山羊痘病毒TK基因功能缺失对其增殖的影响

Effect on Proliferation of Goat Pox Virus of TK Gene Deficiency

为了研究山羊痘病毒TK基因功能缺失对其增殖的影响,同源重组构建TK基因缺失型重组山羊痘病毒,检测其增殖特性。试验在通用转移质粒pTKfpgigp内插入不同长度的X1~X4片段,构建了4个重组转移质粒;重组质粒转染已感染山羊痘病毒的羔羊睾丸细胞,经同源重组产生4株TK基因缺失型重组山羊痘病毒rGPV/tk-,其TK基因内分别插入了长度约为2 800、4 000、5 200、7 700bp的外源DNA片段;筛选纯化重组毒株,测定rGPV/tk-病毒滴度。结果显示,TK基因失活后,rGPV/tk-病毒滴度明显降低;随着插入DNA片段的延长,重组毒株的病毒滴度下降程度更加明显。研究表明,TK基因功能的缺失对山羊痘病毒的增殖具有一定的抑制作用,并与外源DNA的长度具有相关性。

In this study,the proliferation of goat pox virus（GPV）was researched using the recombinant GPV of TK gene insertion inactivation.Different length DNA fragments X1,X2,X3 and X4were inserted into the transfer plasmid pTKfpgigp to constructed the recombinant transfer plasmids and these plasmids were transfected into lamb testis cells infected GPV.Recombinant GPVs of TK gene inactivation by inserting foreign DNA fragment of 2 800,4 000,5 200and7 700 bp,respectively,were generated via homologous recombination.Four recombinant GPVs（rGPV/tk-）were obtained by selective culture and the virus titer were mensurated by 50% tissue culture infective dose.The results showed that the rGPV/tk-virus titers were decreased obviosuly which was comparable with the wild type GPV AV41.Furthermore,the virus titer of the recombinant virus was decreased more obviously with the extension of the foreign DNA fragment.The result indicated that the proliferation of the GPV was depressed when GPV TK gene was deficient.