Screening and obataining of aptamers for the blood group antigen-binding adhesin (BabA) to block Helicobacter pylori (H.pylori) colonization in the stomach of mice.
- Author:
Yuan YUAN
1
;
Weipeng LI
1
;
Xiaojing ZHOU
1
;
Weili SUN
1
;
Xiaolei TANG
2
,
3
Author Information
1. Department of Nuclear Medicine, First Affiliated Hospital of Bengbu Medical College, Bengbu 233030, China.
2. Translational Medicine Centre, Second Affiliated Hospital of Wannan Medical College, Wuhu 241000
3. Clinical Pathogen Detection Engineering Center of Wuhu, Wuhu 241000, China. *Corresponding author, E-mail: 278471655@qq.com.
- Publication Type:Journal Article
- MeSH:
Animals;
Mice;
Helicobacter pylori/genetics*;
Interleukin-4;
Interleukin-6;
Interleukin-8;
Tumor Necrosis Factor-alpha;
Stomach;
Oligonucleotides;
Adhesins, Bacterial/genetics*;
Blood Group Antigens
- From:
Chinese Journal of Cellular and Molecular Immunology
2023;39(9):793-800
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the aptamer specific binding blood group antigen-binding adhesin (BabA) of Helicobacter pylori (H.pylori) for blocking of H.pylori adhering host cell. Methods H.pylori strain was cultured and its genome was extracted as templates to amplify the BabA gene by PCR with designed primers. The BabA gene obtained was cloned and constructed into prokaryotic expression plasmid, which was induced by isopropyl beta-D-galactoside (IPTG) and purified as target. The single stranded DNA (ssDNA) aptamers that specifically bind to BabA were screened by SELEX. Enzyme-linked oligonucleotide assay (ELONA) was used to detect and evaluate the characteristics of candidate aptamers. The blocking effect of ssDNA aptamers on H.pylori adhesion was subsequently verified by flow cytometry and colony counting at the cell level in vitro and in mouse model of infection, respectively. Meanwhile, the levels of cytokines, interleukin 6 (IL-6), IL-8, tumor necrosis factor α (TNF-α), IL-10 and IL-4 in the homogenate of mouse gastric mucosa cells were detected by ELISA. Results The genome of H.pylori ATCC 43504 strains was extracted and the recombinant plasmid pET32a-BabA was constructed. After induction and purification, the relative molecular mass (Mr) of the recombinant BabA protein was about 39 000. The amino acid sequence of recombinent protein was consistent with BabA protein by peptide mass fingerprint (PMF). Five candidate aptamers were selected to bind to the above recombinent BabA protein by SELEX. The aptamers A10, A30 and A42 identified the same site, while A3, A16 and the above three aptamers identified different sites respectively. The aptamer significantly blocked the adhesion of H.pylori in vitro. Animal model experiments showed that the aptamers can block the colonization of H.pylori in gastric mucosa by intragastric injection and reduce the inflammatory response. The levels of IL-4, IL-6, IL-8 and TNF-α in gastric mucosal homogenates in the model group with aptamer treatment were lower than that of model group without treatment. Conclusion Aptamers can reduce the colonization of H.pylori in gastric mucosa via binding BabA to block the adhesion between H.pylori and gastric mucosal epithelial cells.
