鸽痘病毒TK基因的扩增、克隆及序列分析

根据3个鸡痘病毒株的TK基因序列,借助基因分析软件设计合成了引物H1 、H2一。对PPV地方分离弱毒株PPVR的3个型PPVD(大)、PPVZ(中)、PPVX(小)和强毒株PPVY及鸽痘病毒疫苗株VVG、鸡痘病毒疫苗株VVJ进行TK基因的PCR,均成功地扩增出预期大小的目的片段。对PCR产物用限制性内切酶NcoI进行酶切分析,结果酶切产物得到两条条带,大小分别为628 bp和732 bp,与3个已发表的TK基因分析结果一致。分别将6个禽痘病毒TK基因克隆至pGEM-T Easy载体中,重组质粒经PCR和酶切鉴定后,进行测序。结果表明,本试验获得的TK基因长1 360 bp,存在一个NcoI酶切位点,两个XbaI酶切位点。序列分析表明:PPVD和PPVX同VVG和VVJ的TK基因同源性为100％;在TK基因的编码区内PPVY有一个碱基与其它毒株不同;各毒株TK基因的侧翼都存在15 bp的正向重复序列和8 bp的倒转重复序列;PPVD、PPVX和PPVZ虽然来源于同一个毒株,但TK基因存在差异。

Cloning and Sequence Analysis of Pigeonpox virus TK Gene

The TK gene fragment (1. 36 kb) of the six APV strains was amplified by polymerase chain reactioin (PCR) using the primers H1 and H2-. The result indicated that the size of the PCR products was the same as expected. When the PCR products were digested with the restriction enzyme Ncol, two DNA fragments (628 bp and 723 bp) appeared in digested products. The fragment of TK gene of the six APV strains were respectively cloned to pGEM-T Easy vector, and six recombinant plasmids (pGEM-T-TK) were obtained. The result of sequencing revealed that the TK gene fragment consised of 1 360 bp. The TK gene of the six APV strains were compared with that of FPV strains published previously. The result indicated that the six APV strains had very high homology in TK gene one another, and there was 100% homology in TK genes of PPVD, PPVZ,PPVX and PPVY. In the open reading frame of TK gene,the PPVY strain contained a single mutation of T at position 434 nt to C. The genes that flank the TK gene in the six APV strains had 15 bp direct repeats and 8bp inverted repeats. PPVD,PPVZ and PPVX came from the same PPV strains, but had differences in TK gene.