Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein

Abstract

Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constrain in large cardamom production in India. Purification of LCCV from large cardamom tissues is difficult and therefore immunodiagnostic reagents are not available. In the present study, we have successfully expressed coat protein (CP) gene of LCCV in Escherichia coli. The purification of expressed protein by Ni-NTA affinity chromatography was inefficient due to precipitation of protein during renaturation. We have optimized a simple, inexpensive and efficient method for purification of the expressed CP through gel extraction with 5 % SDS followed by renaturation in Milli-Q water, which resulted in high yield (4.7 mg/ml) and good quality of the protein. A higher titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced, which strongly recognized LCCV in crude leaf extract and showed minimal background reaction with the healthy leaf extract in enzyme linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The sensitivities of the ELISA and DIBA were 5 and 0.1 ng of expressed protein, respectively. Both the ELISA and DIBA were validated with 100 % accuracy in detecting LCCV in field samples. The PAb differentiated Cardamom mosaic virus, another close relative of LCCV. Our study is first to report highly efficient immunodiagnosis with PAb to E. coli expressed recombinant CP of a virus under the genus Macluravirus. The antigen expression construct and PAb developed in the present study will be useful in production of virus free planting materials of large cardamom.

Efficient immunodiagnosis of Citrus yellow mosaic virus using polyclonal antibodies with an expressed recombinant virion-associated protein

“…The supernatant containing protein was collected and the step was repeated for 2-3 times to remove SDS. Finally, the protein was dialyzed against Milli-Q water for 12 h at 4 °C (Vijayanandraj et al 2013).…”

A Simplified Multiplex PCR Assay for Simultaneous Detection of Six Viruses Infecting Diverse Chilli Species in India and Its Application in Field Diagnosis

“…Large cardamom chirke virus, a member of the genus Macluravirus in the family Potyviridae , which naturally infects large cardamom was recorded to infect C. annuum (landrace Dalle Khursani), a solanaceous crop, for the first time, exhibiting the cross-species transmission capability of macluraviruses [ 21 , 41 ]. The LCCV, a positive sense single stranded RNA virus, is vectored by aphids, Rhopalosiphum maidis , Brachycaudus helichrysi , and Myzus persicae in a non-persistent manner [ 42 , 43 ]. Its natural cross-transmission to chilli has raised concerns over a possible new outbreak in solanaceous crops.…”

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

“…Continuous production of polyclonal antibodies (PAb) to virus using purified preparation is difficult and considered as a major limitation in immunodiagnosis. To overcome this problem, the viral capsid protein is expressed as a recombinant fusion protein and used as an antigen for raising PAb to a number of plant viruses (Rani et al 2010;Khatabi et al 2012;Tatineni et al 2013;Vijayanandraj et al 2013). Recently, Sharma et al (2014) identified a putative coat protein gene of BSMYV based on in silico analysis and developed recombinant antibodies for detecting the virus from leaf samples having typical streak symptoms.…”