Generation Of Recombinant Rabies Virus RERA-Luc Strain Expressing Luciferase And Its Biological Characteristics

In order to establish a rabies virus(RABV)to clearly the infection mechanism of RABV,we based on the recombinant RABV r ERA vector,using reverse genetic technology to construct a full-length r ERA genome cDNA,and then,appraisal the recombinant plasmid by Pme I to determine luciferase(Luc)gene is correctly identified.The plasmid and the helper plasmid are co-transfected into BSR cells,collected supernatant 4 days later,then blinded 3 generations in BSR cells,collect supernatant for identification.RT-PCR amplification results showed that the target band of Luc gene was obtained;IFA identification results showed that recombinant virus and the parent virus infected cells showed green fluorescence;the recombinant virus was purified and observed under electron microscope,the results showed that both the recombinant virus and the parent virus were bullet-shaped.Western blot results showed that the protein expression of the recombinant virus and the parent virus was consistent.The recombinant virus was named r ERA-Luc.The recombinant virus was passed continuously in vitro for 15 generations,and each generation was identified by RT-PCR and sequencing.The virus titer was determined by IFA to study its inheritance as qualitative.At the same time,the samples were processed by the Dual-Luciferase(?)Reporter 1000 Assay System and the samples were measured.The luciferase activity of the recombinant virus of the generations;the RT-PCR and sequencing results of each generation of recombinant virus showed no gene mutation;IFA results showed that after F5 generation,the virus titer of the recombinant virus and the parent virus in vitro was same as parent virus and could be stably passed down to the 15th generation;the recombinant virus fluorescein the enzyme activity gradually increased with time and from the 5th generation onwards,the recombinant virus of each generation can stably express luciferase,and the parent virus has always maintained a low luciferase activity.The recombinant virus and the parent virus were respectively infected with BSR cells and NA cells,determination the virus titer at different times by IFA,the growth curve was drawn and the luciferase activity was measured.The results showed that the growth characteristics of the recombinant virus is same as the parent virus,the luciferase activity of the recombinant virus gradually increased over time,and the parent strain maintained a low luciferase activity,indicating that the insertion of the Luc gene did not affect the growth characteristics of the recombinant virus.Recombinant virus and parental strain were injected intramuscularly(i.m.)and intracranially(i.c.)6-week-old BALB/c SPF mice at a dose of 10~4 ffu/mouse respectively,and PBS control groups inoculated by these two methods were set up at the same time,respectively record the weight of mice(i.m.group)and observe the clinical symptoms of mice(i.c.group).The results showed that the body weight changes of the recombinant virus i.m.group mice and the parent virus i.m.group mice were consistent,and the clinical symptoms and survival rates of the recombinant virus i.c.group mice is same as the parent virus i.c.group mice.However,the mice in the control group had no clinical symptoms,and the results showed that the insertion of the Luc gene did not affect the pathogenicity of the virus.The recombinant virus r ERA-Luc constructed in this study laid the foundation for the study of the pathogenic mechanism of RABV.