Transient recombinant expression of highly immunogenic CagA, VacA and NapA in Nicotiana benthamiana

Abstract

Interest in the plant-based transient production of recombinant immunogenic antigens has tremendously progressed because plants are cost-effective, easily selectable, free of mammalian contamination, and support complex post-translational modifications. Nicotiana benthamiana is a convenient system for transient expression of recombinant antigens. The present study documented a platform for rapid production of Helicobacter pylori CagA, VacA and NapA antigens three days (first harvest, FH) and six days (second harvest, SH) after agro-infiltration using a syringe. In this study, CagA, VacA and NapA antigen genes from Helicobacter pylori were cloned into the binary vector pBI121 and transformed into Nicotiana benthamiana by the Agrobacterium-mediated process. Leaves of four to five weeks old Nicotiana benthamiana plants were agroinfiltrated with EHA105 subtype of Agrobacterium tumefaciens strain containing cloned CagA (pBI121-CagA), VacA (pBI121-VacA) and NapA (pBI121-NapA) constructs. The transient expression and accumulation of the recombinant genes containing CagA, VacA and NapA expression cassettes were confirmed using qRT-PCR by comparing the relative expression at FH and SH post-infiltration with the non-infiltrated (control) samples and using ELISA at 1/5 and 1/10 dilution ratios. The qRT-PCR findings showed that Agrobacterium-mediated syringe infiltration of leaves of four to five weeks old Nicotiana benthamiana plants produced significantly higher transcript levels of CagA (about 8-fold and 7-fold), VacA (38-fold and 24-fold) and NapA (7-fold and 5-fold) genes at FH and SH compared to the control sample. Besides, the maximum amount of CagA, VacA and NapA antigens were detected at the FH stage compared to the SH stage, when the antibody concentrations of the agro-infiltrated leaf extracts containing these recombinant antigens were diluted in a 1/5 ratio. This study has developed evidence to support that recombinant CagA, VacA and NapA can be transiently produced in Nicotiana benthamiana plants.

Keywords: CagA; ELISA; Helicobacter pylori; NapA; Nicotiana bethamiana; VacA; qRT-PCR.

Fig. 1

PCR amplification of CagA (a) VacA (b) and NapA (c) genes . M: DNA ladder; lane 1: Desired PCR amplicons.

Fig. 2

Plasmid construction by restriction digestion of the gene constructs. M: DNA ladder; lane 1: undigested gene construct; lane 2: digested gene construct.

Fig. 3

Syringe-assisted agro-infiltration of Nicotiana benthamiana. (a) Four to five weeks old Nicotiana benthamiana plant. (b) Agro-infiltrated Nicotiana benthamiana leaflet.

Fig. 4

Presence of engineered pBI121-CagA, pBI121-VacA and pBI121-NapA constructs. (a) t-DNA sections of the pBI121-CagA, pBI121-VacA and pBI121-NapA vectors; LB: left border and RB: right border of t-DNA; nos: nopaline synthase terminator from Agrobacterium; 35S: promoter of the cauliflower mosaic virus RNA. (b) Colony PCR of Agrobacterium tumefaciens containing pBI121-CagA, pBI121-VacA and pBI121-NapA plasmids; M: DNA ladder and lane 1: Desired PCR amplicons.

Fig. 5

Relative gene expression of the pBI121-CagA, pBI121-VacA and pBI121-NapA constructs in agro-infiltrated and non-infiltrated (control) Nicotiana bethamiana leaves at first and second harvest post-infiltration; FH: first harvest; SH: second harvest; y-axis: fold change analyzed by qRT-PCR; x-axis: harvest stages; the bars indicate the mean ± SD of the experiments, statistical significance was represented by single, double and triple asterisks (P < 0.05, < 0.01 and < 0.001).

Fig. 6

ELISA assay with respect to 1/5 and 1/10 dilution ratios of recombinant CagA, VacA and NapA in agro-infiltrated and control Nicotiana bethamiana leaves after first and second harvests. FH: first harvest; SH: second harvest; OD: optical density; y-axis: antibody density; x-axis: serial dilution ratios; the bars indicate the mean ± SD of the experiments, statistical significance was represented by single, double and triple asterisks (P < 0.05, < 0.01 and < 0.001). Expression of highly immunogenic CagA, VacA and NapA antigens of Helicobacter pylori recombinantly in Nicotiana benthamiana.