Agrobacterium-mediated Transformation of Spinach using Outer Membrane Protein C (OmpC) gene from Salmonella

Abstract

Salmonellosis is a bacterial infection caused by Salmonella. Severe Salmonella infections lead to the development of colon cancer. The currently available vaccines against this infection are Ty21a and Vi CPS, which work only against Salmonella typhi and not the others serovars of Salmonella. Outer membrane protein C (OmpC) is an immunogenic protein of Salmonella and is a potential candidate for the development of sub-unit vaccine. Plants can be used as bio-factories for the expression of vaccine antigens. The present research work aimed to optimize the tissue-culture conditions for Spinacia oleracea leaf explants and to develop an efficient transformation protocol for Agrobacterium-mediated stable transformation of Spinacia oleracea with OmpC antigen. Sterilization of spinach seeds with 0.2% mercuric chloride provided good results. Full MS media gave maximum regeneration efficiency for spinach plant. For tissue culture, full MS media supplemented with 1 mg/L BAP and 0.5 mg/L IAA showed the highest callus formation efficiency. 20 mg/L concentration of hygromycin antibiotic was found optimum for selection of transformed leaf explants. Successful stable transformation of spinach was carried out with OmpC antigen. Transformed explants were grown on selection media containing suitable concentration of hygromycin. Explants with 3 days co-cultivation time showed 50% callus formation efficiency. Transformation was confirmed through PCR using gene specific primers. Transgene expression in plants was analyzed by quantitative real-time PCR (qRT-PCR) in comparison with β-actin gene as control and copy number calculated was 1. Protein expression was confirmed through dot Blot, western blotting and ELISA. Taken together, successful expression of Salmonella OmpC antigen in plants could facilitate the development of edible and cost-effective subunit vaccine against salmonellosis. Keywords: Spinacia oleracea, OmpC gene, Salmonellosis, Agrobacterium-mediated stable transformation, PCR, qRT-PCR, Dot blot, Western blotting