| 1. Establishment of Pseudorabies Virus Ea Strain gD gene transferred cell linesThe retrovirus mediated transfer gene method and Eukaryotic expression recombinant plasmid mediated transfer gene method were applied to introduce PrV Ea gD gene into PK-15 respectively, and cell lines designated as PKD and PKPD expressing gD were established. By observation the fluorescence, it is discovered that the expressed gD by PKD and PKPD distributed in the cell membrane and demonstrated the same immunoreactivity as the original gD. In addition to that, it was noticed that the gD was in apparent polar distribution. Each other 5 passages, PCR technique and indirect immunofluorescence assay were applied to detect PKD untill the 30lh passage, gD gene and its expression product always can be detected. The results demonstrated that the transferred gD gene was genetically stable. In the same culture condition, the growth speed and the shape of PKD and PKPD did not show apparent difference compared with normal PK-15. Using PKD and PKPD as host cells, by lipofeltin mediated method, PrV Ea genome was transferred into them respectively, a typical cytopathic effect was observed after 30 hours which prove PKD and PKPD had the endurance to lipofeltin and could be used as the cells to select recombinant virus. The detection results of TCID5o demonstrated that although PKD and PKPD showed apparent suppression to PrV Ea strain amplification, it still could be amplified in PKD and PKPD normally. All this research results suggested that PKD could be used to establish PrV Ea strain gD deleted mutant and thus provided a essential tool for it.2. Construction of gD Gene Deleted Pseudorabies Virus MutantsPlasmid SK-gG-2.4 that gD gene was deleted was constructed firstly, Plasmids SK-gG-2.4 -. p6.6BEIT and genome of PrV Ea TK7gE7LacZ+ were contransfered into PKD, two recombinant virus TK7 gD7gE\ TK7 gD7gE"B were obtained. The sequencing results of PCR amplified fragments proved that the recombinant fragments were in the recombinant virus indeed, But the two recombinant viruses both can amplify normally in PK-15 ^ BHK> He^ IBRS-2 and the virus liters of the first and second passage showed no apparent difference, and no apparent difference was observed between the two recombinant viruses and TK7gE7LacZ+. The result showed that the two recombinant viruses could amplify in nocomplemented cell lines normally. The biological characteristics of the two recombinant viruses were different compared with the gD gene deleted mutants established by foreigner.In order to find out the resean causing the difference, we checked the possibility that gD gene still exist in the recombinant viruses.The results proved that gD gene still exist in the recombinant viruses,and in the original location.The recombinant viruses adopted another site to recombine with TK7gE7LacZ+ instead of the expected site.To find out another possible identity arm, the transfer plasmids and TKVgEVLacZ^ genome were compared, we finally focus on the LacZ gene. After identification of the direction of LacZ expression box in TK7gE7LacZ+ and sequencing of PCR amplified fragment, we finally found out that the recombinat viruses were obtained by adopting LacZ expression box in TK7gE7LacZ+ and LacZ gene a fragment (451-626bp) in transfer plasmids as one of the two identity arms.The unique EcoRl site which is brought into TK7gE7LacZ+ genome by LacZ expression box is located in the tail of the LacZ expression box, when TK7gE7LacZ+ genone was cut into two fragment by EcoRl, if adopt the LacZ gene a fragment (451-626bp) in transfer plasmids as one of the two identity arms, the two identity arms were located in the same fragment of the cuted genome, in this condition, it is almost impossibility to obtain such kind of recombinant virus, but, we obtained two strains of such kind of recombinant virus and 95% of the first passage of one of them?TK7 gDVgE" is positive. The results are hard to understand and worthy of further studies. The discovery that LacZ gene a fragment (451-626bp) in transf...
|
PDF Full Text Request