Research On Vero Cells And Influenza A Virus (H1N1)under Serum-low And Free Circumstances

Influenza is a kind of acute respiratory disease infected by influenza virus, which leads to serious damage to human life and health.At present, inoculating influenza vaccines is the effective way to prevent influenza. Chicken embryos are most commonly used as the culture matrix of Influenza virus and they are also often used to produce influenza vaccines and to separate the influenza virus.Influenza vaccine from chicken embryos has many disadvantages, which are as follows:The process is susceptible to microbial contamination; people with severe allergies to egg protein can not inoculate the vaccine; it has a low genetic stability; it is difficult to supply enough chicken embryos to fulfill the production demand while it’s out breaking. Owing to the disadvantages of chicken embryos, it is very urgent to develop influenza vaccine based on mammal cells.Vero cells are recommended to produce influenza vaccines by WHO and they can be cultivated in a large scale by fermentation with microcarrier.Therefore, it has been widely used for the production of human vaccines. The cultivation of Vero cells with serum-low and free medium on the one hand can avoid the neutralization of pancreatic enzymes,which is indispensable for effective infection of influenza virus, by the residual bovine serum in normal cell cultures of the influenza virus, and a high production strains of influenza virus adapted to Vero cells can be obtained; On the other hand, a biologically safer influenza vaccine is gained due to the reduction or even elimination of the unclear composition and unknown pathogens in the bovine serum.Partl The cultivation of Vero cells and H1N1influenza virus with serum-low medium.The newborn calf serum at various concentrations were added into different basic medium separately, in which Vero cells were cultured and observed for growth. According to the result, we decided to add2%the newborn calf serum to the medium to continue our experiments lately.We selected the DMEM/F12as the most suitable basic medium for the cultivation of Vero cells with MTT method.In addition,the optimal incubation conditions of the H1N1influenza virus were optimized.Part2The cultivation of Vero cells and H1N1influenza virus using serum-free medium.We preliminarily optimized a serum free medium for Vero cells by adding a certain percentage of proteins, growth factors, pro-adherent cell growth factors and other substances to basic medium. We optimized the cultivation conditions of H1N1influenza virus by adapting different cell age、pH、different contents of TPCK-Typsin and so on. In the end we made it sure that the best culture conditions were as follows:the cell age36hours, pH between7.2-7.4, TPCK-Typsin0.8μg/ml, and basic medium DMEM/F12: MEM (1:1) its proliferation time48hours.