Development of nanobodies against the coat protein of maize chlorotic mottle virus

Abstract

Maize lethal necrosis (MLN) is a maize disease caused by the maize chlorotic mottle virus (MCMV), a potyvirus which causes yield losses of 30-100%. The present study aimed to isolate nanobodies against the MCMV coat protein (CP) for the diagnosis of MLN. MCMV CP expressed in Escherichia coli was used for llama immunization. VHH (i.e. variable heavy domain of heavy chain) gene fragments were prepared from blood drawn from the immunized llama and used to generate a library in E. coli TG1 cells. MCMV specific nanobodies were selected by three rounds of phage display and panning against MCMV CP. The selected nanobodies were finally expressed in E. coli WK6 cells and purified. Eleven MCMV-specific nanobodies were identified and shown to detect MCMV in infected maize plants. Thus, our results show that nanobodies isolated from llama immunized with MCMV CP can distinguish infected and healthy maize plants, potentially enabling development of affordable MCMV detection protocols.

Keywords: MCMV; MLN; antigen; nanobodies; protein expression; single domain antibody.

Fig. 1

EcoR1/Ndel test digest after plasmid preparation for MCMV CP constructs run on 1% agarose gel. MM is the molecular marker in base pairs (bps), while lanes 1, 2, 3 and 4 are the samples. The correct insert size of approximately 750 bp was observed on constructs loaded on lanes 1 and 3.

Fig. 2

Expression of MCMV CP in E. coli BL21 DE3 cells. Protein purification was achieved by chitin column affinity purification and MonoS ion exchange chromatography. (A) 15% SDS/PAGE was used for analysis after chitin column affinity purification of MCMV coat protein. The gel lanes were loaded as follows: MM, molecular weight marker in kDa; l and 2; products after the overnight self‐cleavage induced by dithiothreitol. MCMV CP was observed at 25 kDa. From the gel image, other proteins co‐eluted with the desired protein. (B) 15% SDS/PAGE analysis after MonoS ion purification of MCMV coat protein, with the protein of interest eluted using NaCl salt gradient. MM is the molecular weight marker; 1–6 are the fractions eluted during the chromatographic process. 1 to 5 are the eluted NaCl gradient fractions. MCMV CP eluted when the concentration of NaCl was at 1 m.

Fig. 3

Representative immune monitoring showing the strong immune response to MCMV coat antigen. (in this response measure, both conventional and heavy chain antibodies are measured). The serum of the llama from the blood drawn before and after immunization was serially diluted from 1 : 10 up to 1 : 1 771 470 (n = 12 for each dilution loaded in triplicate, geom_bar drawn using ggplot2 with the SD used to indicate the error bars).

Fig. 4

Identification of MCMV CP specific nanobodies by periplasmic ELISA. From the analysis of 95 clones randomly picked from the second and third round of panning, 76 clones were identified as positive for nanobodies that bind MCMV CP. The absorbance read at 415 nm was at least twice in the antigen coated well compared to the control well. However, clone 29 had a higher reading in the control well, which could be a result of contamination.

Fig. 5

Colony PCR to test for colonies containing an insert with the desired MCMV VHH genes. The PCR products were analyzed on a 1% agarose gel. A positive nanobody clone produces an amplicon of approximately 400 bp. MM is the molecular marker in base pairs (bps) while 1 to 32 are the samples. Of the 32 selected clones, 28 of them had a fragment that was approximately 400 bp.

Fig. 6

Phylogeny tree of the protein sequences of the selected nanobodies demonstrating the homology between the different VHH sequences against the MCMV CP. The sequences group to four families.

Fig. 7

Soluble expression of MCMV CP specific nanobodies in E. coli WK6 cells. Protein purification was achieved after osmotic shock lysis and IMAC purification. In the first well, MM is the molecular marker and the nanobodies were loaded in the remaining wells. A good quality band of approximately 15 kDa is observed on 15% SDS‐PAGE gel indicating high quality nanobodies.

Fig. 8

A bar chart representing absorbance at 415 nm of the nanobodies developed against MCMV CP in indirect ELISA. The test wells were coated with 100 μL of sample material prepared from MCMV infected maize plants. The control wells were coated with extraction buffer. All six tested nanobodies were detecting MCMV antigens with a high signal compared to the control well (n = 17 loaded in duplicate, geom_bar drawn using ggplot2 with the SD used to indicate the error bars).