ABSTRACT
To develop a new recombinant hepatitis E vaccine, we used Hansenula polymorpha expression system to express recombinant hepatitis E virus-like particles (HEV VLPs), to construct a recombinant engineered strain HP/HEV2.3. The fermentation conditions and purification process were studied next. The first working seed lots were fermented in liquid culture, and the fermentation products were collected, then crushed, clarified, purified by ultrafiltration, silica gel adsorbed and desorbed, concentrated by ultrafiltration, purified by liquid chromatography and sterilized by filtration. The purity reached 99% with a yield of 33%. Electron microscopy analysis revealed that both the purified recombinant HEV VLPs from HP/HEV2.3 and natural hepatitis E virus particles appear identical of being 32 nm. The resulting DNA sequence obtained from VLPs is identical to the published HEV sequence. The SDS-PAGE analysis has revealed that the protein molecular weight of the HEV VLPs is 56 kDa, and the expression product HEV VLPs were accumulated up to 26% of total cellular protein. The expression level is 1.0 g/L. Western blotting, enzyme-linked immunosorbent assay (ELISA) results of the protein and ED₅₀ of the vaccine showed that the HEV VLPs have good antigenicity and immunogenicity. In summary, the recombinant HEV VLPs from Hansenula polymorpha can be used in the manufacture of a new genetically engineered vaccine against hepatitis E.
ABSTRACT
Objective To construct the double hairpin siRNA and green fluorescent protein(GFP) expressing vector pU6/double-siRNA/Neo/GFP/1A/2A to interfere 1A and 2A gene of Coxsackie virus B4.Methods 21 bp fragments of the Coxsackie virus B4 2A and 1A gene were chosen as the targets and 65 bp complimentary fragments were synthesized,then the target gene fragments were cloned into pSilencer2.1U6 Neo and pGCsi-U6/Neo/GFP/siNeGative,respectively,then the double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A /2A was constructed by restrict endonuclease digestion,elctrophoresis isolation and reclaimer,ligatied by T4 DNA ligase;then the double siRNA expressing plasmid was transfected into Hela cells,and the GFP was observed under fluorescent microscope.Results The correct results showed that the recombinant plasmid had the correct special fragments and DNA sequence detected by restrict endonuclease digestion, electrophoresis and DNA sequencing;and GFP was also observed in Hela cells tansfected with pU6/double-siRNA/Neo/GFP/1A /2A under fluorescent microscope more than 15 d after transfection.Conclusion The double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A/2A is constructed successfully;it has the correct target viral gene sequences and can express GFP gene in Hela cells more than 15 d after transfection.