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

Abstract

Availability of adequate quantity of purified virus preparation from plant tissue is the major limitation in producing polyclonal antibodies (PAb) to begomovirus. Very few examples show successful utilization of E. coli expressed recombinant coat protein (CP) for immuno diagnosis of begomoviruses. In the present study, *771 bp CP gene (*29.0 kDa) of Pumpkin yellow vein mosaic virus (PYVMV) was expressed as a *71.0 kDa fusion protein with maltose binding protein (MBP) (*42.0 kDa) in E. coli. The MBP-CP was obtained in soluble state. The PAb to the purified fusion protein successfully detected PYVMV and other bipartite and monopartite begomoviruses in the field samples at 1:250 dilution in enzyme linked immunosorbent assay. Our study for the first time showed that MBP-tag fusion CP was suitable to produce diagnostic antibody to begomoviruses. Keywords Pumpkin yellow vein mosaic virusBegomoviruses (family Geminiviridae) are emerging constrain in the production of numerous crops [14]. Both, immune-and nucleo-based techniques have been employed for the detection of begomoviruses [5]. Begomoviruses are phloem limiting, generally not sap transmissible and many of them do not have suitable propagative hosts. As a result, transmission and purification of begomoviruses are difficult and yield of purified virus is usually low. However, tedious method of virus propagation in large number of host plants through whitefly (Bemisia tabaci) inoculation and purification of virus from large batches of infected plant tissues were performed to produce polyclonal antibodies (PAb) against a few begomoviruses such as squash leaf curl virus (SLCV) [3], tomato yellow leaf curl virus (TYLCV) [9] and tomato leaf curl Bangalore virus (ToLCBaV) [4]. The traditional method of begomovirus antigen preparation from infected plant tissue generally results into contamination with host proteins and thus limits their use in serological diagnosis. Begomovirus purification by conventional method further limits the renewable production of PAb. As a result, routine detection and identification using serology has been less commonly used for begomoviruses compared to the other plant viruses. 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]. There is an increasing incidence of PYVMV and many other begomoviruses in the Indian subcontinent [14], but PAb to begomoviruses for immunodia...