Construction And Identification Of A Live Vaccine With GE&TK Genes Deletion Of Porcine Pseudorabies Virus Variant

Pseudorabies,also known as Aujesky's disease,is an acute infectious disease caused by Pseudorabies virus(PRV),which can cause pruritus?fervescence?and encephalomyelitis of livestock and other animals.The main clinical manifestations of infected pigs are fervescence,the newborn piglets will get nervous symptoms,the fattening pigs will get respiratory symptoms,sows often abortion,the infected adult pigs will get growth arrest,so PRV makes great harm to the swine industry.At present,there is no effective drug for the treatment of pseudorabies.Vaccination is the main measure to prevent and control the disease.In 2011,a lot of pigs farms immunized with Bartha-K61 vaccine appeared pseudorabies in the east of China,and the epidemic spread gradually to the pig farms all over the country.The PRV isolated from dead piglets of the pig farms immunized with Bartha-K61 vaccine is a variant strain of pseudorabies virus and it's pathogenicity has been enhanced.Indicating that the attenuated vaccine Bartha-K61 can't provide full protection to pigs in the face of Pseudorabies variant strain.Therefore develope a vaccine against the Pseudorabies variant strain is extremely urgent and necessary for the pig industry in china.This research is based on BACPRV-G which is constructed by National Research Center of Veterinary Biologicals engineering and technology,using En Passant method to constructe double gene deletion mutants PRV ATK&gE-AH02 by means of homologous recombination,and carried out a preliminary study of the growth characteristics in vitro and immune protection of this mutant.1 Construction of PRV TK&gE double gene deletion strainUsing the genomic DNA of PRV AH02LA strain as template to amplify the sequence of TK gene's both sides as recombinant arms.Then transferring the KAN resistance gene with both sides of the TK gene into the competent cells of BACPRV-G to replace the TK gene to construct BACPRV ?TK/gE/gI/KAN+.The results of PCR and RFLP showed that KAN gene successfully replaced the TK gene in BAC genome.Knocking down the KAN gene in the BAC genome after the induction of Arabia sugar to construct the BACPRV?TK/gE/gI,and PCR and RFLP identification results showed agreement with expectations.Using DNA of LA-AB strain as template to amplify the transfer vector by primers PRV BAC H1 short and PRV BAC H2 short R.Then the transfer vector and the genomic DNA of BACPRV ?TK/gE/gI was transfected together into CEF cells and no fluorescence spots were observed under fluorescence microscope,that means we successful rescue of the double gene deletion recombinant virus(named PRV ?TK&gE-AH02)without mini-F and regaining of g1 gene.The results of PCR identification showed that we successfully delete 347bp of TK gene and 1418bp of gE gene.2 The growth characteristics of PRV ?TK&gE AH02The study found that the highest content of PRV ?TK&gE-AH02 and virulent strain PRV AH02LA in ST cells were 10-6 29TCID50/0.1ml and 10-7.19 TCID50/0.1ml.Compared with the parent strain,although the deletion of both TK and gE gene makes a certain effect on the proliferation,the virus content of the double gene deletion strain could reach a high level in vitro.We also measured the growth curve of the proliferation of PRV ?TK&gE-AH02 and PRV AH02LA strain in ST cell,repeat the experiment three times,and take the average of the results to plot the growth curve.3 Study on the safety and immune efficacy of PRV ?TK&gE AH02In this study,we investigated the safety and immunogenicity of the double gene deleted virus(PRV ?TK&gE-AH02).Immunize the 28 day old healthy susceptible pigs with PRV?TK&gE-AH02 by intramuscular injection,the inoculation dose was 106TCID50.At the same time set pigs immunized with Bartha-K61 as control and inoculate the same dose.All immunized pigs showed no clinical signs of PR.7 days after immunization,for the experimental pigs immunized with PRV ?TK&gE-AH02 by intramuscular injection and immunized with Bartha-K61 and the blank control group,inoculate with the virulent strain PRV AH02LA.No clinical symptoms were observed on the pigs immunized PRV?TK&gE-AH02 and did not detect detoxification,all pigs get complete protection.But the body temperature of the pigs immunized Bartha-K61 increased and we detecte detoxification after poison attack.This indicates that PRV ?TK&gE-AH02 has good immunogenicity and is safe for pigs,also indicates that PRV ?TK&gE-AH02 can protect pigs against.PRV mutant strains.