Heterologous expression of pepper mild mottle virus coat protein encoding region and its application in immuno-diagnosti
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ORIGINAL ARTICLE
Heterologous expression of pepper mild mottle virus coat protein encoding region and its application in immuno-diagnostics Nidhi Kumari1 • Vivek Sharma1 • Priyankaben Patel1 • P. N. Sharma1
Received: 11 November 2019 / Accepted: 29 April 2020 Indian Virological Society 2020
Abstract Pepper mild mottle virus (PMMoV), a tobamovirus of family Virgaviridae affects the quality and quantity of Capsicum. PMMoV is highly contagious, capable of transmitting through infected seeds and soil. Symptoms are more severe when crop is infected at young stage but remain unnoticed when infection takes place at maturity. Therefore, cost effective diagnostic techniques are required for timely and accurate detection of virus. In present study, coat protein encoding region of PMMoV-HP1 isolate was cloned into expression vector system, pET28a and expressed in BL21, a protease deficient strain of Escherichia coli. The PMMoV-HP1 pathotype was identified as PMMoV-P12 on the basis of coat protein amino acid sequence analysis in our previous study. The overexpression of recombinant coat protein of 26 kDa, corresponding to the expected 6X Histidine tag fused recombinant protein was purified using Ni–NTA columns from insoluble fraction. For antisera production, the purified recombinant protein was dialyzed * 24 h to remove urea and then used for raising polyclonal antisera. The specificity and sensitivity of antiserum obtained was demonstrated using different dilutions of antiserum for western blot assay and direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). In Western blot assay, the test antiserum reacted strongly both with PMMoV-CP in purified protein and native CP in crude sap from PMMoV infected pepper plants, whereas no reaction was observed with healthy plant sap. In DAC-ELISA antiserum dilution up to 1:1000
& P. N. Sharma [email protected] 1
Department of Plant Pathology, CSK HP Agricultural University Palampur, Palampur, Himachal Pradesh 176062, India
was capable of detecting the virus in infected sample. The absence of any cross reactivity of test antiserum was confirmed against tobacco mosaic virus, cucumber mosaic virus, tomato spotted wilt virus, pepper veinal mottle virus, potato virus Y and tomato yellow leaf curl virus antigen, known to infect capsicum. Keywords PMMoV Coat protein Capsicum Western blot assay DAC-ELISA
Introduction Capsicum annuum L. var. grossum Sendt (Bell pepper or Sweet pepper), cultivated across the world under various climatic and environmental conditions [20] is an important spice and vegetable crop. Capsicum spp., originated in Mexico, South Peru and Bolivia [22, 42, 53], in India, popularly known as ‘‘Shimla Mirch’’ is one of the premier vegetable crops being cultivated in open as well as protected conditions. As per the records of National Horticultural Board, the total area and production of capsicum in India is 24,000 ha and 306,000 MT, respectively (http:// nhb.gov.in/statistics/State_Level/2016-17(Final). The production of capsicum is being impeded
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