伪狂犬病病毒Bartha株TK-/LacZ+突变株的构建及其生物学特性研究

本研究将伪狂犬病病毒Bartha株基因组与含有LacZ标志基因的TK基因转移质料pUEKPZ共转染猪肾传代细胞PK－5，细胞出现病变后，反复冻融3次收毒，按1：5稀释接种于IBRS－2细胞。在X－gal存在下挑取蓝斑，蓝斑筛选3次，再进行空斑试验，同时用PCR扩增LacZ基因，经3次空斑纯化，随机挑取的空斑均能扩增出LacZ基因，证实所获得的重组病毒为伪狂犬病病毒Bartha株TK^-/LacZ^ 突变株。TCID50试验表明，TK失活对Bartha株在细胞上增殖无影响；Balb/C小鼠试验表明，该突变对Balb/C小鼠的安全性明显高于Bartha亲本毒株。

Construction of TK-/LacZ+ Mutant Strain of Pseudorabies Virus Bartha Strain and the Study on its Biological Property

In this study,the TK gene transfer vector pUEKPZ containing LacZ gene and PRV Bartha genome DNA were cotransfected onto PK 15 cells.When cytopathogenic effect appeared,virus contained supernatant was harvested.Transfection progeny was frozen and thawed for three times,diluted as 5 fold volume,and plated onto IBRS 2 cells at the presence of X gal.The picked blue plaques were purified by plaque technique for three times.Then plaques were picked randomly,used as templates and LacZ gene fragment could be amplified from all of them.This result showed that the mutant virus was PRV Bartha TK -/LacZ + strain.TCID 50 assay indicates the mutant strain grows well on PK 15 cells;Inoculation to Balb/C mice indicates it is safer to Balb/C mice than the parent virus Bartha.