口蹄疫病毒WFL株2C-P3-3′NCR片段的克隆及适于设计siRNA的序列分析

目的筛选出口蹄疫病毒(FMDV)基因组上适于设计siRNA的基因片段。方法以FMDV WFL株RNA为模板,用3′RACE法扩增出2C-P3-3′NCR序列,将PCR扩增片段克隆到pMD18-T载体上,转化E.coli DH5α,筛选阳性重组子,进行序列测定,并与参考毒株序列比较。结果扩增的基因片段大小为4033bp,其2C、P3和3′NCR序列的核苷酸序列与参考毒株的同源性分别为87·11%～94·23%、84·91%～96·66%和66·98%～82·35%,2C和P3与参考毒株的氨基酸同源性分别为94·97%～99·39%和91·84%～98·55%,WFL株P3基因的第1150～1270、1680～1840和2080～2490bp以及2C基因的第354～470bp范围内与参考毒株具有高度保守的特性。因此,可以依据siRNA设计原则,在这4个区域内筛选抑制效果好的siRNA。结论成功克隆了FMDV WFL株2C-P3-3′NCR基因的序列,并筛选出4个适于设计siRNA的基因区段。

Cloning of 2C-P3-3'NCR of Foot-and-Mouth Disease Virus WFL Strain and Screening of Gene Fragment for Design of siRNA

Objective To screen the gene fragment suitable for design of siRNA of foot-and-mouth disease virus(FMDV).Methods Amplify 2C-P3-3'NCR sequence by 3'RACE method using the RNA of FMDV WFL strain as template and clone into pMD18-T vector.Transform the recombinant plasmid to E.coli DH5α and screen positive recombinants for sequencing.Compare the sequence of cloned gene fragment with that of reference strain.Results The length of amplified gene fragment was 4 033 bp.The homologies of nucleotide sequences of cloned 2C,P3 and 3'NCR genes to those of reference strains were 87.11%-94.23%,84.91%-96.66% and 66.98%-82.35% respectively.The homologies of deduced amino acid sequences of 2C and P3 genes to those of reference strains were 94.97%-99.39% and 91.84%-98.55% respectively.The 1150-1 270,1680-1840 and 2080-2490 bp of P3 gene as well as the 354-470 bp of 2C gene were highly conserved and suitable for the design of siRNA.Conclusion The 2C-P3-3'NCR sequence of FMDV WFL strain was successfully cloned,and four gene fragments suitable for design of siRNA were screened.