山羊痘病毒疫苗株TK基因的克隆与序列分析

Cloning and Sequence Analysis of TK Gene of the Attenuated Capripoxvirus Vaccine Strain

用绵羊睾丸细胞培养山羊痘病毒疫苗株G14-STV44-55,提取其基因组为模板,设计TK基因的特异性引物并进行PCR扩增,获得了2405bp的DNA片段,并将PCR产物克隆至pGEM-TEasy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基因内存在唯一的ACC65I酶切位点和3′末端早期转录终止信号TTTTTN(T)T。核苷酸和氨基酸同源性分析表明,弱毒疫苗株G14-STV44-55TK基因与基因Bank中发表的13个参考毒株的同源性在96%和95%以上,说明羊痘病毒TK基因具有高度的保守性。

The capripoxvirus live attenuated vaccine strain G14-STV44-55 was cultured with sheep testicular cell,and the genomic DNA was extracted from the virus strain,and a pair of specific primers were desined in order to amplify a TK gene.The PCR product approximately 2 405 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 ACC65 I site and a transcription termination signal TTTTTN （ T） T were found in 3＇ side of TK gene.Analysis showed that G14-STV44-55 strain shared 96 % and 95 % identity rates with the reference strains in levels of nucleotide and amino acid.It indicate that TK gene is very conservative.