口蹄疫病毒WFL株基因组全长cDNA的克隆及序列分析

根据口蹄疫病毒基因组的结构特点以及GenBank上公布的全序列，用DNAMAN分别设计了涵盖整个基因组序列的3对引物，从接种口蹄疫病毒WFL株的细胞培养液中提取了病毒基因组RNA，采用RT-PCR方法和RACE法分别扩增了3条基因片段，并将扩增片段分别与T载体连接，在体外分别进行了5-半分子和3’半分子的构建。最后将5’半分子和3’半分子连接成基因组全长cDNA分子。经PCR鉴定、酶切鉴定及全长cDNA测序，证实成功构建了口蹄疫病毒wFL株基因组全长eDNA分子。序列分析结果表明，供试口蹄疫病毒基因组全长为8155nt，5＇UTR长1059nt；具有一个大的读码框，其核苷酸长度为6969nt。包括201aa的前导蛋白基因和2122aa的聚合蛋白基因；3＇UTR长127nt，包括34nt的polv（A）。

Construction of the genomic full-length cDNA of foot-and-mouth disease virus WFL strain

In order to establish a reverse genetic system of foot-and-mouth disease virus(FMDV), the full-length cDNA of FMDV WFL strain was constructed. Using the DNAMAN, three pairs of over-lapping primers, which cover the whole genome of FMDV WFL strain, were designed according to the nucleotide sequence reported. The total RNA extracted, three over-lapping fragments were amplified by the RT-PCR or the RACE method, and cloned into T-vector, respectively. The three fragments were ligated into full-length cDNA molecule of WFL strain in vitro. It was showed that the full-length cDNA molecule of FMDV WFL strain has been constructed successfully, which was proved by PCR, restriction analysis and sequencing. The result of sequence analysis showed that the full-length cDNA of WFL strain was 8 155 nt, including 1 059 nt of the 5'UTR, 6 969 nt of the ORF and 127 nt of the 3'UTR.