| Pseudorabies virus (PrV), or suidherpesvirus I, is a member of Alphaherpesvirinae, herpesviridae, which can cause Aujeszky's disease in swine, bovine, sheep and wild animals.Pseudorabies is an acute infectious disease and characterized by high fever, extreme itching(not in swine) and encephalomyelitis. PrV infection could 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 except Classic Swine Fever and Food and Mouth Disease. Immuned by PrV's inactivated and attenuated vaccines could in some extent prevent pig from the infection of Aujeszky's disease, but is not so effective as to both completely inhibit PrV's replication at the primary replication site and abortion-inducing spread of the virus via the blood. With the development of molecular biological techniques, several gene deleted PrV vaccines were produced and played an important role in prevention and eradication of this disease. In some developed countries, such as European Union and America, the rigid regulation were created, which said: only the gE gene deleted PrV vaccines and the relevant immune and molecular diagnosis methods can be used in prevention of pseudorabies disease.The local PrV Shanghai isolate (PRV-SH), isolated from the pigs with the clinical symptom in a swine farm at Shanghai suburbs, can form tiny and irregular plaques in PK15 cells.TCIDso is 10~66s/0.1mL.It could induce the typical syndromes of pseudorabies in rabbit and weaning piglets after inoculation, just as the PrV virulent strain Ea . Those results show that PRV-SH is a virulent PrV strain. The analytical comparison of gE and gl genes 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 nucleotide and deduced amino acid sequences. So we chose PRV-SH to construct the relevant gene deleted vaccine, which may has great application prospective and economic value in China.gE gene is an important virulent PRV gene , plays a key role in PrV's nerve penetration. Researches have proved that the PrV with the valine (125) and cysteine (126) codons deleted gE gene has reduced virulence and intact immunogenicity. gl is also a PrV virulent gene. Its product can bind to that of gE gene in forming an gE/gl protein complex to function. Swine immunized by attenuated strain with completely deleted gE and gl genescould not resist the attack from the Prv virulent strains, gl gene is necessary for inducing complete immune protection for pig. The relevant protective antibodies can't be induced in body if the 33th-100th amino acids of N terminal of gE glycoprotein had been deleted, which includes its main antigenic epitopes. This fact could serve to distinguish virus infection from vaccine injection.The 238-amino acids green fluorescent protein (GFP) from jellyfish Aequoreu Victoria does not require any cofactors or additional gene products to work. In a reporter gene system, its gene mutation derivative EGFP, which has been used as a versatile biological marker in various organisms and cell lines, has the fluorescence intensity of the 45 times sensitivities as much as itself (wide-type GFP). The merits of EGFP are convenient to detected, with stable fluorescence, no toxicant for living cells.In our research here, we amplified the gE and partial gl genes from PRV-SH isolate with PCR method. Then the PRV-SH gene recombinant transfering vector pgEI-GFPZ was constructed through inserting the GFP gene expressing cassette and LacZ reporting gene into the locus of the virulence-related gE's 5' terminal fragment, which had been digested with endonuclease from the amplified gene sequence.The repeated screening and the purification for gene deleted recombinant virus were done through blue plaques selection as well as green fluorenscenc in RK cell line, which had been transfected with pgE-GFPZ using DOTAP transfecting kit. The LD50 virulence test for mice and the safety assay for rabbits showe...
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