Serological Studies Using Polyclonal Antisera Prepared Against the Viral Coat Protein of Four Begomoviruses Expressed in Escherichia coli

Abstract

Polyclonal rabbit antisera were produced to the coat protein of Bean golden mosaic virus Brazil isolate (BGMV), Cabbage leaf curl virus (CabLCV), Tomato yellow leaf curl virus (TYLCV), and Tomato mottle virus (ToMoV), all expressed in Escherichia coli by the pETh expression vector. The expressed coat protein of each virus was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for use as an immunogen. The antisera to BGMV, CabLCV, TYLCV, and ToMoV reacted in indirect (plate-trapping) enzyme-linked immunosorbent assay (ELISA) with extracts from begomovirus-infected tissue. The antisera to BGMV, CabLCV, TYLCV, and ToMoV also reacted specifically with the test begomovirus antigens in leaf imprint blots and Western blots. The CabLCV and TYLCV antisera were used to detect Bean golden yellow mosaic virus antigens by immunogold labeling of thin sections of infected bean tissues. In tissue blot immunoassays, the TYLCV antiserum reacted well with TYLCV antigens but not with ToMoV antigens, while CabLCV antiserum reacted well with ToMoV antigens and weakly with TYLCV antigens. The results indicate that polyclonal antisera prepared to expressed begomovirus coat proteins were useful for the detection of begomoviruses in an array of assays.

Fusion coat protein of pumpkin yellow vein mosaic virus with maltose binding protein: Applications in immunodiagnosis of begomoviruses

“…Maximum expression of the fusion protein from pMal-PY-CP construct was achieved in E. coli TB1 cells. Higher induction was observed with 0.4 mM IPTG at 37°C for 3 h. Unlike other vector systems [1,6], the fusion protein in the present study was found in the soluble fraction. Whereas the pET-PY-CP clone did not show any visible expression under above conditions.…”

“…To overcome these limitations, molecular biology techniques are currently being used to express the viral genes of interest in E. coli and the recombinant antigen is used for the production of PAb. Recombinant antigens for several RNA plant viruses were successfully produced in E. coli [6,7,10], whereas, very few DNA plant viruses have been used for successful expression of recombinant antigen in E. coli [1].Pumpkin yellow vein mosaic virus, a bipartite begomovirus is known in India since 1950s [13] and the virus was characterized based on host range and serology [8]. Further, phylogenetic analysis of DNA-A sequence revealed that PYVMV was synonymous to squash leaf curl China virus (SLCCNV) reported from China [11,12].…”

“…To overcome these limitations, molecular biology techniques are currently being used to express the viral genes of interest in E. coli and the recombinant antigen is used for the production of PAb. Recombinant antigens for several RNA plant viruses were successfully produced in E. coli [6,7,10], whereas, very few DNA plant viruses have been used for successful expression of recombinant antigen in E. coli [1].…”

“…The use of rCP as an immunogen to develop antibodies has been shown as of great value particularly for viruses that are difficult to propagate and purify (Lee and Chang, 2008). PAb to geminiviral proteins expressed in E. coli using recombinant technology was demonstrated earlier by using expression vectors pETh-3c [1] and pQE-30UA [4]. In both these previous studies the expressed protein was in insoluble state, which was difficult to purify.…”

“…In the present study also, low titer PAb was obtained against MBP-CP (71.0 kDa) of PYVMV, a bipartite begomovirus. However, relatively higher titer (1:10,000) was reported against His-tag fusion CP of cabbage leaf curl virus [1]. Although, the titer of the PAb to PYVMV was low, it was useful in detecting both monopartite and bipartite begomovirus affecting various plant species.…”