Molecular cloning and high-level expression of G2 protein of hantaan (HTN) virus 76-118 strain in the yeast Pichia pastoris KM71.

Hantaan viral G2 envelope gene, which is known to be one of major antigens and induce neutralizing antibodies, was cloned into expression vector pHIL-S1 which consists of AOX1 promoter, PHO1 signal sequence, HIS4 gene and other components. The recombined plasmid was transformed into methylotropic yeast, Pichia pastoris of KM71 and recombinant strains harboring multi-copy of G2 gene were selected. Expression of the cloned G2 gene was confirmed with Western blot analysis using anti-sera of guinea pig immunized with the carboxyl terminal region of G2 protein expressed in Escherichia coli. The expression of G2 gene from the recombinant strain was tightly repressed by dextrose and effectively induced by methanol, an inducer of AOX1 promoter. The highest expression level was observed from 1 day after induction and maintained at the same level for up to 4 days.

Nucleotide sequence of nucleocapsid protein (N) of Hantaan virus isolated from a Korean hemorrhagic fever patient.

The nucleotide sequence of the nucleocapsid protein (N) coding region of a Hantaan virus strain (CFC94-2) isolated from a Korean Hemorrhagic Fever (KHF) patient was determined by sequencing a series of deletion mutants. Comparison of the N coding sequence of CFC94-2 to the sequence of the prototype of Hantaan virus, strain 76-118 reveals a 4.97% difference in the nucleotide sequence and a 1.6% difference in the deduced amino acid sequence. The rate of amino acid sequence variation in N protein of different Hantaan viruses (1.6%) is quite similar to that in G1 and G2 envelope proteins (1.7%). These results suggest that N protein may be under a similar selection pressure to G1 and G2 envelope proteins against host immune system.