| Summary: | 博士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 107 === Classical swine fever virus (CSFV) infection is a severe swine disease, often
causing large economic losses. Pichia pastoris yeast-expressed CSFV glycoprotein E2
(yE2) has been shown to induce a protective immune response against the virus. To
improve the expression level of yE2, the first codon of E2 gene (CGG) was optimized
to the most favorite codon (AGA) and deleted transmembrane domain. Three
recombinant E2 glycoproteins N342, N330 and N301 which composed the N-terminal
342, 330 and 301 residues respectively, were constructed. The yield of E2 protein was
remarkably increased in the codon optimized strain (N342). The immunogenicity of
each recombinant E2 subunits was confirmed by the immunization of pigs, and all
immunized groups demonstrated high neutralizing antibody titers after boost
immunization, which lasted for a long period of time. In addition, the envelope
glycoprotein Erns has been shown to bind to the sulphated-heparin like
glycosaminoglycans (GAGs) on cell surface and participates in cell attachment of the
virus. The CSFV Erns gene was codon optimized for the expression in Pichia pastoris.
The C-terminally truncated Erns recombinant protein lacking the previously identified
heparin-binding domain (HBD) bound to heparin column, suggesting the presence of
another HBD in CSFV Erns. Sequence analyses of the CSFV Erns coding region revealed
a common potential N-terminal HBD at residues 34-44. Site-directed mutagenesis of the
basic amino acids at K36 and K39 significantly reduced the heparin-binding affinity of
the protein. Further mutations of both T43 and H44 had little effect. Thus, a novel
potential heparin-binding site near the N-terminus of CSFV strain TD96 Erns has been
detected, and the two basic amino acids K36 and K39 are crucial for binding activity to
heparin matrix and cell-surface GAGs.
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