Research Of Split And Purified Technics For Influenza Inactivated Vaccine

Influenza viruses have a highly infectivity and can cause actue respiratory disease. Its epidemic feature is outbreaking in short term and quickly spreading. Influenza causes various degree epidemics including pandemic, seasonal epidemic and difusing, yet can cause higher morbility and mortality than other common diseases. Now there is not specific medicine to cure anyone from getting the flu, but vaccination is regarded as the best method of influenza prevention. Fertilized hens' eggs is the common choice in manufacturing influenza vaccine.At first we inoculate different three types of influenza virus (H1N1, H3N2 and B) separately into the embryonated egg of SPF level, after cultivation, harvest their viral original fluids, then carries on ultrafiltration, concentration, purification, split, inactivation, twice purification,etc. Finally we get three types of monovalent influenza virus bulk and detect some of their main parameters. In this process, we first make sure the best dilution multiple and harvest time, then find the appropriate OD value of ultraviolet Spectrometry using the sucrose density gradient centrifugation when purification samples should harvest. Simultaneously select splitter and inactivator for the researching, we finally determine that the craft techniques of the production.The purified fluid were lysed with Triton X100 and Triton N101 at different concentrations, and the lysates were observed under electron microscope to determine the best disrupt time, examine the HA content by single radial immunodiffusion test(SRID) is the evaluating indicator. The result indicated: Triton X100's splitting is superior, the concentration of 0.5%～1%, effect 120min can completely decompose the influenza virus. We introduce BEI to inactive the fluid in our study, compared with formaldehyde. The best deactivation time of BEI is between 24-36h while formaldehyde is 3h. The antigenicity examination and statistics analysis proved that BEI is surpasses formaldehyde obviously. Use RT-PCR method to detect the 8 of specific genes before or after the viral inactivate process with two inactivators. This indicates that BEI deactivate the virus and destroy the 8 gene orders completely, however the formaldehyde can't. This can prove that BEI inacticate influenza vaccine is more safe and realiable in Microbiology level. After the single radial immunodiffusion test(SRID) to the two inactivator's deactivation effects, discovered that although the deactivation time made by BEI is longer, but the HA content in its sample campares higher than in formaldehyde's sample. But the wether BEI can be used in human vaccine is still needs further experiments and clinical test to confirm.Our research is aim at exploring the crucial steps in produce influenza virus splitting vaccine which fertilized in embryonated hen's eggs. This can build the foundation of further improvement.