Abstract
Background: Cryptosporidium is a protozoan parasite that has medical and veterinary importance, and causes diarrhea and vomiting in a vast range of vertebrates. Some surface antigens, such as gp40/15, play important roles in adhesion and invasion of the parasite to host cells and consequently stimulate immune responses. Cloning and expression of the gp40/15 gene to provide recombinant proteins of the parasite antigens is valuable. Objectives: This study aimed at cloning and expression of the gp40/15 gene in Escherichia coli. Methods: In this experimental study, the gp40/15 gene sequence was extracted from GenBank (No. AF155624) and cloned in the PET28a+ plasmid. Colony polymerase chain reaction (PCR) and enzyme digestion methods by restricting enzymes, including BamHI and XhoI, were applied for verification. The recombinant plasmid was transferred to the Escherichia coli and the protein expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and enzyme linked immunosorbent assay (ELISA) techniques using serum containing antibodies against Cryptosporidium parvum. The protein was further purified by column chromatography. Results: The gp40/15 gene was successfully cloned in the PET28a+ plasmid. The colony PCR and enzymatic digestion methods showed a 921-bp fragment. Furthermore, expression of pEgp40/15 in Escherichia coli demonstrated a 43-kDa band. Antibody titrations in sera of test groups were significantly (P < 0.0001) higher than that of the control groups. Furthermore, antibody titration in test groups with four injections was significantly higher than that of the three injections (P < 0.05). Conclusions: The gp40/15 gene, which was cloned in the PET28a+, was successfully expressed and produced in E. coli. Therefore, this protein can be used in future studies to develop recombinant vaccines and diagnostic kits.
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