Studies On Recombinant Subunit Vaccine Of Respiratory Syncytial Virus (RSV)

Respiratory syncytial virus (RSV) is a kind of non-segmental RNA virus of negative strand, and it is classified as a member of the genus Pneumonia virus in the family of Paramyxoviridae. RSV genome has 15,222 nucleotides mainly coding 10 specific proteins, the fusion protein F on membrane surface and adhesion protein G, as the important aspect of research and development in vaccination, are the mainly surface antigens of RSV to induce the production of protective antibodies. RSV is one of the most common pathogen which cause the lower respiratory tract infection called asthmatic suffocating pneumonia(ASP) of newborns and infant widespread over the world.As the major respiratory pathogen in China, it makes ASP outbreak in every years.So far, RSV infection is still the primary cause of hospitalization of the infants so that the production of RSV Vaccines had been included in the global plans by WHO as one of the priority products. There is no licensed RSV vaccine after 40 years development. In this study, RSV fusion protein F and adhesion protein G have been successfully produced as RSV subunit vaccine candidate with baculovirus-Insect expression system. The immunogenicity and dffectivity of the RSV subunit vaccine candidate have been evaluated after inoculation in Balb/c mice. Amino acids of 205-223 and 255-278 from F ptotein, and 142-204 from G protein were selected to be epitopes analyzed by bioinformatic software. Epitope fusion protein of F and G have been expressed and purified successfully. It is helpful for further devolepment of RSV subunit vaccine in China.Recombinant plasmid construction, expression and purification of RSV complete F protein and G protein objective expression and purification of F and G gene of RSV (A type, Long strain). Methods F and G full gene of RSV (A type, Long strain) were amplified by RT-PCR, and the plasmid of F-pFastHT A and G-pFastHT A were constructed.The positive purified recombinant plasimid was transformed into DH10Bac E. coli for transposition into the bacmid to get F-Bacmid and G-Bacmid, respectively. The positive purified recombinant bacmids have been transfected into insect cell for successful virus packing. Product of the transfected cells were purified by Ni2+ Ion affinity chromatography and the expression of protein were detected by SDS-PAGE assay, Indirect immunofluorescence and and western-blot assay. Results The interest protein ( F and G) were expressed of specificity and absorbed efficiently by the Ni2+ affinity chromatograph column.Conclusion F gene and G gene were cloned successfully and proteins were expressed in insect cells. The purity was up to 85% by Ni2+ Ion affinity chromatography purification that it was fit for immunization to Balb/c mice.Evaluation of immune effect of recombinant protein of complete F protein and G protein and RSV subunit vaccine candidate Objective to exam the immunogenicity and safety of the vaccine candidates of F and G protein of RSV. Methods F protein and G protein were immuned intranasally to Balb/c mice with Mf59 respectively as vaccine candidates and intramuscularly with Freund's adjuvant respectively as control. The specific IgG and neutralizing antibody titers of immune mouse serum were determined Separately by indirect ELISA and micro neutralization test; the titers of specific IgG and IgA of lung lavage fluid and nasal lavage fluid were detected by indirect ELISA in immune mouse; multiplication of splenic lymphocyte was detected by MTT assay; secretion ofγ-IFN,IL-2 and IL-4 were detected by ELISPOT assay. Results Serum IgG, neutralizing antibodies and lung local antibodies of high titers were produced in Balb/c mice by induction F protein and G protein expressed in insect baculovirus expression system. Balanced IgG1/IgG2a was induced by F protein also. Conclusion F protein could induce the humoral immunity and cell-mediated immunity, G protein only induce humoral immunity and the advantage response of Th2 cells.Recombinant plasmid construction, expression and purification of RSV F-G epitope fusion protein Objective Choosing the protein epitope that could induce humoral immunity and cell-mediated immunity simultaneously and induce the balanced Th1/Th2 response also.Methods The spatial structure of F and G protein were analyzed and forecasted and epitopes of F and G protein was found by using software of bioinformatics.205aa-223aa and 255aa-278aa of F protein and 142aa-204aa of G protein were selected and their coding gene of F and G epitope were synthesized and linked with G2SG2 by overlapping PCR. Then F-G fusion epitope protein was expressed in insect cells and purified by Ni2+ affinity chromatograph. Results Specific recombinant fusion epitope protein F-G was expressed in insect cells, and absorbed efficiently by the Ni2+ affinity chromatograph column for purification. Conclusion RSV fusion epitope gene was cloned successfully and the fusion epitope protein F-G was expressed in Sf9 insect cells. The putity of the inteset fusion protein was up to 85% by Ni2+ Ion affinity chromatography.