Expression And Processing Of Foot-and-mouth Disease Virus Empty Capsid Proteins And The Assessment Of Its Immunogenicity

Foot-and-mouth disease(FMD) is a highly contagious viral disease of cloven-hoofed animals and severely threatens animal husbandry.FMD is endemic in large areas of Africa,Asia and South America and has shown an extraordinary ability to cross international boundaries and to cause epidemics in previously free areas. Vaccination is the major method to prevent FMD.However the conventional inactivated vaccines have many defects.Therefore,it is necessary to develop a safe and efficient FMD vaccine.These empty capsids are able to induce neutralizing antibodies of the same specificity as the whole virus and appear to be a good candidate for the development of a recombinant vaccine.In this study,A series of deletion mutation vectors of P1 coding sequence were constructed and were tested whether they would effect empty capsid assembling.A retroviral vector containing the P1-2A and 3C coding sequences was constructed and MDBK cells line stable expressing empty capsid proteins was established. Subsequently,the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus were used to immunize guinea pigs.All guinea pigs immunized with the lysate of MDBK cells infected by pBABE puro-P1-2A-3C virus developed specific anti-FMDV antibody and neutralizing antibody.These guinea pigs were completely protected from viral challenge.These results laid a foundation of development of FMD subunit vaccine.(1) A series of deletion mutations were introduced into P1 coding sequence. These deletion sequences included sequence which 11 amino acids at VP3/VP1 junction were deleted and sequences which 33,100,167 amino acids at C-terminal of VP1 were deleted,respectively.The FLAG tag was introduced at C-terminal of VP1 by PCR in order to accurately test target proteins.A series of deleted sequences were obtained by PCR and were subcloned into expression vectors.Processing of the P1-polyprotein was verified by in vitro transcription/translation using T7 RNA polymerase transient expression system.The plasmids containing deleted P1 sequences and the plasmids containing 3C sequence were transferred into BHK-21 cells with liposome-mediated method.The transferred cells were harvested and lysated after 24h transfection.The products were analysed using Western-blot and ELISA.The result demonstrated deletion mutation of P1 sequence impaired its ability to act as substrates for 3C proteinase activity.The assembling efficiency was high when the ratio of plasmids was 40:1.(2) P1-2A and 3C sequences were obtained by PCR.A recombinant retroviral vector pBABEpuro-P1-2A-3C was constructed by inserting P1-2A sequence and 3C sequence by turns.Both this vector and pVSV-G envelope vector were co-transfected into GP2-293 cells by liposome-mediated method.The recombinant pseudovirus were produced after 24h transfection.The pseudovirus infected the interesting MDBK cells. The infected MDBK cells were selected by puromycin(2.5μg/ml) for 10d and the monoclonal cells were selected using cloning rings.The expression of capsid proteins was detected by indirect immunofluorescence and sandwich-ELISA.The empty capsids of FMDV were observed under electron microscope.The results revealed the target gene sequences had been integrated into genome of MDBK cells and OD values of target proteins were lower than OD values of positive control.The expression levels in screened cells of various passages showed no significant difference.(3) Subsequently,the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus were used as antigen to immunize guinea pigs and evaluated for its ability to induce a humoral response of FMDV in guinea pigs.In addition,the ability to protect guinea pigs against homologous virus challenge was examined.Guinea pigs were given booster vaccination twice and guinea pigs were challenged 22 days after the third vaccination.Control groups included animals immunized with commercial vaccine,lysate of MDBK cells infected by pBABEpuro virus and lysate of uninfected MDBK cells.Anti-FMDV antibodys were elicited and reached a high level 14d after the second inoculation.Furthermore,guinea pigs developed a strong neutralizing antibody responses and antibodies increased further after the third inoculation.The neutralizing antibody titers of two guinea pigs immunized with the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus reached 1:64.These guinea pigs were completely protected from viral challenge.In conclusion,this study may provide the experimental bases of research for subunit vaccine.