The Construction Of Gene-deleted Pseudorabies Virus Which Express Green Fluorescence Protein

The Pseudorabies(PR) is by the a-herpes virus branch Pseudorabies Virus(PrV) causes one kind of acute infectious disease which also contracts includingthe many kinds of domestic animals and the wild animal. Swine was the natural host and reservoir of PRV ,this sickness may cause the serious reproduction barrier, has created the huge economic loss to the pig industry. The prevention, the control and finally eradicate this sickness is arduous errand which our country current faces. Uses the safe and effective vaccine to carry on the immunity prevention,is main measure to control this disease. Traditional diedvaccine and attenuated live vaccine count for much function to Pseudorabies control, but it has also brought the enormous barrier during application which eradicates to the disease, it causes in the immune hoggery the PRV immune body complication, the conventional serology method very difficult to differentiate the vaccine immunity swine and the wild poisonous infection swine. The PRV genetic engineering gene-deleted vaccine is uses the genetic engineering technology to insert the person or delete section of sequences in the PRV gene group causes PRV certain genes can't be expressed, thus attenuated PRV, simultaneously maintains its stronger immunogenicity, mainly its toxicity gene thymidine kinase (TK), protein kinase (PK), ribotide reductase (RR) and deoxyuridine triphosphate kinase(dUTPase) as well as some immunogenicity glycoprotein gG, gE,gC, gD and so on. Europe and America and so on the developed country adotp Pseudorabies vaccine gene-deleted PrV vaccine currently, therefore, our country researches and applications the Pseudorabies gene-deleted vaccine.The Pseudorabies virus gene group is a double strand, filamentary DNA,has many the non-prerequisite gene in the long approximately 150kb genegroup which has nothing to do with the viral duplication, but delete these genes for the exogenous gene insertion, gG located the gene group US area, the length are 1.494bp, codes 498 aminophenol. The gG gene belongs to the later period gene, its promotor is stronger,is the (?)level promotor. The gG gene is the PRV one kind of non- prerequisite gene, not in the viral granule, but is by the infection cell secretion, by molecular weight 99kD form polymerization to ininfection cell culture medium. gG regarding virus's adsorption, penetration as well as from cell to cell proliferation non- prerequisite. gG is a Kind of glycoprotein which at present be discovered all wildpoisonous expressed, and has very strong antigenicity, after the gG flaw is not expressed, like this can distinguish the wildpoisonous infection and vaccine immunity.According to Genebank the announcement Plasmid pGFPuv sequence, the plasmid pUSK sequence and PRV the gene gG promotor, designed a pair of primers which contain restriction enzyme PstI and the NotI sites, Containing green fluorescent protein gene pGFPuv plasmid as a template, amplified a complete GFPuv gene, Amplified fragment length assay, Then PstI and NotI-digested and the amplified fragment by PstI and Notl-digested the cloned pUSK vector and transformed into E. coli DH5a, constructing a recombinant plasmid pUSK-pGFPuv transfer. Was identified by PCR and the recombinant plasmid correct size, and insert a green fluorescent protein, methods and proved to insert the right direction. Further sequencing, sequencing results showed the footage inserted in line with expectations, there was no mutation, the correct time frame.The use of lipofectamine method, The plasmid pUSK-pGFPuv and PRV genome were transfected VERO homologous recombination and observed that the cells fluorescence, for further screening and identification of recombinant virus laid the foundations Construction for pseudorabies vaccine gG gene deletion lay the groundwork.