Study On Oral Liposomes Freeze-dried Live Vaccines Of RSRV₉Attenuated Strain And Its Immune Potency Determination

There were some differences identified in the genome structure of Rabies virus between rSRV9attenuated strain and parent strain using RT-PCR method, cytoc genes (369bp) inserted in the genome of rSRV9attenuated strain were amplified. For30-generation parent SRV9virus through continuous culture and rescued rSRV9virus, viral titer and virulence determination results showed the differences of its average viral titer were0.65logFFD50/0.1mL (P<0.01), which indicated that rSRV9attenuated strain modified by reverse genetics technology had the similar cell proliferation with parent SRV9strain. Viral virulence tests suggested that rSRV9attenuated strain reduced by2.99logLD50/0.03mL (P<0.01) in compare with parent SRV9strain. Specific Negri bodies were detected in the neonatal brain cells of dead neonatal rats intracerebrally injected with parent SRV9strain and rSRV9attenuated strain by histopathological and immunohistochemical methods, it indicated that both of them had the pathogenicity for neonatal rats.In this study, cationic blank liposomes were made using hydrogenated soybean lecithin, cholesterol, organic amines and other "substances through the film dispersion method, and then mixed the same volume of cationic liposomes and rSRV9virus using freeze-drying technology, oral liposomes freeze-dried live vaccines of rSRV9attenuated strain were developed through adding Lyoprotectant freeze-dried again. The formed blank liposomes were large single room liposomes under electron microscopy, its average diameters were378nm measured by MASTER2000laser particle size analyzer, and the vaccine antigen encapsulation efficiency was determined as71.19%by the BCA protein quantitative method.According to different immunization dose, different immune times and different immune forms, oral liposomes freeze-dried live vaccines of rSRV9attenuated strain were vaccinated in average25g weight mice. The oral mucosa secreted SIgA, serum rabies-specific IgG and spleen lymphocyte transformation rate of mice vaccinated after1-7weeks were detected by double-antibody sandwich ELISA and MTT assay. After three-time oral immunization and35days later, and1.0mLper time, SIgA antibody production level of mice was38.63μg/mL and rabies-specific serum IgG concentration was18.70ng/mL; while spleen lymphocyte proliferation OD value was1.86, which showed this assay had good immune effects. To summary, we got a conclusion that the best immunization dosage of oral liposomes freeze-dried live vaccines of rSRV9attenuated strain was1.0mL (7.4logFFD50/0.1mL), and the best immune times were three time (the first immunization time was one day, and then the strengthened immunization time were on7th and14th day, especially).