Discipline: Biological Sciences
Subcategory: Biomedical Engineering
Shaiyenne Miller - Alabama State University
Co-Author(s): Carrie Sanders, Timothy Egbo, and Alain B. Waffo, Alabama State University, Montgomery, AL G. W. Nchinda, The Chantal Biya International Reference Center for Research, Yaoundẻ-Messa, Cameroon
Background: Malaria is an infectious disease caused by the Plasmodium parasite, and is transmitted through the bite of female mosquitoes. An antigen from Plasmodium falciparum known as the UBO5 region has been found to elicit antigen specific antibodies in semi-immune adults. Antibodies to UBO5 play a role in limiting the invasion of the parasites into the red blood cells, indicating the UBO5 region can be a useful biomarker. We have developed an original, non-traditional phage display technology based on RNA coliphage Qβ with key feature: quasispecies. Hypothesis: We will be constructing hybrid phages that expose the UBO5 gene on the surface, in hopes that these hybrids will stimulate an immune response leading to the synthesis of a vaccination and/or a tool for detection or determining the efficacy of possible vaccine candidates. Methods: Sequence specific primers were used to amplify the UBO5 region. The insert was digested with unique restriction enzymes (REs) and cloned into our vector plasmid producing recombinant pQβUBO5. The recombinant pQβUBO5 was used to transform E. coli HB101 and produce phage hybrids that expose the UBO5 insert. Results: PCR results show successful amplification of UBO5 fragment. Digestion using Res, AflII and NsiI, cleaved our UBO5 fragment and vector plasmid, separately, preparing for ligation of the two. Transformation yielded clones, and when digested, showed expected band sizes. Conclusions/Future Research: With sequencing proving successful construction of recombinant plasmids, we expect to have synthesized hybrid phages with UBO5 exposed. Western Blot and ELISA assays will be conducted to determine the efficiency of the QβUBO5 in antigen-antibody studies. In addition, antibodies from patients infected with Malaria will be used to determine the antibody titer and binding specificity of QβUBO5 hybrid. This could ultimately lead to point of care system that will have the ability to detect the early stages of malaria, which is a key aspect of successful treatment. Furthermore with this phage displaying technology, we would like to explore the characterization and randomization of the hybrids to select the best candidate for in vivo studies.
Funder Acknowledgement(s): MSEIP Program of the Center of NanoBiotechnology Research at Alabama State University
Faculty Advisor: Alain B. Waffo, firstname.lastname@example.org
Role: I preformed all physical laboratory experiments associated with this project such as PCR, Electrophoresis, Digestion, Ligation, Transformation, Purification, Gel Extraction, Making Competent cells, Plating cells, Making media etc., as well as any writing on this project. The only aspects I was not participating in was sequencing of the clones and any testing done with animals.