山羊痘弱毒疫苗株TK基因的克隆及序列分析

Cloning and sequence analysis of TK gene of the attenuated capripoxvirus vaccine strain

以提取的山羊痘弱毒疫苗株HLJ—V9 DNA为模板，设计特异性引物并进行PCR扩增，获得了2078bp的DNA片段，并将PCR产物克隆至pGEM—T Easy载体。酶切鉴定、PCR鉴定和测序结果表明，成功获得了TK基因，获得的TK基因内存在一个Kpn Ⅰ酶切位点和编码区的早期转录终止信号TTTTTN（T）T。核苷酸和氨基酸同源性分析表明，弱毒疫苗毒株HLJ—V9 TK基因与参考毒株的同源性在95．5％～100％之间，说明羊痘病毒TK基因具有高度的保守性。

The genomic DNA was extracted from the capripoxvirus live attenuated vaccine strain HLJ- V9,and a pair of specific primers were designed in order to amplify a TK gene. The PCR product approximately 2 078 bp in size was cloned into pGEM-T Easy vector. Restriction enzyme assay,PCR and sequencing confirmed that the TK gene was obtained successfully. Only one Kpn I site and a transcription termination signal TTTTTN（T）T were found in the gene. Analysis showed that HLJ-V9 strain shared 95.5% --100% identity rates with the reference strains in levels of nucleotide and amino acid,indicating that TK gene is very conservative.