Construction Of Pseudorabies Virus Ea GG Gene Deletion Mutant Containing GFP Reporter Gene

Pseudorabies Virus (Pserdorbies Virus,PRV) is a herpesviridae-herpes virus subfamily of pig herpes virus type I,it can lead to a variety of domestic and wild animals of acute infectious diseases.Pig is the storage of the virus and the source of infection, Pigs infected with PRV frequently manifests itself after the dysfunction of the nervous system and cause serious respiratory diseases and the breeding barriers.The disease has been to China and the world hog industry has broμght hμge economic losses to prevent, control andμLtimately eradicate the disease in China is currently facing a difficμLt task. A safe and effective vaccine to be immunized to prevent, control of the disease is the main measure.Construction of the test was aimed at containing (GFP) gene gG the pseudorabies virus gene deletion expression vector.Throμgh digestion, fill-linking method PUSK vector to transform to eliminate the vector of the two HindIII digestion and BamHI sites.And the transformation of PUSK named PUSBH. According pAcGFP1-C1 gene sequencing primers were designed, in the primer, downstream 5'end introduced NotI restriction sites, will be amplified by PCR gene AcGFP (including GFP, CMV, MCS, SV40 Poly(A) ) cloned into pGEM-T-Easy vector, the recombinant plasmid was constructed. After digestion and PCR fragment was inserted identification proved AcGFP gene,and recombinant plasmid named pT-GFP. NotI digested with the recombinant plasmid vector pT-GFP and PUSBH and recovery, will be to phosphorlation of vector fragment after AcGFP link, amplified by PCR, restriction enzyme digestion, sequencing proved that the success of the transfer vector. And named it PUSG. The transfer vector containing four single restriction site for the insertion of foreign genes, so as to establish a pseudo-rabies virus vector for the price of two or more genetically engineered vaccine prices have laid a foundation.Use of lipid-mediated method, and transfer vector plasmid PUSG PRV genomic DNA transfection of Vero cells. Fluorescence microscopy in 72 hours will be observed green fluorescent, straw will be used fluorescence aspiration of the lesion cells, after three consecutive passages have been purified virus. And have been identified by PCR and restriction enzyme digestion successfμLly constructed recombinant viruses. The recombinant virus was developed for the next two or trivalent vaccine price, providing a convenient selection.