Prokaryotic expression, purification and targeting property identification of fusion peptide SAM-GFP targeting M cells
10.13200/j.cnki.cjb.004520
- VernacularTitle:M细胞靶向融合肽SAM-GFP的原核表达、纯化及靶向性质鉴定
- Author:
WU Jing
- Publication Type:Journal Article
- Keywords:
Green fluorescent protein(GFP);
M cell-targeted peptide;
Prokaryotic expression;
Fusion protein
- From:
Chinese Journal of Biologicals
2025;38(07):796-801
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express M cell-targeted fusion peptide SAM-GFP in prokaryotic cells, purify it and identify its targeting properties, so as to lay a foundation for the research of M cell-targeted oral vaccine against Helicobacter pylori.Methods The GFP gene was synthesized by PCR-based accurate synthesis(PAS) and cloned into the vector pCzn1-SAM to construct the recombinant plasmid pCzn1-SAM-GFP. The recombinant plasmid was transformed into competent E.coli Arctic Express, induced by IPTG, and then purified by Ni-NTA affinity chromatography. The obtained fusion protein SAM-GFP was identified by 12% SDS-PAGE, Western blot and fluorescence microscopy. The M-cell targeting properties of SAM-GFP fusion protein was verified by immunofluorescence staining using mouse ileal loop model.Results The recombinant plasmid pCzn1-SAM-GFP was constructed correctly as identified by double digestion and sequencing. The fusion protein SAM-GFP had a relative molecular mass of about 54 900, and was mainly expressed in soluble form with a purity of 94. 5% after purification. It showed specific binding to anti-6 × His/GFP monoclonal antibody, and green fluorescence was observed under fluorescence microscope. SAM-GFP could be specifically uptaken by small intestinal M cells. Conclusion The fusion protein SAMGFP with M-cell targeted function was successfully expressed and purified, which lays a foundation for the subsequent research and development of oral vaccine against Helicobacter pylori.
