Cloning The Pseudorabies Virus Genome As A Bacterial Artificial Chromosome In Escherichia Coli And Its Application For Construction Of PRV Mutants

Pseudorabies virus is known by its taxonomic name, suid herpesvirus 1, or by its original name, Aujeszky's disease virus. PRV is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. PRV has been of interest to virologists and neurobiologists as well as those concerned about disease control in swine agriculture. PRV provides an attractive model for detailed analysis of the pathogenesis of this virus group because of its ease of laboratory manipulation, its ability to infect and cause similar disease in a wide variety of animals, and its inability to infect humans.Molecular techniques are often used to examine the roles of virally encoded gene products in viral growth and pathogenesis. The primary method for investigating the function of individual herpesvirus genes is mutagenesis. Mutated viruses are usually constructed by homologous recombination following the cotransfection of viral genomic DNA and a mutated allele on a separate DNA fragment. Recombinant viruses are either screened or selected during several sequential rounds of plaque purification. However, if the mutation results in a growth defect relative to wild-type parental virus, the mutant virus may be difficult or impossible to purify. To overcome some of these limitations, several laboratories have cloned entire herpesvirus genomes in Escherichia coli. Because of the large size of the viral genomes, the clones comprise either overlapping cosmid sets or single full-length clones in F plasmids. Because the entire viral genome is maintained in a single E. coli plasmid, these clones do not require repair or homologous recombination following transfection into susceptible eukaryotic cells. Furthermore, F-plasmid cloning technology has gained widespread acceptance for the construction of mammalian genomic libraries due to their stable maintenance of large foreign DNA inserts in E. coli. Therefore, F-plasmid-based clones of herpesvirus genomes have three important benefits: (i) they are stable in E. coli, (ii) they are amendable to E. coli genetic methods, and(iii) they result in productive viral infection without the need for repair or homologous recombination following transfection of eukaryotic cells.Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. Cre and loxP both come from P1 bacteriophage. No other proteins or auxiliary factors are needed when recombination, and only the amount of nmol can combine with the loxP site, then the DNA recombination can be finished in—integrate outside gene to a fixed site in the chromosome or delete the target DNA. Cre/loxP site-specific system has been effectively used in these aspects such as gene target operation, identification of gene function, and integration of outside gene and so on. Also, it has been proved as a useful tool in DNA recombination of transgenic yeasts, plants, insects and mammals. In this study, we constructed a bacterial artificial chromosome contain full length genomic DNA of PRV by using these newly developed technologies.A GFP expression cassette flanked by two loxP sites with the same orientation was amplified by PCR and inserted into the EcoRI site of pSKLR. Based on homologous recombination, the GFP expression recombinant virus S03109 was constructed using PRV RongA as a parental strain. Fluorescence microscope, western blot analysis and PCR were employed to detect the expression of GFP. The results showed that S03109 infected cells could express GFP. Multi step growth curves test indicated that S03109 has a slightly reduced tite in cell culture when compared with PRV RongA strain. The LD₅₀ of S03109 for BALB/c mice was exceeded 3×10⁵ PFU. Using S03109 immunized BALB/c mice; the mean survival rate was 67.5% after wild virus challenge.After serial passage of S03109 on HEK-293T cells which were transfected with Cre encoding plasmid pOG231, the recombinant virus S0419 was obtained after plaque purification. Fluorescence microscope, western blot analysis and PCR were employed to detect the expression of GFP. The results showed that S03109 infected cells could express GFP but S0419 could not. PCR amplification and sequencing of S0419 TK gene indicated that the GFP expression cassette between two LoxP sites were deleted and only one LoxP site was left in TK locus. The sequencing result was submitted to GenBank (AY822465). The LD₅₀ of S03109 and S0419 for BALB/c mice both exceeded 3×10⁵ PFU. Using S03109 and S0419 immunized BALB/c mice; the mean survival rate was 67.5% and 70% respectively after wild virus challenge. As a conclusion, the report gene GFP expression cassette was removed successfully from PRV genome by using cre/LoxP recombinant system.A pair of primer was designed according to Cre reeombinase gene sequence. Cre coding sequence were obtained by PCR and then cloned into bicistronic vector pIREShyg. The resulting recombinant vector pIREShyg-Cre was transfected into BHK-21 cells using Lipofectamine 2000. After selection with 400ug/mL hygromicin B and PCR identification of 50 stably transformed cell clones, one positive clone were denominated as BHK-Cre. Using the same methods, we have obtained another cell line named 293A-Cre which derived from HEK-293A cells. BHK-Cre and 293A-Cre were infected by PRV S03109 strain which containing a green fluorescent protein (GFP) expression cassette flanked by two LoxP sites with the same orientation in TK locus. Fluorescence microscope, PCR and western blot analysis were employed to detect the expression of GFP. The results showed that after three generation serial passage of PRV S03109 strain on BHK-Cre or 293A-Cre cells no GFP expression were detected. PCR amplification and sequence of the third generation progeny virus TK gene indicated that GFP expression cassette between two LoxP sites were deleted and only one LoxP site were leaved in TK locus. In conclusion, the report gene GFP expression cassette was removed successfully and easily from PRV genome by using the Cre expressing cell lines BHK-Cre or 293A-Cre.Using PRV S0419 to infect 293A-Cre cells which transfected with pGS275 previously, a LacZ expression recombinant virus clone S06293 was obtained after plaque purification on RK13 cells. PCR and X-Gal in suit stain assay indicated that S06293 genomic DNA contain LacZ gene and can expressionβ-galactosidase when infect mammalian cells. Circular genomic DNA of S06293 was extracted and electroporated into DH10B. The obtained PRV-BAC clone DNA was extracted and transfected into BHK-21 cells. All resulted virus plaques express LacZ. This result suggested that the PRV-BAC was constructed successfully.US7 and US9-US2 regions of PRV RongA .genomic DNA were amplified by PCR respectively and Sub-cloned into pGS284 to from a transfer vector pGS284-gE which US8(gE) was deleted during the concatenation of US7 and US9-US2 fragments. Cc118 which contain pGS284-gE and GS500 which contain PRV-BAC were cross-streaked on a LB plate. After pick up bacterial clones from intersection, the allelic exchanged BAC clones were isolated with antibiotics selection and one clone was named PRV-BAC-gE. After transfection PRV-BAC-gE DNA into HEK-293 cells, the LacZ expression recombinant virus S0645 was obtained. Western blot assay suggested that S0645 is gE minus.As a conclusion, we constructed PRV-BAC successfully.