,plaque assay and other detection method.The results showed the infectious cDNA clone was obtained.RNA transcripts in vitro were transfected into BHK21cells and serially subcultivated,causing BHK21to produce cytopathic effects.In conclusion,the constructed cDNA can be used as a carrier of genetic manipulation,which is of great help to the research of genetically engineered vaccines with enhanced security,good stability and improved immunogenicity.