Study On The Promotion Of The Proliferation Of Rabies Virus By L-arginine And L-leucine And On The Process Of Antigen Purification Of Rabies Virus

In order to enhance the inactivated vaccine immune effect and increase the titer of rabies virus in cell culture, we carried a research on the subjects of the promotion of the proliferation of rabies virus by L-arginine and L-leucine and of the process of antigen purification of rabies virus. We found that 1.2 mmol / L L-arginine, 0.8 mmol / L L-leucine significantly increased rabies virus titers by 0.68㏒ LD50/0.03ml (P <0.05), and 0.53㏒ LD50/0.03ml (P <0.05) than the control group, respectively. According to the detection of the concentration of glucose, ammonia and lactic acid, the accession of L-arginine and L-leucine had no effect on cell metabolism. Adding 1.2 mmol / L L-arginine in the test, it was observed that nitric oxide (NO) concentration was significantly increased by 3.09, 3.97, 10.8, 7.54μmol / L (P <0.05) than the control group after 24 h, 48 h, 72 h, 96 h, respectively. However, there is no difference by adding 0.8 mmol / L L-leucine at above time points. By the polyclonal fluorescent antibody staining, we found that, compared with the control group, in the group of L-arginine and L-leucine respectively, virus adsorption time shortened from 60min to 30 min, and the virus membrane wearing time shortened from 90 min to 60 min. By the nucleoprotein monoclonal antibody fluorescence staining, it was domonstrated that the time of synthesis of the virus nucleoprotein shortened from 21 h to 18 h. By using the rabies virus antigen test kit , we found that the time of the virus detected was ahead from 54 h to 42 h.Establishing the rabies virus fluorescence quantitative PCR detection method (RV FQ-PCR), it was observed that the glycoprotein gene expression levels of rabies virus in cell culture were 5.16,7.36,8.91,7.50 fold by adding L-arginine, and 2.87, 6.13, 6.9, 5.01 fold by adding L-leucine after 24 h, 48 h, 72 h, 96 h, respectively, when compared with the control group. By the detection of rabies virus in the mice and dogs brain tissue infected artificially, it was domonstrated that FQ-PCR is more sensitive than conventional PCR, fluorescent antibody test (FAT) and the mouse inoculation test (MIT) about 10, 100, 1000 fold, respectively.Through the screening of concentration methods and the comparison of the gel purification, it was showed that application of membrane reactor increased the rabies virus concentration by 50 to 60 fold, with the utilization of Sepharose 4 FastFlow gel used for purification, the achieved removal rate of impurity was more than 99%, and the antigen recovery rate was more than 77%, and the antigen activity was increased 25.59 fold. The process above can be amplified linearly, easy to operate, and suitable for industrial production.