| HFMD (hand, foot and mouth disease) is a common infectious disease in infants and children. EV71 (enterovirus 71) is one of the major causative agents associated with this disease. EV71 infection may result in HFMD and a variety of neurological diseases, such as aseptic meningitis, brainstem encephalitis and poliomyelitis-like paralysis and other nervous system related diseases, which lead to serious concerns about children's health. The prevention of EV71 outbreak is becoming very important. In 2009, statistical data showed that there were approximately 1,155,000 new cases of HFMD in mainland of China, and 353 deaths. HFMD actually showed a highest prevalence rate in infectious diseases of third class. There is no preventive vaccine available on market. Pandemic monitorations simply rely on early discovery and early treatment. Development of preventive vaccines against EV71 is therefore quite important.Saccharomyces cerevisiae was adopted as expression system to produce EV71 VLP (virus-like particle). EV71 P1 and 3CD genes were optimized according to the codon bias of Saccharomyces cerevisiae to avoid the rare codons and improve protein expression. The P1 and 3CD gene fragments were subcloned into expression vector and recombinant plasmid was further transformed into yeast. Moreover, four genes encoding EV71 VP1, VP2, VP3 and VP4 were amplified and subcloned into two different expression vectors and two constructs were co-transformed into yeast for VLP formation. Yeast cells were collected by centrifuge and lysed by laboratory homogenizer. VLPs were isolated by PEG protein precipitation and cesium chloride density gradient centrifugation. The purified samples were examined by Western Blot and electron microscope imaging.Two methods were used to produce EV71 VLP in Saccharomyces cerevisiae: 1) Co-expression of EV71 VP1, VP2, VP3 and VP4 genes in yeast; 2) Co-expression of P1 and 3CD genes in yeast; our data demonstrated that EV71 VLP formation can be successfully realized by both methods. |
