Preliminary Study On F2 Protein Subunit Vaccine Of Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is one of the major pathogens of lower respiratory tract infection in infants worldwide. Almost 95% children are infected before age of 2, cause bronchiolitis, pneumonia, bronchitis. Since the virus was found, some RSV vaccine has been developed, However, a completely safe and effective vaccine is not available yet.In this study, we tried to establish the prokaryotic expression system for F2 peptide of RSV envelope protein, and explore the immunogenicity of expressed F2.To clone and express RSV F2 protein, the viruses were grown in Hela cells. When more than 80% cells showed cytopathic effect, the cells were collected for total RNA extraction. F2 cDNA was obtained by reverse transcriptional-PCR, and then was inserted into pGEX-6p-1 vector. F2/pGEX-6p-1 expressed in E.coli Rosetta, the correct size of GST-F2 fusion protein was detected, and the amount of GST-F2 was less than 10% of total proteins. Further study showed that the GST-F2 proteins formed inclusion bodies, in which 50% were GST-F2 proteins. Inclusion bodies were extracted, washed, denaturated, renaturated and purified by GST affinity chromatography, after that the purity of fusion protein reached 85%.To test the immunogenicity of the prokaryotic expressed F2 proteins, mice experiment has been performed. Thirty six mice were divided into 6 groups, each group were immunized at week 0,2,4 using the following combinations:A, PBS group; B, LT (heat-labile enterotoxin, LT)group; C, GST group; D, GST+LT group; E, GST-F2 group; F, GST-F2+LT group. Sera were collected from the tail vein of all groups of mice at week 1,3,5, respectively. Washings of respiratory tract were harvested at the final immunization. IgG, IgG1, IgG2a, IgA and mucosal secretory IgA (sIgA) of respiratory tract were detected by ELISA. The results showed that, the concentration of IgQ IgG1, IgG2a, IgA and sIgA of F and E groups were significantly higher than the other control groups. The fusion protein could augment the levels of antibodies from the serum and the mucosa of the respiratory tract. IgG2a levels of the E group were higher than F group, therefore, the immune response inducted by F2 protein was mainly the type of Th2. The levels of sIgA of the F and E groups were slightly higher than the other control groups, there were no significantly difference between E and F groups. GST-F2 could augment the levels of antibodies from the mucosa of the respiratory tract,however, the effect of LT as a mucosal adjuvant was limited.In summary, GST-F2 protein was expressed and purified, the mice was coimmuned with GST-F2 protein and LT. Levels of detected antibodies demonstrated that this protein exhibited high immunogenicity, whereas the effect of LT was not significant. This will lay the groundwork for further study on pathology, vaccine development and immune adjuvant of RSV.