The Eukaryotic Expression Of Foot-and-mouth Disease Virus Structural Protein VP4 Gene

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals,including cattles, pigs and sheep.This disease is widely endemic all over the world and often results in considerable economic losses. Current FMD vaccines are mainly based on inactivated viruses, although effective, but outbreaks of FMD have been directly associated with incomplete inactivation of virus, and even some time contribution to the escape of virus from vaccine manufacturing facilities. FMDV capsid contains 60 copies of each of four structural proteins, VP1, VP2, VP3, and VP4. An anti-FMD virus type AsiaⅠpeptide vaccine is obtained from the epitope protein encoded by DNA sequence from the B cell and T cell epitope amino acid of FMD virus type AsiaⅠVP1 and VP4 or linking epitope amino acid with the vector ,in which B cell epitope amino acid was chosen from FMD virus type AsiaⅠV P1 80-101,133-160 and 200-213 and T cell epitope amino acid was chosen from FMD virus type AsiaⅠV P4 20-34 or VP1 20-34 and 35-48.In my study, the expression vector pcDNA3.1(+) and pUC57-VP4 gene were digested with restriction endonucleases, EcoRI and BamHI, respectively. The VP4 gene was inserted into the eukaryotic expression vector pcDNA3.1. Positive clone, a recombinant plasmid named as pcDNA3.1-VP1 with interest gene, was identified by restriction analysis and DNA sequencing. Then the recombinant plasmids were transfered into E.coli DH5αfor VP4 expression. The interest gene was identified by restriction analysis and DNA sequeneing. The homology of the sequences of VP4 gene among foot-and-mouth disease virus O isolate O/NYOO of FMDVs registered in GenBank was compared(AY333431.1). It was found that the pcDNA3.1-VP4 gene shared highest homology with foot-and-mouth disease virus O isolate O/NYOO strain up to 100%. In order to identify whether the interest gene of the VP4 expresses in eukaryotic cells, eukaryotic expression vector pcDNA3.1-VP4 were used to transfect HeLa with pcDNA3.lA empty vector transfected HeLa as a negative control. SDS-PAGE results showed that there is an obvious protein of 8 kD, and empty vector had no hybrid zone in the corresponding position of gel.The recombinant plamids, pcDNA3.1-VP4, was transfected into HeLa cells for the detection of VP4 expression in vitro. The results show that plasmid was successfully constructed and it can be expressed in HeLa cells as VP4 protein in vitro, which paves the way for further studying its antigen biology, and the immune response stimulated with VP4 that are necessary both for antigenic modification and adjuvant screening.