关键词: 葡萄病毒B, 外壳蛋白, 原核表达, 抗血清

Abstract:

In this study，the coat protein gene（cp，594 bp）of Grapevine virus B（GVB）was amplified
from grapevine by RT-PCR using primers designed according to previous reported GVB sequences. After
sequence determination and analysis，the cp gene of preponderant isolate GVB-JFL was inserted into
expression vector pET-28a and constructed recombinant plasmid pET-GVB cp，pET-GVB cp was then
transformed into E. coli BL21（DE3）and induced by IPTG. SDS-PAGE analysis showed that the coat
protein（approximately 26 kD）was induced at a high level. The purified coat protein was used as antigen
for raising antiserum in rabbits，and the specificity of the antiserum was tested by ELISA and dot-ELISA.
The results showed that the antiserum could be successfully used to detect GVB in the infected Nicotiana
occidentalis and grapevine，but had no reaction with the healthy plants and Grapevine virus A（GVA，
another member of vitiruses）infected plants，the antibody titer for positive samples could be up to 1︰4 000，
and 9 of the 16 field samples detected after inoculated in tobacco were positive，these results indicated that the antiserum obtained in the study could be used for efficient and specific detection of GVB.

Key words: Grapevine virus B, coat protein, prokaryotic expression, antiserum