Construction And Identification Of Rebies Virus Glycoprotein Recombinant Lentiviral Vector And Immuinty Test

Rabies virus belongs to genus Lyssavirus within the family Rhabdoviridae. The virion, involving a bullet-shaped structure, is about 170 nm in length and 75nm in cross-section diameter. A typical virion is composed of a molecule of negative, single strand genomic RNA and five structural proteins respectively named as nucleoprotein, phosphoprotein, matrix protein, glycoprotein and large protein.Rabies which is nearly always fatal is a kind of neuroinvasive infectious disease induced by rabies virus. The rabies viruses mainly invade the host include warm-blooded animals and human by bruised skin or mucosa, and enter the central nervous system (CNS) via nerve terminal. The clinical symptoms of rabies are characterized by anxiety, agitation, paralysis, wild aggressiveness and final death.Rabies firstly known 3000 years ago occasionally breaks out in more than 80 countries even today especially in Asia and Africa. Rabies cases in human being occur focusing in developing countries, leading into 50000 to 55000 deaths annually (3000 in China), above 95% of which transmitted by canines.The best way of controlling rabies is to immunize canines with effective vaccines. However, although commercial vaccines with high safety and efficiency play a significant role in preventing and controlling rabies, yet in developing countries, they are not widely applied because of high expenses and repeat immunization to get effective protection. Besides, there are various problems with late-model vaccines that they can't be practically used till now. As a consequence, developing a kind of high-efficiency and low-cost rabies vaccine becomes urgent.A recombinant lentivirus system derived from HIV-1 was used as vaccine vector in this project because of large extent of exogenous gene, wide infection range, ability to integrate into target gene, deficiency of gene necessary for viral replication, high safety and low immunologic rejection.A recombinant lentivirus system derived from HIV-1 was used as vaccine vector in this project because of large extent of exogenous gene, wide infection range, ability to integrate into target gene, deficiency of gene necessary for viral replication, high safety and low immunologic rejection. The gene of interest was amplified from standard rabies virus 9 (SRV9) strain by RT-PCR. After a subsequent sequencing, the glycoprotein gene was cloned into lentivirus shuttle vector pLVX-IRES-ZsGree to form recombinant expression vector pLVX-IRES-ZsGreen-SRV9G which was then transfected into 293T cell line with envelop plasmid pMD2.G and package plasmid psPAX2 together. The detection of expression of reported gene ZsGreen by fluorescence microscope show that cells could be infected by recombinant viruses. The detection of mRNA of glycoprotein gene by RT-PCR proved the exogenous gene was transcripted. After immunization. Specific antibody against glycoprotein was detected using Western blot in mice previously immunized with recombinant viruses by intramuscular injection. Neutralization antibodies could be detected two weeks after immunization by fluorescent antibody virus neutralization test (FAVN) and reached the peak value 0.5IU/ml which was above the threshold value providing protection three weeks after immunization.In this project, the recombinant virus pLVX-IRES-ZsGreen-SRV9G expressing rabies glycoprotein that could induce the synthesis of neutralization antibody against rabies virus after intramuscular injection was harvest, which paved way for the development of recombinant virus vaccine of rabies virus in the near future.