伪狂犬病病毒Min-A株TK基因的克隆与序列分析

参考GeneBank收录的伪狂犬病病毒TK基因的序列设计了1对引物，对PRV Min-A株进行了PCR扩增，扩增产物克隆于pGEM-T Easy载体．对重组质粒进行限制性内切酶分析和基因测序，证实了克隆片段的可靠性．测序结果表明目的片段包含1个957bp的开放性阅读框(ORF)，编码318个氨基酸组成的多肽．在TK ORF上游-22，-166，-199位存在3个GC框样序列，在终止密码子下游第110个核苷酸处的l306位存在有多聚腺苷加尾信号．PRV Min-A株与PRV Ea株、NIA-3株TK的核苷酸同源性分别为98．4％，97．9％；推导氨基酸的同源性分别为97．8％，97.2％．通过对α疱疹病毒TK氨基酸进行多序列比较．获得了在PRV TK氨基酸序列上的6个保守区间．推测这些保守氨基酸对于胸苷激酶(TK)结构的稳定性和催化活性的发挥是必需的。

Cloning and sequence analysis of the TK gene of pseudorabies virus Min-A strain

A pair of primers was designed according to the sequences pulished by the GeneBank in order to amplifiy TK gene of the pseudorabies virus Min-A strain.The TK gene was obtained by polymerase chain reaction(PCR), and then cloned into the pGEM-T Easy vector. The recombinant plasmid of pGTE-TK was identified by restriction enzyme analysis and sequencing ,which proved completely its validity. Nucleotide sequencing revealed that this fragment contained an open reading frame of 957 bp encoding a 318 aa protein.Upstream of the TK ORF,three putative GC boxes are located at positions -22,-166 and -199. A potential poly A signal begins 110 nucleotides downstream from the termination codon at position 1 306. The homology of the nucleotide sequence of PRV Min-A strain TK with PRV Ea strain,NIA-3 strain was 98.4%,97.9%,respectively ;and the homology of predicted amino acids among them was 97.8%,97.2%,respectively.We have identified six conserved domains by multiple sequence alignments of the thymidine kinase proteins of the alphaherpesviruses. It is speculated that these conserved amino acid residues are necessary for structural stability or involved in functional domains responsible for catalytic activities.