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.
Background: Plant biotechnology revolutionized the branch of plant sciences contributing to improving crop production. Helicobacter pylori was predominantly seen among all the sectors of the human population, closely stuck to stomach ulcers and cancer. Even though traditional vaccines are effective in preventing H. pylori, edible and non-edible parts of special transgenic plants were also being generated to express its antigens which could elicit an immune response after consuming. The induce by contact with epithelium (iceA1) protein of H. pylori is associated with virulence and stated as predisposing factor to stomach cancer. Objectives: This study intends to study the expression levels of iceA1 gene in the brinjal plant. Materials and Methods:The present study, magnified the iceA1 gene from H. pylori strain 26695 and transformed it into callus of brinjal (from leaf explants) through Agrobacterium tumefacians (EHA105). pBI121 vector was used to construct the plant expression vector, and the transgenic plants generated were further validated through gene amplification and protein expression studies. Results: Out of the 46 plants obtained from this study, five of them were found to be positive for the iceA1 expression as seen on validation assays like gene amplification and protein expression studies. Conclusion: The study could beneficially produce a vaccine candidate against H. pylori from transgenic brinjal. In Addition, it could provide, valid scientific data which can further be used by researchers.
Highlights A platform for rapid expression of genes through agro-infiltration is a suitable alternative to the laborious process of generating stable transgenic lines for producing recombinant immunogenic antigens in plants. This study has successfully developed and demonstrated a reproducible transformation protocol for rapid expression of Helicobacter pylori recombinant CagA, VacA and NapA antigens in Nicotiana benthamiana . This is the first study of recombinant expression CagA, VacA and NapA gene of Helicobacter pylori in Nicotiana benthamiana via syringe-assisted A grobacterium infiltration. A syringe infiltration of a four to five weeks old Nicotiana benthamiana plant with Agrobacterium tumefaciens subtype EHA105 was optimal to produce the maximum mRNA levels of CagA, VacA and NapA mRNA in leaf at the third-day post-Agro-infiltration.
Background:The advancement of plant biotechnology improved crop production by revolutionizing plant science. Humans were commonly infected by Helicobacter pylori, and it was closely linked to stomach ulcers and cancer. In addition to traditional vaccines for H. pylori, transgenic plants have also been produced to produce its antigens as well as edible and non-edible parts that can produce an immune response after consumption. The protein present in H. pylori associated with virulence (iceA1) is believed to predispose to stomach cancer. Objectives: The current study was aimed to evaluate the iceA1 gene expression within brinjal plant to produce novel transgenic lines LC420461-B7, -B10, -B15, -B22 and -B27. Materials and Methods: In the present study, amplified iceA1 gene from H. pylori strain 26695 was transformed into callus of brinjal (from leaf explants) through Agrobacterium tumefacians (EHA105). pBI121 vector was used in constructing the plant expression vector, and the transgenics generated were further evaluated by quantitative Real-Time PCR and western blot analysis. Results: Out of the 46 plants obtained five of them were found to be positive for the iceA1 expression. Both real time and western blot confirmed of the presence of expressed gene iceA1 within the plant sample leaves. By studying transgenic brinjal, the study may result in an H. pylori vaccine candidate. As well, the data can be used by researchers to get valid scientific information.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.