Extraction, Cloning and Expression of RTB, as a Vaccine Adjuvant/Carrier,in E. coli and Production of Mouse Polyclonal Antibody (Anti-B chain Abs)
Ricin, the toxic lectin extracted from the castor bean plant (Ricinus communis), consists of an A chain (RTA) and a B chain (RTB). Anti-A chain Abs and anti-B chain Abs can neutralize toxins in vivo and in vitro via blocking the binding of the toxin to the cell. Also, RTB protein is able to serve as an antigen deliver to the mucosal immune system and act as an immunoadjuant. Here, the genomic DNA was extracted from the fresh leave of the castor plant. The RTB gene was amplified by PCR. The prokaryotic expression vector pET-28a (+)- RTB was constructed, and used to transform E. coli Rosetta(DE3). The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of RTB-His by a rabbit monoclonal antibodies against His-tag. The SDS-PAGE profile exhibited the constructed prokaryotic expression efficiently produced RTB at the 1 mmol/L of IPTG. Addition of glycine and Triton X-100 enhanced native extracellular protein excreted into the culture medium. Anti-RTB polyclonal serum was generated by repeated immunization of mice with recombinant RTB protein. Finally, the antigenicity of recombinant RTB was identified by Western blot and indirect ELISA. A relative high titer of anti-RTB antibody was detected after the fourth injection. Western blot analysis was carried out with the polyclonal antibody revealed almost a 32-kDa band which corresponds to RTB protein. In conclusion, we herein report the expression of fully biologically active RTB as a plant lectin by a new strategy. This recombinant Ricin protein could be a promising drug for cancer therapy, vaccine as an immune response enhancement and even viral infected cells.
extraction, cloning and expression of rtb, as a vaccine adjuvant/carrier,in e. coli and production of mouse polyclonal antibody (anti-b chain abs)
ricin, the toxic lectin extracted from the castor bean plant (ricinus communis), consists of an a chain (rta) and a b chain (rtb). anti-a chain abs and anti-b chain abs can neutralize toxins in vivo and in vitro via blocking the binding of the toxin to the cell. also, rtb protein is able to serve as an antigen deliver to the mucosal immune system and act as an immunoadjuant. here, the genomic d...متن کامل
Cloning and Expression of the Variable Regions of Anti-EGFR Monoclonal Antibody in E. coli for Production of a Single Chain Antibody
Background:Epidermal growth factor receptor (EGFR) overexpression is a characteristic of several malignancies and could be considered as an excellent target for designing specific inhibitors such as anti-EGFR monoclonal antibodies for cancer therapy. Drawbacks exerted by large sizes of full-length antibodies have lead to the development of single chain antibodies, which benefit from having smal...متن کامل
Cloning and Expression of Recombinant Helicobacter pylori Urease A and B Subunits as a Putative Vaccine
Helicobacter pylori infection is among the most prevalent infections in the world involving more than half the adult population. H. pylori infection results in active chronic gastritis, peptic ulcers and enhances the risk of gastric malignancies. It is of utmost importance to prevent H. pylori infection particularly in highly prevalent countries including Iran. The urease holoenzyme produced by...متن کامل
cloning and expression of the variable regions of anti-egfr monoclonal antibody in e. coli for production of a single chain antibody
background:epidermal growth factor receptor (egfr) overexpression is a characteristic of several malignancies and could be considered as an excellent target for designing specific inhibitors such as anti-egfr monoclonal antibodies for cancer therapy. drawbacks exerted by large sizes of full-length antibodies have lead to the development of single chain antibodies, which benefit from having smal...متن کامل
Introduction: Hepatitis E virus (HEV) is a fecal-oral transmitting virus which causes a chronic liver disease. ORF2 is an immunogen capsid protein of HEV that has been proposed to be used for Hepatitis E vaccine design. It is a 660-amino acid protein which includes an immunogenic region (residues 112-607). This protein has been expressed in complete and truncated forms, using different expressi...متن کامل
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دوره 7 شماره 4
صفحات 247- 254
تاریخ انتشار 2011-10-01
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