1. Construction, Cloning, and Expression of CagA Recombinant Protein of Helicobacter pylori.
- Author
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Moghaddam, Abbas Shapouri, Mansouri, Shamseddin, Neshani, Alireza, Firoozeh, Farzaneh, Matinpur, Azade, Khaledi, Azad, and Ghazalibina, Mehran
- Subjects
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BIOLOGICAL assay , *BIOTECHNOLOGY , *ELECTROPHORESIS , *ESCHERICHIA coli , *GENES , *GENETIC techniques , *HELICOBACTER pylori , *HYDROCARBONS , *IMMUNOGENETICS , *IMMUNOGLOBULINS , *POLYMERASE chain reaction , *RECOMBINANT proteins , *MICROBIAL virulence , *WESTERN immunoblotting , *BIOINFORMATICS , *SULFUR acids , *GENE expression profiling , *SEQUENCE analysis - Abstract
Background: This study aimed to assess construction and expression of CagA recombinant protein of Helicobacter pylori (H. pylori) in Escherichia coli (E. coli) BL21. Methods: Bioinformatics was used in designing the desired gene by Gene Runner. Next, the construct was subcloned to pET21b vector and this process was confirmed by Polymerase Chain Reaction (PCR), enzyme digestion and sequencing techniques. Then, it was cloned in the Escherichia coli BL21 as an expression host. Expression of protein was verified using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting technique. For purification of the protein, the Ni- NTA column was used. Protein concentration was determined by the Bicinchoninic Acid Protein Assay Kit (Parstoos). Finally, Western blotting was performed using CagA antibodies and normal human serum for determining immunogenicity feature with human antiserum. Results: According to the results of the present study, CagA construct was cloned into the pET21b vector and after confirmation and cloning in host expression, recombinant protein with the size of 38 kDa was successfully expressed and purified. The recombinant CagA protein showed immunogenicity characteristics with human antiserum. Conclusion: In conclusion, only 5'-end of recombinant protein CagA with high immunogenicity effects was successfully constructed, cloned and expressed. Also, CagA recombinant protein showed good immunogenicity activity with human antiserum. [ABSTRACT FROM AUTHOR]
- Published
- 2020