A Study Of Avian Polyomavirus Late Gene Expression Regulation

Avian polyomavirus(APV), early say Budgerigar Fledgling diseases,(BFDV).Can cause a variety of parrot chicks died of acute viral infections. BFDV belonging topapovaviridae polyoma viruses. In order to study the APV-1polycistron late genemRNA translation of downstream VP1gene regulatory mechanism, including AUGstart coding of seven amino acids of the code will have the best translation start signalof nucleotides is designed to be inserted into the upstream of agno-1a mRNA andstart codon AUG artificial mutation, to observe its downstream VP1agno-1a mRNAtranslation effect.This experiment in view of eukaryotic gene polycistron mRNA expression of theregulatory mechanism of translation "Scan-Jumping and Internal Ribosome EntrySite (IRES)" model, using modern molecular biology methods in APV late gene indesign and construction of genome structure and wild type virus has the samemutation agno APV-1a region of the new type of recombinant virus. Such asfragment insert (multiple ATG area) alkaline lysis method to extract the APVpHL1003cDNA cloning of DNA length after the Acc I part of the enzyme recoverylinear purpose fragment, insert the synthesis of agno fragment phosphorylation andpurpose fragment after get recombinant plasmid with T4ligase connection, and thenconvert to the E.coli DH10b competent cells, screening positive clones, verified byPCR and PAGE detection and sequencing by APV-1mutation cDNA cloning; Pointmutations (ATG to ACG or CTG with initiative, a key protein binding site change,key double-stranded RNA secondary structure formation) pHL1003DNA by the Acc Ipart of the enzyme and Nco I after enzyme digestion by PCR technology will agno-1a regional gene initiation codon ATG mutation for ACG and CTG two cDNA cloningand verified by sequencing. Preparation of plasmid DNA, respectively, by the methodof calcium phosphate and multiple gene transfection of single gene mediated chickenembryo fibroblast (CEF), using Western blot detection of recombinant virus geneexpression and observe the purpose protein, as well as the similarities and differences of analysis and research polycistron mRNA expression of translation regulationprocess.This study aims to research Avian Polyoma Virus late molecular mechanisms,gene regulation polycistron translation more understanding eukaryotic genespolycistron mRNA expression of translation course and regulation mechanism of thecomplex and the establishment of the replication ability of infectious cDNArecombinant Virus and transfection and infection of chicken embryo fibroblast cellssystem. Due to avian polyoma virus can cause chicks acute fatal diseases and otherbirds of acute or chronic disease, to prevent the spread of avian polyoma virus diseaseof the large area, reduce the great economic losses, it is necessary in domestic poultrybirds of polyomavirus infection epidemiology investigation, according to the keysecurity and development of genetic engineering vaccine. The preparation of geneticengineering vaccine research can provide theoretical guidance and geneticengineering vaccine in the early development; Associated with poultry farms andveterinary station, monitoring method and system for standardization the outbreak ofthe disease.