Cloning and Expression of Vacuolating Cytotoxin A (VacA) Antigenic Protein in Nicotiana benthamiana Leaves a Potential Source of the Vaccine against Helicobacter pylori

Abstract

Background: The applications of transgenic plants in the healthcare system are immense. They offer an alternative strategy for the fabrication of antigenic determinants of medically important pathogens. Objectives: Cloning and transient expression of the vacuolating cytotoxin A (vacA) gene of Helicobacter pylori in Nicotiana benthamiana is undertaken in the present study. Methods: The vacA gene of H. pylori was amplified. The vacA and pBI121 vectors were digested with BamHI and SacI and the vacA gene was cloned in pBI121 by T4 ligation. The vacA-pBI121 construct was transformed into Escherichia coli DH5α and the transformants were confirmed by isolation and sequencing of vacA-pBI121. Further, the vacA-pBI121 was transformed into Agrobacterium tumefaciens EHA105 by electroporation. The transformants were used for agroinfection of N. benthamiana by agroinjection technique and the transgenic plant was screened for vacA gene expression by Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE). Results: The vacA gene amplification was confirmed by observing an intense DNA band in agarose electrophoresis. Sequencing of vacA gene of E. coli DH5α transformants indicated a gene size of about 2877bp which revealed 99.82% sequence similarity with online available H. pylori vacA gene sequence. The A. tumefaciens EHA105 transformants were confirmed by amplification of the vacA gene. The screening of transgenic leaves of N. benthamiana for vacA gene expression by SDS-PAGE showed VacA protein with a molecular weight of 105kDa. Conclusion: A novel transgenic plant expressing VacA protein was developed as a source of eco-friendly-based synthesis of antigenic determinants for various medical applications.