Expression of virus-like particles of enterovirus 71 in Hansenula polymorpha

Objective To express virus-like particles （VLP） of enterovirns 71 （EV71） in Han- senula polymorpha. Methods The coding sequences of P1 and 3CD genes of EV71 were optimized accord- ing to codon usage bias of Hansenula polymorpha for achieving high expression, and then cloned into the ex- pression vector PMV of Hansenula polymorpha. The recombinant expression vector PMV-P1-3CD was trans- formed into Hansenula polymorpha AU0501. The transformants were stably cultured in selective medium （Yeast Nitrogen Base） and screened for strains with positive P1 and 3CD genes by PCR. Then an induced cultivation on the recombinant strains were performed in a medium supplemented with methanol to a final concentration of 1.0% and the expressed products were analyzed by SDS-PAGE and Western blot assays to select high expression strains. The high expression strains were cultured in 30 L fermentor and its fermenta- tion products were analyzed by electronic microscope after purification. Results EVT1 recombinant expres- sion strains were successfully constructed. The results of SDS-PAGE showed that the expressed products had obvious VP3, VP1, VP0 protein bands with molecular weights of 26x103, 33x103 and 35x103, respective- ly, which were consistent with the expected molecular weight of the fusion proteins. Western blot demonstra- ted that the expressed products could be specifically recognized by the polyclonal antibody against EVT1-VP1 at 33x103, indicating its high immunoreactivity. ELISA confirmed that the expression level of EV71 fermen- tation products was reached to 200 mg/L. Electronic microscope analysis showed that the VLP of recombi- nant EV71 were 24-30 nm in diameter with normal structure. Conclusion The virus-like particles of human enterovirus 71 are successfully expressed in Hansenula polymorpha, which provides a foundation for the fur- ther development of EV71 VLP vaccine. Key words: Enterovirns 71 ;  Hansenula polymorpha;  Virus-like particle;  Expression