Detection Of Rabies Virus With Electron Microscopy And Cloning, Sequential Analysis Of Glycoprotein Gene On The Virus

Rabies virus (RV), is a member of the genus Lyssavirus within the familyRhabdoviridae. It appears a clear bullet-like features which are easily identifiableunder electron microscope. Therefore, the electron microscope examination hasprovided rapid visual diagnosis technology for the rabies virus's morphologicalidentification. Rabies virus glycoprotein is the important element of virus particles,has hemagglutinin activeness, not only glycoprotein virus particle and host cellbinding to the receptor sites for bridge, but the glyeoprotein is only can produce theneutral immune body viral ingredient. It can affect the rabies vaccine immunogenicity.The experiment of the rabies virus electron microscopy analyze its characteristics,provides the basis for the diagnosis of rabies virus, so as to investigate the applicationof electron microscope in virology. Rabies virus glycoprotein cloning and sequenceanalysis has provided the research foundation for Genetical Engineered vaccinedevelopment and the gene immunity.We adopted the negative stain electron microscopy techniques, ultra-thinsectioning and high-speed centrifugation of sample preparation methods in theexperiment. It is observated that rabies virus configuration, particle size,proliferation, tissue distribution with microscopy techniques. According to GenBankof the rabies virus glycoprotein gene sequence, using Oliga software design two pairsof specific primers. Amplified the full-length cDNA fragment of GP gene of strainFlury by reverse transcription-polymerase chain reaction(RT-PCR) and cloned intopMD-18T vector for sequencingResults show:①Typical virus particles were observed using transmissionelectron microscope;②It occurred that agglutination reaction with rabies virus andgeese erythrocyte, the scanning electron microscope observation of the rabies virusattachment and geese erythrocyte surface;③Sequence and homology analysis showedthat the cDNA of RGP consisted of 1574 nucleic acids encoding 524 amino acids.The homologies of nucleic acid and amino acid sequences to those reported abroadwere 82.3%～96.3%and 88.4%～94.3%.In this experiment, it is feasible and effective for identifing the rabies virus withelectron microscopy. Without positive serum or antibody, combined with the clinical symptoms, an epidemiological study and electron microscopy, we cancharacterization of the virus. The GP gene of Flury-lep have successful cloned andsequenced. The study provided a theoretical and scientific basis for molecularepidemiology investigate and selected a candidate vaccine strain. Meanwhile, laid afoundation of molecular biology.