伪狂犬病病毒Fa株gE基因的克隆与序列的比较分析

根据已发表的伪狂犬病病毒（ＰＲＶ）的基因序列，在ＰＲＶｇＥ基因阅读框外的上、下分别设计一对引物Ｐ１和Ｐ２，在引物的 ５’端分别加上 ＫｐｎＩ和 ＥｃｏＲＩ位点。以 ＰＲＶ Ｆａ株的 ＤＮＡ为模板成功地扩增得到一条长的 １．７ｋｂ的片段，经酶切鉴定，初步确定为ｇＥ基因。将此ＰＣＲ产物经ＫｐｎＩ、ＥｃｏＲＩ消化后与同样酶切的ＰＵＣ１８质粒连接、转化，得到了明显大于载体的重组质粒ＰＰｇＥ。重组质粒ＰＰｇＥ经酶切鉴定确定为含有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。在对 ＰＲＶ Ｆａ（来自福建）、Ｒｉｃｅ（来自俄国）、ＴＮＬ（来自台湾）、Ｅａ（来自武汉）、ＳＨ（来自上海）的ｇＥ同源性比较分析发现毒株间同源性最低也高达９７％，这一定程度上也体现ｇＥ基因组的保守性。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。本实验认为 ｇＥ序列在 １０４０－ １４１０碱基的高同源性区域作为ＰＣＲ检测或核酸探针是非常有用的。

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

By means of polymerase chain reaction, about 1. 7kb 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 protains in virto.