伪狂犬病病毒Fa株gE基因的克隆与序列的比较分析

Cloning of Pseudorrabies Virus Fa Strain gE Gene and Comparative gE Sequence of Five PRV Virus Strains

根据已发表的伪狂犬病病毒(PRV)的基因序列,设计合成一对引物PCL1和PCL2,以PRVFa株的DNA为模板成功地扩增得到预期的长约1 7kb的特异片段,经酶切鉴定,初步确定为gE基因。将扩增产物经KpnⅠ、EcoRⅠ消化形成粘末端克隆到pUC18质粒,得到了明显大于载体的重组质粒PpgE。重组质粒PpgE经酶切鉴定为含有PRV的gE基因,测序PpgE得到完整的gE基因片段,并与4株不同来源的毒株进行了比较分析。5毒株的gE同源性比较分析发现毒株间同源性最低也高达97%,这体现了gE基因的保守性。分析还表明Fa与TNL同源。同时99%的高同源性也显示Fa、Ea、SH可能是同一毒株。gE序列在1040-1410碱基的高同源性区域作为PRV的PCR检测或作为核酸探针是非常重要的。

By means of polymerase chain reaction,about 17 kb fragment was amplified from PRV Fa strain.The PCR product was proved to be true by NcoⅠ?SmaⅠ and SphⅠ restriction enzyme analysis,and then cloned into PUC18 plasmid.Through restriction enzyme digestion and Dotblot,the recombinant PpgE plasmid was indicated containing gE gene.Sequencing of gE gene in PUC18 plasmid,Comparative gE sequence of five PRV virus strains,the homologue among these virus strains was at least 96%.Therefore it was proved that gE gene is a conservative gene.Taiwan strain was higher homologue with China strains in gE gene.Nucleotides 10371407 sits were identical in all strains.It provided a useful PCR region or probe to detect the latency of PRV.In addition,this reaseach was a foundation to get gE proteins in vitro.