Expression of Heat-labile Enterotoxin of Escherichia coli in Biolistic Transformed Hairy Roots of Daucus carota L.

Sharada Laxman Deore,Someshekhar S Khadabadi,Bhushan A Baviskar

doi:10.5530/pj.2020.12.198

Abstract

Expression and secretion of recombination proteins in transgenic hairy roots have opened door to simple, feasible and economic option compared to animal and bacterial systems. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heatlabile enterotoxin (LTB) in Daucus carota L hairy root where transformation is achieved by biolistic gene gun method. Agrobacterium rhizogenes strain (R-1000) cells containing synthetic LTB gene in pMYO51T plant expression vector under the CaMV 35S promoter introduced to Daucus carota L roots by biolistic gene gun method. PCR amplification confirmed the integration of synthetic LTB gene in carrot hairy roots. Western blot analysis confirmed production of LTB pentamer in similar banding pattern to the native bacterial derived LTB. 0.50-0.70% of LTB protein estimated in carrot hairy roots. GM1-ganglioside assay indicated formation of biological active pentamers. LTB protein with biochemical properties identical to native LTB protein in the hairy roots of edible carrot roots opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

Highlights

Production of antigens and antibodies in plant provide a potential approach for the development of vaccines for oral delivery.[1]
The integration of LTB gene in the carrot hairy roots is confirmed by polymerase chain reaction (PCR) (Figure 2)
Western blot analysis of hairy roots carried out to confirm immunogenicity of the soluble proteins from the transgenic group against anti-LTB antiserum which revealed that specific signal from transgenic carrot hairy roots compared to no signal from untransformed roots

Summary

INTRODUCTION

Production of antigens and antibodies in plant provide a potential approach for the development of vaccines for oral delivery.[1]. Heat-labile enterotoxin (LT) protein composed of one A-subunit (LTA, 27 kDa) and five noncovalently associated B-subunits (LTB, 11.6 kDa each) forming a ring-like pentamer from Escherichia coli causes diarrhea.[6] Wild-type LT is toxic and unsuitable for clinical use. Previous research confirmed that recombinant LTB could potential mucosal and parenteral vaccine adjuvants to stimulate strong serum and mucosal immune responses against LT. Nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been expressed in a number of plant species with favourable quantity, including potato (17 lg g-1 FW), tomato (38 lg g-1 DW), tobacco (75 lg g-1 FW) and carrot FW).[7] present study reports biolistic gene gun mediated expression of LTB proteins in Daucus carota L hairy roots first time

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION

CONFLICTS OF INTEREST