Expression and Characterization of Synthetic Heat-Labile Enterotoxin B Subunit and Hemagglutinin–Neuraminidase-Neutralizing Epitope Fusion Protein in Escherichia coli and Tobacco Chloroplasts

Abstract

Synthetic enterotoxigenic Escherichia coli heatlabile enterotoxin B subunit-HN-neutralizing epitope fusion protein was expressed in E. coli and tobacco chloroplasts. Bacterial and chloroplastic recombinant LTB-HN-neutralizing epitope (LTB-HNE) fusion proteins showed the pentameric structures by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a strong affinity for G M1 -ganglioside. Bacterial and chloroplastic recombinant LTB-HNE was detected by Western blot analysis using polyclonal antibodies to HN-neutralizing epitope. Insertion of the gene encoding LTB-HNE protein into the chloroplast genomic DNA of spectinomycinresistant plants was confirmed by polymerase chain reaction. The presence of the LTB-HNE specific transcript in the total RNA of transgenic plant leaves was verified by reverse transcriptase polymerase chain reaction. The highest level of expression of recombinant LTB-HNE fusion proteins in the leaves of transplastomic plants was about 0.5% of the total soluble protein. Growth rates, flowering, and seed setting were not affected, and the cassette for homologous integration and gene expression in the transplastomic T1 plant was subsequently inherited.

Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa)

“…After quantifying soluble protein, test samples (100 μg in 100 μl PBS) were intraperitoneally injected into ten mice on day 1, followed by four booster injections on days 8, 15, 22, and 29. As positive controls, purified LTB (10 μg/mouse) and purified CTB (30 μg/mouse) expressed in E. coli as previous our report (Sim et al 2009 ). To express CTB in E. coli , codon-optimized CTB gene was cloned at the N-terminus at Nde I site and at the C-terminus at Xho I (pET21a-CTB).…”

“…Similarly, mammalian cell cultures are expensive, hindering production of large quantities of vaccines. The development of various plant expression systems has enabled the expression of foreign antigens in plant tissue as edible vaccine vehicles (Haq et al 1995 ; Du et al 2005 ; El Adab et al 2007 ; Ko et al 2009 ; Sim et al 2009 ; Unni and Soniya 2010 ; Cui et al 2011 ; Guo et al 2012 ). Plant-based edible vaccines offer advantages of longer shelf-life, stability at room temperature without loss of immunogenicity, and reduced production costs (Streatfield et al 2001 ).…”

Chloroplast-Derived Therapeutic and Prophylactic Vaccines

“…Since the realization of the chloroplast's ability to produce vaccine antigens, considerable accomplishments regarding the expression of bacterial antigens have been achieved. Vaccines against enterogenic E. coli (Kang et al 2004(Kang et al , 2003Rosales-Mendoza et al 2009 ;Sim et al 2009 ) , Clostridium tetani (Tregoning et al 2003(Tregoning et al , 2005 , Bacillus anthracis (Gorantala et al 2011 ;Koya et al 2005 ;Watson et al 2004 ) , Yersinia pestis (Arlen et al 2007(Arlen et al , 2008 , Borrelia burdoferi (Glenz et al 2006 ) , as well as a multi-epitope vaccine against diphtheria, pertusis, and tetanus ) have been produced through the chloroplast. The yield of these vaccines is typically antigen-dependant and therefore contingent on accumulation and susceptibility to proteolysis.…”

“…The yield of these vaccines is typically antigen-dependant and therefore contingent on accumulation and susceptibility to proteolysis. Examples ranging 31% total soluble protein (TSP) (Molina et al 2004 ) to as low as 0.5% TSP (Sim et al 2009 ) have been reported. While a multitude of bacterial antigens have been effectively expressed through CTT, only some of these studies instituted an in vivo functional evaluation of the recombinant antigen.…”

Towards Generation of Transplastomic Tobacco, Expressing a 35 kdaProtein as an Antigen: A Step towards Affordable Plant made Vaccine against Mycobacterium

“…Immunogenicity against a hepatitis antigen was achieved in mice fed with transgenic potatoes developed by various groups [23,24]. Successful experiments with plant derived vaccines have also been carried out with Norwalk virus [25], Rabies [26], Human Papilloma Virus [27], Newcastle disease virus [28] and many more. Immunogenicity has been reported in humans by consumption of transgenic potatoes containing enterotoxin subunit B from E.coli [29].…”