Stable Expression Of P12A Gene Of Foot-and-Mouth Disease Virus In BHK-21 Cell And Studied On The Immunogenicity Of Products

In this study,subunit vaccines of FMD were developed by employing molec-ular biological tools,and its immunogenicity had been primarily evaluated. The main research process as following:1.The expression vector pBABE-P12AEGFP was constructed In this study, according to the ORF sequence of cloned P12X of serotype A foot-and-mouth disease virus (FMDV) and positive clone plasmid of P-EGFPN1,the primers were de-signed with different restriction sites and P12A and EGFP were get by PCR m-ethod following multiple cloning sites(MCS) of Expression Vector. The two am-plified DNA fragments were subsequently ligated into the pBABEpuro vector,and then the recombinant vector plasmid named pBABEpuro-P12AEGFP was ide-ntified by digested with the appropriate restriction enzymes , PCR and sequence analysis. It was named pBABEpuro-P12AEGFP. The results showed that the retroviral vector with P12A and EGFP gene can be constructed successfully.2.Recombinant plasmid pBABEpuro-P12AEGFP was constructed , and then pseudovirus was obtained by cotransfection of recombinant retroviral vector pBABE-puro-P12A-EGFP and pVSV-G envelope vector into the GP2-293 package cells. BHK-21 cells lines were infected by pseudovirus mediated by po-lybrene,and thepositive cell clones were selected by puromycine.Detection of Antibody sandwi-ched ELISA,indirect immunofluorescence,EGFP f-luorescence, SDS-PAGE, Western blot showed that P1 structural protein was expressed in BHK-21 cells.The results of indirect immunofluorescence and Western blot showed that P1structuralprotein could be recognizedby positive serum of type A FMDV,which indicated that positive cel-ls could stably carry target genes in subsequent passaging and P1 expressed protein had good immunoreactivity.3.Plenty of harvested cell culture were emulsificated by Freund`s adjuvant to immunize Balb/C mice,and efficiency of P1 antigen were evaluated by blocking ELISA. The results indicated that mouse antibodies were detected after 15 days of the first immunization. In addition,there were significant differences between experimental group and control group. Antibody titres of experimental mouse reached 1:8,even up to 1:64 after the second immunization and there was an increase of antibody level with the antigen dose increased.Antibody titres of control group were below 1:4 all the time. The studies indicated that p1 expressed protein had good immunogenicity.In conclusion, the above results showed that transferred cell which could sta-bly carry target genes in subsequent passaging had been successfully constructed.It provided a new experimental method for further FMD subunitvaccine research.