Sequence Comparisons Of The VP1 Protein And Development Of Epitope-based Recombinant Vaccines For Foot-and-Mouth Disease Virus Type O

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, caused by foot-and-mouth disease virus (FMDV). Vaccination of the susceptible livestock with safe and effective vaccine is the primary requirement to control foot-and-mouth disease in an epidemic country. Nowadays, main prevention of the disease is vaccination with chemically inactivated virus vaccines. These conventional vaccines, although generally effective, do have some disadvantages such as the possibility of incomplete inactivation, escape of live viruses from vaccine workshops.Thus, the development of a safe alternative, epitope-based recombinant protein vaccines against bovine O FMDV, have been explored in our study.In order to sequence the VP1-coding region of an epidemic FMDV strain and compare the variations among FMD type O viruses which is the most prevalent of the seven serotypes.Total RNA of an suspectible bovine O stain of FMDV was extracted using Trizol reagent (Invitrogen corporation) according to the manufacturer's protocol. Reverse transcription-polymerase chain reaction (RT-PCR) was successfully conducted.The products of RT-PCR was cloned into pMD18-T vector and transformed to E.coli (JM109) and sequenced. The results indicate the epidemic strain have high similarity with a strain named O/HKN/3/75 (NCBI accession number: CAC20175) which is another type O foot-and-mouth disease virus in China.The most important outer-papsid polypeptide of FMDV is VP1. In our study, VP1 structure of the Tibet strain (Panasia) was predicted using the Swiss Model system on line. The crystal structure identified that G-H loop is on the surface of the FMDV particle. The conserved RGD motif, located within the G-H loop, is visable.In order to get the knowledge of the variation in the VP1 protein, the software BIOEDIT was used in the comparisons of the VP1 sequences in China. Accoding to the results and situations in China, two bovine O stains of FMDV were chosen as targets, by the name of XJ1 and Tibet.According to the B and T cell epitope, we have successfully developed two FMDV recombinant protein vaccines separately, including tandem repeats of different length of B cell and T cell epitopes within the VP1. They were expressed in Escherichia coli. The recombinant fusion proteins named (HA)3 and (BN)3 were characterized by SDS-PAGE and in Western Blot analysis. Purified proteins mixed with 206 adjuvant were used separately for immunization in guinea pigs with 200 micrograms per animal.FMDV specific humoral immune response was observed at the 21th day post vaccination.The immune responses were assessed by indirect ELISA and Serum neutralization test. The results showed that the recombinant protein, (HA)3 and (BN)3 cound induce specific antibodies, but fail in inducing high levels of neutralizing antibodies against FMDV of the same type in, guiea pigs.