马疱疹病毒1型主要毒力基因分析及TK基因缺失载体的构建

为了解新疆马疱疹病毒1型（EHV-1）主要毒力基因遗传进化情况并构建TK基因缺失株，本研究以EHV-1 XJ2015株DNA为模板，对其主要毒力基因TK、gI和gE全长进行克隆、测序及生物信息学分析，并扩增TK基因左右重组臂TKL和TKR，构建质粒pUC-TKLR，将扩增后的增强绿色荧光蛋白（EGFP，含有CMV+polyA）插入pUC-TKLR质粒，构建TK基因缺失打靶质粒。TK、gI和gE基因同源性分析结果显示，XJ2015株与国外EHV-1分离株TK、gI和gE基因同源性均较高，分别为99.8%~100.0%、99.6%~100.0%和99.9%~100.0%；与EHV-3分离株同源性均最低，分别为72.9%、59.4%和62.1%；遗传进化分析显示，3个基因均与国外EHV-1同属于一个遗传进化分支，与EHV-9和EHV-4进化关系较近，但与EHV-3进化关系较远，表明XJ2015毒株与国外EHV-1毒株TK、gI、gE基因核苷酸上差异不明显，没有明显的地域性特征，功能基因保守且进化缓慢，同源基因功能相同或相近；经PCR扩增、酶切、测序及转染鉴定，本试验成功构建了用于TK基因缺失的打靶质粒pUC-TKLR-EGFP。通过对EHV-1主要毒力基因的分析及TK基因缺失打靶载体的构建，为新疆地区马鼻肺炎流行病学调查分析、TK基因缺失株的构建提供理论依据。

Analysis of Major Viral Genes of Equine Herpesvirus Type 1 and Construction of TK Gene Deletion Vector

In order to understand the genetic evolution of the major virulence genes of equine herpesvirus 1 (EHV-1) of Xinjiang (XJ2015 strain) and construct TK gene-deleted strain,we cloned,sequenced and bioinformatics analyzed the full-length of TK,gI and gE genes of XJ2015 strain in this study.The DNA of the XJ2015 strain was used as a template,the TK gene recombination arms TKL and TKR were amplified to construct plasmid pUC-TKLR,and then inserted the amplified EGFP (CMV+polyA) into the pUC-TKLR plasmid.The results showed that it had higher homology between TK,gI and gE genes of XJ2015 strain and foreign EHV-1 isolates,which were 99.8% to 100.0%,99.6% to 100.0% and 99.9% to 100.0%,respectively,and had the lowest homology with foreign EHV-3 isolate,which were 72.9%,59.4% and 62.1%,respectively;Genetic evolutionary analysis showed that all three genes were genetically related to foreign EHV-1 isolates,the evolutionary relationship with EHV-3 was far away,but it was closely related to EHV-9 and EHV-4.There were no significant difference,no obvious regional features,the functional genes were conserved,the evolution was slow and homologous genes had the same or similar functions of TK,gI and gE genes of XJ2015 strain between foreign EHV-1 strains.After enzyme digestion,sequencing and transfection,the plasmid pUC-TKLR-EGFP for TK gene deletion targeting was successfully constructed.Through the analysis of EHV-1 main virulence genes and the construction of TK gene deletion targeting vector,it provided a theoretical basis for the epidemiological investigation and analysis of the TK gene deletion strain in Xinjiang.