Application Of Cre/loxP System In Pseudorabies Recombinant Virus

Pseudorabies virus (PRV), or suid herpes virus I, is a member of Alphaherpesvirinae, herpesviridae, which can cause Aujeszky's disease in swine, bovine, sheep and wild animals. PRV infection can cause its natural and main storing host-swine (also its main infectious origination) a huge loses, the most huge economical lose in all swine infectious diseases besides Classic Swine Fever and Food and Mouth Disease. It has been classified in list B by OIE in infectious diseases. The local PRV Shanghai strain (PRV SH) is isolated from the pigs with the clinical symptom on a swine farm in Shanghai suburbs. The analytical comparison of TK gene of PRV SH with that of the virulent Ea and Fa strains, both isolated from China, shows that there are great homologue and similarity between their nucleotides and deduced amino acid sequences. So the PRV SH strain we chose may has great applicational prospectivity and economic value in China.Cre/loxP system has the character of site specific, time specific and tissue specific in recombination with high efficiency, which makes this system universal in vivo and in vitro recombinations of bacteria, fungus, plants, insects and mammals. Cre and loxP both come from P1 bacteriophage. No other proteins or auxiliary factors are needed when recombinating, and only the amount of nmol enzyme can combine with the loxP site, then the DNA recombination can be finished -integrating outside gene to a fixed site in the chromosome or deleting the target DNA. Cre/loxP site-specific system has been effectively applied in these aspects such as gene target operation, identification of gene function, integration of outside genes and so forth. Also, it has been proved as a useful tool in DNA recombination of transgenic yeasts, plants, insects and mammals.A pair of primers were synthesized according to the pEGFP - Cl sequence published on GeneBank, and were used to amplify the EGFP gene expression cassette with one loxP site flanking each side. This target gene was cloned into pSKLR to abtain the transfer vector pSKLR-GFP-loxP. And then pSKLR-GFP-loxP were contransfected into 293 T cell with PRV SH strain virus. The recombinant virus rPRV₁ was selected and purified in TK143 cell by fluorescent plaques under the environment of BrdU. Then contransfect rPRV, and pPOG231 which was expected to express Cre enzyme. Through the function of Cre/loxP system, a second recombinant...