Heterologous expression of pepper mild mottle virus coat protein encoding region and its application in immuno-diagnostics

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 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: Capsicum; Coat protein; DAC-ELISA; PMMoV; Western blot assay.

Fig. 1

a RT-PCR amplification of PMMoV-HP1 infected “California wonder” plants; b Amplification of PMMoV-CP gene using primers with built in restriction sites for EcoR1 and Sal1; c A diagram showing PMMoV-CP cloned in expression vector, pET-28a; d Nucleotide sequence of coat protein region of PMMoV cloned in pET-28a

Fig. 2

Alignment of amino acid sequence of pET-28a + CP with that of PMMoV-HP1 generated through multiple alignment tool clustalW

Fig. 3

15% SDS-PAGE presenting recombinant coat protein gene expressed using 1 mM of IPTG (a); recombinant protein purified through Ni–NTA affinity chromatography, Lane L: Benchmark unstained protein ladder (Fischer Scientific, Cat. No.: 10747-012) Lane 1: uninduced sample, Lane 2: induced sample, Lane 3: flow through fraction collected, Lane 4-6: three elution fractions collected separately (b); Solubility analysis of target protein. L: unstained protein ladder, Lane 1: uninduced sample, Lane 3: crude extract A (soluble protein fraction), 4: crude extract B (insoluble protein fraction) (c); Western blot analysis following electrophoresis in SDS-PAGE, electroblotting of purified recombinant protein using 6XHis-tag specific monoclonal antibodies Lane M: Benchmark prestained protein ladder (Fisher Scientific, Cat. Number: 10748-010), Lane 7- represents purified recombinant protein, Lane 8- represents the uninduced culture (d) and purified recombinant protein and crude extract of PMMoV infected plants using 1:100 dilution of polyclonal antiserum. L: Benchmark pre-stained protein ladder (Cat. No.:10748-010), Lane 1: purified recombinant protein, Lane 2: crude extract isolated from PMMoV infected plants (e)