Expression of Shigella flexneri ipaB gene in tobacco

Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotypespecific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System [TTSS] of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli [E. coli], the feasibility of the expression of this protein in tobacco has been investigated. The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens [A. tumefaciens] strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum [N. tobaccum] leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting

Enteroaggregative Escherichia coli, a heterogenous, underestimated and under-diagnosed E. coli pathotype in Iran

The main features of enteroaggregative Escherichia coli [EAEC] pathogenesis include attachment of bacteria to the intestinal mucosa, production of various toxins and cytotoxins, and stimulation of mucosal inflammation. 'Virulence' genes encode these features. Comparison of different EAEC isolates has shown that the virulence gene content of these isolates varies considerably. The heterogeneity of EAEC strains was concluded from the results obtained from the volunteer as well as other studies. Although the underlying mechanism behind the apparent increase in O104:H4 virulence is not known, several bacterial factors have been implicated. In this review, the known virulence factors involved in pathogenesis of EAEC pathotype are summarized

Priming hepatitis B surface [HBsAg]- and core antigen [HBcAg]- specific immune responses by chimeric, HBcAg with a HBsAg 'a' determinant

We developed an immunogen to stimulate multivalent immunity against hepatitis B surface antigen [HBsAg] and hepatitis B core antigens [HBcAg]. Immune responses specific for both HBsAg and HBcAg play an important role in controlling the infection. HBsAg-specific antibodies mediate elimination of virions at an early stage of infection and prevent the spread of virus. The immunogen was constructed by inserting the immunodominant, antibody-binding 'a' determinant [aa 111-149] of HBsAg [with or without a poly-glycine [PG] linker] into the e2 epitope of HBcAg. Only the constructs in which the HBsAg 'a' determinant was inserted into HBcAg, flanked by PG linkers, expressed a chimeric protein in human embryonic kidney cells with HBsAg and HBcAg antigenicity. Both glycosylated and non-glycosylated forms of the chimeric protein were immunoprecipitated from cell lysate. Intramuscular DNA vaccination of mice with plasmids expressing chimeric HBcAg primed antibody responses against well-defined serologically-defined determinants of both, native HBcAg, and native HBsAg. In addition, CD8[+] T cell responses against HBcAg epitopes were primed by this chimeric HBV antigen. The e2 sequence of HBcAg can thus be used to present heterologous epitopes without loss of immunogenicity of the HBcAg protein

Expression of a Chimeric protein containing the catalytic domain of Shiga-like toxin and Human Granulocyte macrophage colony-stimulating Factor [hGM-CSF] in Escherichia coli and Its recognition by reciprocal antibodies

Fusion of two genes at DNA level produces a single protein, known as a chimeric protein. Immunotoxins are chimeric proteins composed of specific cell targeting and cell killing moieties. Bacterial or plant toxins are commonly used as the killing moieties of the chimeric immunotoxins. In this investigation, the catalytic domain of Shiga-like toxin [A1] was fused to human granulocyte macrophage colony stimulating factor [hGM-CSF] gene and the fused gene was then expressed using an expression vector containing arabinose promoter. The protein thus obtained could be recognized by two different ELISA system designed for detection of hGM-CSF and Shiga-toxin and reconfirmed by Western-blot. The recognition of the chimeric protein by specific antibodies could be indicative of the proper form of the protein, which justifies further steps to be taken to evaluate the potential effects of the chimeric protein

Construction of hybrid gene of hepatitis B surface antigen carrying heat-stable enterotoxin of escherichia coli and its expression in mammalian cell line

Hepatitis B surface antigen is the first genetically engineered vaccine licensed for human use. Various strategies have been proposed to obtain a vaccine that would bypass the need for injection. In this study, a non-toxic portion of heat-stable enterotoxin of Escherichia coli that is capable of adhering to epithelial cells was inserted at amino acid position 112 of hepatitis surface antigen. The construct was used for transfection of human embryonic kidney cells in order to assess the expression of the hybrid protein. The data obtained showed a very low level of expression. In vivo antibody production and cytotoxic T lymphocyte response in B6 mice were assessed using DNA immunization. Three out of five injected mice responded with titers 10 mIU/ml anti-HBsAg and cytotoxic T-lymphocyte response was much higher with construct encoding the chimeric protein. Although this study proves that the chimeric protein is capable of eliciting both humoral and cellular responses, but further work is required to fully explore the feasibility of combining the properties of the two proteins