Development of an ID-ELISA for the detection of Rice black-streaked dwarf virus in plants.
The Rice black-streaked dwarf virus (RBSDV) virion is composed of two layers of capsid proteins and 10 segments of double-stranded genomic RNA (S1-S10). Due to the fragility of the RBSDV outer capsid, it is very difficult to obtain intact virus particles for preparation of antiserum needed in virus detection. In this work, the major outer capsid protein (P10) encoded by S10 of RBSDV was expressed in Escherichia coli cells as a glutathione-S-transferase (GST) fusion protein. After purification of GST-P10 through affinity chromatography, P10 was released from the fusion protein by thrombin digestion and the purified P10 protein was used to immunize rabbits. The resulting polyclonal antiserum specifically recognized RBSDV from infected plant tissue and a planthopper vector in Western blotting assays. To facilitate screening of large numbers of samples, an indirect enzyme-linked immunosorbent assay (ID-ELISA) protocol capable of detecting RBSDV in very dilute wheat leaf extracts was developed. Based on the results, we conclude that efficient and economic detection of RBSDV can be performed routinely using polyclonal antiserum against P10 expressed in prokaryotic cells.
Wang
Zhao-Hui
ZH
State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100094, PR China.
Fang
Shou-Guo
SG
Zhang
Zhi-Yan
ZY
Han
Cheng-Gui
CG
Li
Da-Wei
DW
Yu
Jia-Lin
JL
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007106:Immune Sera; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000821:virology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012087:Reoviridae / Q000737:chemistry / Q000302:isolation & purification*; D014908:Triticum / Q000821:virology
D000914:Antibodies, Viral; D036022:Capsid Proteins; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase