In order to determine the most optimal host cell line,inoculation dose and the time of harvest,we studied the growth pattern and cytopathic effect (CPE) of the mutants of Pseudorabies virus (PRV) Ea strain,gG -/LacZ +,in different cell lines.A clone virus of gG -/LacZ +,the titer of which could be up to 10 -8.0 /0.1 ml,after purification four times by plaques,was obtained.The clone virus was inoculated into different cell lines,BHK-21,IBRS-2,MDBK and PK-15,respectively.Infected BHK-21 could be induced CPE at 12 hours p.i..However,The CPE of MDBK appeared after 24 hours p.i..In terms of the growth pattern,the titer of IBRS-2 could reach the peak level at 40 hours p.i. and the peak titer was up to 10 -8.0 /0.1 ml,which was maintained until 64 hours p.i..In contrast,the peak of BHK-21 was only 10 -6.5 /0.1 ml during the 96 hours p.i.. We also studied the titer of IBRS-2 when it was inoculated with different dose virus from 0.001 PFU/cell to 100PFU/cell.It was discovered that the titer could reached over 10 -7.5 /0.1 ml when inoculated with 0.1PFU/cell,0.01PFU/cell after 30,48 hours p.i.,respectively.These data indicate that IBRS-2 is the most optimal cell and that the titer could reach the peak when inoculated with 0.01PFU/cell and harvested at 40-50 hours p.i..
Using pseudorabies virus Ea strain as material,we inserted LacZ gene expression cassette into gE gene.After blue plaque and plaque purification,a recombinant virus PRVEa TK+-/gE+-/LacZ++ generated.Utilizing EcoR I site in LacZ gene, digested PRVEa TK+-/gE+-/LacZ++ genome DNA was cotransfected into PK-15 cells with plasmid pFBBS,then PRVEa TK+-/gE+-/gp63+- generated after plaque purification.Four pairs of primers amplification demonstrated the virus was pure TK+-/gE+-/gp63+- mutant virus.PCR product sequence indicates there were 205bp deletion in TK gene;1247bp deletion in gE,gp63 and intergenic region of PRVEa TK+-/gE+-/gp63+- mutant virus genome DNA.Inoculation to Balb/C mice with PRVEa TK+-/gE+-/gp63+- indicates the virulence is reduced greatly.
