In order to develop a simple and safe test for the detection of vaccinated as well as wild type Pseudorabies virus (PRV) infected pigs,the modified gE gene of PRV Ea strain,obtained by cutting the 5’ UTR using PCR an...In order to develop a simple and safe test for the detection of vaccinated as well as wild type Pseudorabies virus (PRV) infected pigs,the modified gE gene of PRV Ea strain,obtained by cutting the 5’ UTR using PCR and DNA recombinant technique,was inserted into baculovirus expression vector pFastBac 1,resulting the trans-position plamid pFE1.75.After homologous recombination,recombinant baculovirus rvBacE1.75 was gained and high level expression of glycoprotein E (gE) was observed after the infection of rvBacE1.75 to Tn-5B1-4 cells.The expression product was 80～88kD and was specific to antisera against PRV Ea strain by Western-blotting.Purified recombinant proteins were used as an antigen in Latex Agglutination Test(gE-LAT) and the test was specific,sensitive,safe and simple.展开更多
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 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..展开更多
文摘In order to develop a simple and safe test for the detection of vaccinated as well as wild type Pseudorabies virus (PRV) infected pigs,the modified gE gene of PRV Ea strain,obtained by cutting the 5’ UTR using PCR and DNA recombinant technique,was inserted into baculovirus expression vector pFastBac 1,resulting the trans-position plamid pFE1.75.After homologous recombination,recombinant baculovirus rvBacE1.75 was gained and high level expression of glycoprotein E (gE) was observed after the infection of rvBacE1.75 to Tn-5B1-4 cells.The expression product was 80～88kD and was specific to antisera against PRV Ea strain by Western-blotting.Purified recombinant proteins were used as an antigen in Latex Agglutination Test(gE-LAT) and the test was specific,sensitive,safe and simple.
文摘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..
