论文部分内容阅读
为揭示PRV tk基因结构与毒力的相互关系, tk基因结构变异对病毒生物学特性的影响和探讨病毒的致弱机理, 以伪狂犬病毒湖北株(PRV HB)为材料, 用BUdR诱变选择, 分离出BUdR抗性的PRV TK-突变株. 在对野毒株和TK-突变株tk基因克隆和测序的基础上, 分析比较了两种毒株tk基因的一级结构. 测定的PRV HB野毒株tk基因片段长1587 bp, GC含量为72.7%. 包含一个1098 bp的ORF. 编码366个氨基酸残基组成的蛋白. ORF内有2个137 bp核苷酸的重复序列, 两者以一个A连接. 测定的TK-突变株tk基因片段长1458 bp, 野毒株中137 bp重复序列之一和连接A被自然删除, 另有一个8核苷酸(GCGCGCCC)重复片段的插入, 其他所有核苷酸与野毒株一致. 在TK-突变株中因核苷酸片段的缺失和插入, tk基因发生移框突变, 导致转译提前终止, 不能产生有功能的TK蛋白. 氨基酸序列分析显示, PRV HB TK具有疱疹病毒TK共有的保守功能区并多出一个保守的DRH功能位点. 实验还证实, TK-突变株具有生物学遗传稳定性, 与野毒株比较, 其毒力显著降低. 用TK-突变株接种小鼠, 能诱导小鼠产生针对PRV强毒株致死攻击的有效保护.
In order to reveal the relationship between the structure and virulence of PRV tk gene and the structural variation of tk gene on the biological characteristics of the virus and to explore the mechanism of virus weakening, PRV HB strain was used as the material and mutagenized with BUdR , And the strain of PRV TK-mutants resistant to BUdR was isolated.On the basis of cloning and sequencing of the tk gene of wild-type strain and TK-mutant strain, the primary structure of tk gene was analyzed and compared.The measured PRV HB The wild-type strain tk gene was 1587 bp in length with a GC content of 72.7%. It contained a 1098 bp ORF encoding a protein of 366 amino acid residues. The ORF contained two 137-bp nucleotide repeats, One A. The tk gene fragment of the TK-mutant strain was determined to be 1458 bp in length and one of the 137 bp repeats in the wild-type strain was deleted naturally with Attachment A and another 8-nucleotide (GCGCGCCC) repeat was inserted, while the other All the nucleotides are identical with the wild-type strain.At the same time, the tk gene was mutated in frame due to the deletion and insertion of the nucleotide fragment in the TK-mutant strain, leading to the early termination of translation and unable to produce the functional TK protein.Amino acid sequence analysis showed that, PRV HB TK has a conserved work common to herpes virus TK And more than a conservative site of DRH function.Experiments also confirmed that TK-mutant strains with biological genetic stability, compared with the wild-type strain, its virulence significantly reduced vaccination with TK-mutant mice, can induce small Mice produce potent protection against lethal challenge with virulent strains of PRV.