[Cloning, expression and identification of hpaA gene from a clinical isolate of Helicobacter pylori]

Area of Science:

• Microbiology
• Molecular Biology
• Immunology

Background:

• Helicobacter pylori infection is a major cause of gastritis and peptic ulcers.
• The adhesin HpaA plays a crucial role in H. pylori colonization and pathogenesis.
• Developing effective vaccines against H. pylori requires identifying and characterizing key antigens.

Purpose of the Study:

• To clone the Helicobacter pylori adhesin (hpaA) gene.
• To construct an expression vector for the hpaA gene.
• To evaluate the immunogenicity of the expressed HpaA fusion protein.

Main Methods:

• High-fidelity PCR amplification of the hpaA gene from a clinical H. pylori isolate.
• T-A cloning and sequencing of the amplified hpaA gene.
• Construction of the pET32a-hpaA expression vector and transformation into E. coli BL21DE3.
• Induction of HpaA fusion protein expression using IPTG.
• Western blot and immunodiffusion assays to assess protein expression and immunogenicity.

Main Results:

• The cloned hpaA gene exhibited high nucleotide (94.25-97.32%) and amino acid (95.38-98.46%) homology with reported sequences.
• The HpaA fusion protein was expressed at approximately 40% of total bacterial proteins in the E. coli system.
• The HpaA fusion protein demonstrated immunoreactivity with anti-H. pylori antibodies and induced a high-titer antibody response in rabbits.

Conclusions:

• A highly efficient expression system for the H. pylori hpaA gene was successfully established.
• The expressed HpaA fusion protein possesses satisfactory immunogenicity and immunoreactivity.
• This HpaA fusion protein can be utilized as a potential antigen in the development of H. pylori vaccines.