FMDV全ORF编码基因的克隆及其腺病毒穿梭质粒的构建

Cloning of whole ORF coding genes of FMDV and construction of recombinant adenoviral pAdTrack-CMV

为研制FMD复制缺陷型腺病毒活载体疫苗,通过RT-PCR方法获得了O型口蹄疫病毒(FMDV)全开放阅读框(ORF)基因片段,将其克隆到pMD18-T载体后测序,再将ORF编码基因定向克隆入腺病毒穿梭载体pAdTrack-CMV中,构建了重组腺病毒穿梭质粒。经PCR、酶切及测序鉴定,所克隆的ORF编码基因序列与原始强毒株Akesu/58的ORF编码基因比较,L、P1、P2、P3基因的核苷酸同源性分别为98.8%、97.9%、99.0%和97.6%,PCR、酶切鉴定重组腺病毒穿梭质粒均获得了长约6.9 kb的目的基因片段。表明,成功获得了含有O型FMDV全ORF编码基因的阳性克隆,并成功构建了含有完整FMDV开放阅读框架的编码基因表达盒的重组腺病毒穿梭质粒。

In order to develop a recombinant adenoviral pAdTrack-CMV vaccine, genes of a whole ORF coding genes of foot-and-mouth disease virus（FMDV） were amplified by RT-PCR and inserted subsequently into a pMD18-T vector and sequenced. The gene was cloned into the vector pAdTrack-CMV to construct the recombinant adenoviral pAdTract-CMV/ORF. The recombinant pAdTrack/ORF was identified by PCR,digestion with restriction endonucleases and sequencing. The sequencing results showed that the amplified L, P1, P2 and P3 gene＇s nucleotide sequences shared 98.8% ,97.9% ,99.0% and 97.6% homology with that of Akesu/58, respectively. PCR amplification and digestion with Xba I ＋Not I confirmed that the recombinant adenoviral pAdTrack-CMV contained the target genes of 6.9 kb. The results showed that the recombinant pAdTrack/ORF containing the whole ORF gene of O type FMDV was constructed successfully.