Cloning, expression, purification and interaction evaluation of 30 amino acids in C terminus of Clostridium perfringens toxin with its receptor Claudin-4

Abstract

Oral vaccine is a strategy being the most interested about treatments of gastrointestinal infections because of many great benefits outweigh conventional injection vaccines. In order to resolve the dispersion of antigens in gastrointestinal surfaces, the immunological tolerance and also be capable to stimulate immune responses effectively, M cells are targeted for antigens delivery. A number of researches reported that 30 amino acids in C terminus of Clostridium perfringens toxin (CPE30) have a high affinity to Claudin-4 receptor presenting on M cells. It is highly indispensable to produce a resource for assessing of CPE30 binding ability so cpe30 gene was cloned into the pET-gfp plasmid by two restriction enzymes BamHI and NdeI on the E. coli DH5α strain. The expression and confirmation of the fusion protein CPE30-GFP which was induced by IPTG in E. coli BL21 (DE3) strain and assessed by SDS-PAGE and Western blot with 6xHis Taq antibody demonstrated that there was the over expression of CPE30 GFP fusion protein in the cytoplasm, mainly in the soluble form. Finally, CPE30-GFP was purified which the purity was approximately 92.3%. In vitro protein interaction measurement using silicon nanowire field-effect transistors (SiNW FETs) showed that CPE30-GFP had a good binding affinity with its receptor Claudin-4 (R4). This result laid the groundwork for the CPE30 interaction study with the M cell in vivo.

Prokaryotic Expression of Murine Cellular Prion Protein for in Vitro Evaluation

“…Current knowledge on M cell has shown that antigen uptake occurs via surface receptors, and antigen transports through dorm structure to beneath lymphoid tissue and present to PP's immunocytes, leading to the production of specific IgA (Neutra et al, 2001, Azizi et al, 2010. Because of these characteristics, orientating vaccine antigen to M cell receptors is a promising strategy and has been carried out in many studies (Huynh et al, 2019, Shima et al, 2014. For research on this strategy, it is necessary to identify the ligands of M cell receptors and verify the interaction between them.…”

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“…Current knowledge on M cell has shown that antigen uptake occurs via surface receptors, and antigen transports through dorm structure to beneath lymphoid tissue and present to PP's immunocytes, leading to the production of specific IgA (Neutra et al, 2001, Azizi et al, 2010. Because of these characteristics, orientating vaccine antigen to M cell receptors is a promising strategy and has been carried out in many studies (Huynh et al, 2019, Shima et al, 2014. For research on this strategy, it is necessary to identify the ligands of M cell receptors and verify the interaction between them.…”