Subcloning and Expression of ML1-stxB Fusion Gene of Mistletoe Lectin in E. coli and Production of its Antibody in Mouse

Abstract

Background and Objectives: Extract of mistletoe lectin (Viscum album L) leaves contains MLI protein that is a type 2 ribosome-inactivating protein (RIP2) with lectin properties. Shiga toxin (STxB) has been considered as an immunoadjuvant and carrier, which binds to its cell surface receptor, Gb3, that is expressed on most of the body cells. In this study, the expression of ML1-stxB antigen and production of its antibody, were investigated in mouse. Methods: In this experimental study, ML1 gene containing pUC57 plasmid with enzymes sites of NdeI and SalI, was subcloned in the pET28a(+)-stxB expression vector, and then was transformed into E. coli BL21 (DE3). The expression of ML1-stxB gene cassette was induced by IPTG. After purification, the MLI–STxB recombinant protein was purified by nickel affinity chromatography and injected into mice four times. Results: In this study, the cloned ML1-stxB gene in expression vector of pET28a(+) was confirmed by PCR and enzyme analysis. The produced recombinant protein were confirmed by SDS-PAGE and Western blotting. Then, the produced antibody was isolated from the serum of mouse and investigated using ELISA method. Conclusion: According to this study, ML1 protein has ribosome inactivating property and STxB has adjuvant and carrier functions, therefore,this recombinant protein can be a candidate vaccine against ML-1 toxin of Shigella dysentery, which its antibody can be used as identifier.

Keywords

• escherichia coli
• subcloning
• shigella
• mistletoe lectin
• shigella