Prokaryotic expression of coat protein of Grapevine leafroll associated virus-3 and its special antiserum preparation.

The coat protein gene of a Chinese isolate of Grapevine leafroll associated virus-3 was amplified by RT-PCR. Sequence analysis showed that the cp was 942 bp,and it had different similarities with that of other reported GLRaV-3 isolates ranging 91%-99% at nucleotide sequence level and 95%-100% at amino acid sequence level. This gene was cloned into the prokaryotic expression vector pET-28a(+),then trans formed into E. coli BL-21 (DE3 plysS),and subsequently induced by IPTG at final concentration of 1 mmol/L. It was shown that the recombinant CP was expressed with the molecular weight of 35 kDa by SDS-PAGE and Western blotting analysis. Antiserum was prepared after the rabbits were immunized with purified recombinant CP. Results showed that the special antiserum against CP could be used efficiently to detect GLRaV-3 in infected grapevine samples by protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA ) and dot-blot immunobinding assay (DBIA).