Molecular Aetiolog Investigation Of Porcine Pseudorabias Virus In Guangxi And Construction Of Bifunctional Molecule For Discriminating Wild Infected Pigs

Pseudorabies disease which is caused by Pseudorabies Virus as one of contagious diseases has made great losses .At present,though there are PCR and ELISA which discriminate vaccine immuned pigs and wild infected pigs,they need any special instruments and empirical personnels who can manipulate them,what's more,they are not used widespread for basement layers,so we must build a new diagnostic method which is more convient,volant and differential diagnosis.Based on the use of PRV gE gene deletion vaccine ,we utilized the erythrocyte agglutination test and spliced monoclonal antibody gene(2E8)against human erythrocyte with PRV gE gene by SOE PCR for building a bifunctional molecule to discriminate PRV wild infected pigs and vaccine injected pigs. If the diagnosis method had been built successfully,it would play an important part in diagnosing rapidly PR.In order to obtain the PRV gE antigen peptide of bifunctional molecule,and master epidemiology and inheritance mutation ,we detected 163 samples from 122 different pig farms in 13 regions of Guangxi by PCR,at last there were 6 samples confirmed as positive ones,which positive rate was 3.7%; We detected 61 samples serum from 8 pig farms by gE-ELISA in Nanning ,Yulin,Guilin,Guigang and so on,and there were 7 positive samples which positive rate was 11.5%.The investigation showed that the incidences of PseudorabiesVirus had been decreasing since two years. The gE genes of 6 strains PRV were cloned,sequenced and compared with MinA strain,GXB strain,GXW strain,Ea strain,SH strain,LA strain in exterior and interior.The results showed that the nucleotide homology were 97.8%～99.0%,amino acids homology were 96.5%～99.0%.And the nucleotide and amino acids homology was lower,nucleotide 96.4～97.0%,amino acids 93.6～95.2%.Study indicated that the genetic relationship between Guangxi PRV strains and domestic strains was intimate,and the mutation was not large.The incidences of PR detection were low,and the investigation also indicated that we had made great performances on preventing PR,but at the same time we found that subclinical infection remain be present in some pig fields ,which told us that the work of PR prevention must not be slacked.Six positive samples were used for the work of virus isolation and Identification.At present,Only one of them was isolated successfully .The virus caused infectedrabbits typical pseudorabies clinical symptom,induced typical cytopatho- genic effect (CPE) after inoculated in PK-15cell,and virus TCID₅₀ was10^7.22 /0.1ml. A specific bind was obtained by PCR amplification, cloned, sequencd and comp- ared with five PR strains and MinA strain,Ea strain,SH strain,LA strain,GXB strain,GXW strain,which the nucleotide homology was 98.4%～99.8%,the amino acids was between 97.8% and 99.4%.Compared with Rice strain ,the nucleotide and the amino acids homology were lower,only 97.0% and 95.2% respectively.At last,the virus was confirmed as Pseudorabies virus,which was named GXBB-1 strain. The gE gene of the virus was used as one arm of bifunctional molecule for our research.We designed a pair of primers to constructe 2E8-gE fusion gene through oligo software, spliced anti-hum un RBC H antigenic monoclone gene 2E8 with GXBB PRV gE gene by SOE PCR method,reclaimed the PCR pruduct ,ligated into PMD18-T simple vector and sequenced.The result showed that the two parts were spliced successfully,and the reading frame was correct.The recombinant plasmid PMD-2E8-gE was digested by BamHI and EcoRI,and the fragment of 2E8-gE digested was reclaimed,and ligated into the plasmid of PET-His which had been digested by BamHI and EcoRI.At last,the recombinant plasmid of PET-His-2E8-gE which was identified by PCR and enzyme restriction analysis was built successfully,which provided elementary for the expression of protein to build the kit of rapid diagnostic reagent.