Construction and expression of 75kDa readthrough protein gene from beet necrotic yellow vein virus

Using DNA recombinant techniques, coat protein (CP) gene and 54kDa fragment from beet necrotic yellow vein virus (BNYVV) RNA2 were ligated to construct 75kDa readthrough protein gene. Comparing with wild-type 75kDa readthrough protein gene, four nucleotides of the constructed gene were replaced, including CP amber termination codon UAG to AUG. Correspondingly, two amino acids were changed. The temperature inducible expression vectors containing 75kDa readthrough protein gene or its 54kDa fragment were constructed and transformed into E. coli BL21 (DE3), respectively. The results of SDS-PAGE and Western blotting showed that (1) the 75kDa readthrough protein gene was expressed specifically by temperature induction and some smaller protein components, one of which is 37 kDa, were observed as well; (2) only a 37kDa protein was produced from the 54kDa fragment.