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Developing aptamer sensors for Bacterial Kidney Disease of salmonids Emily Byrd Berglund Lab Bacterial Kidney Disease • Caused by Renibacterium salmoninarum (Rs) • Common problem in fish hatcheries and farming industries • Disease symptoms: – granulomas and postules on the kidneys – distended belly – exophthalmos – dark coloration of the skin BKD: a problematic disease • No vaccine exists; antibiotic treatments have minimal affect • Transmitted horizontally and vertically – vertical transmission makes it difficult to prevent the disease • Resilient bacteria that can both live and grow in the host’s immune system • Bacteria grows slowly, making it difficult to culture and study Goal • Current detection methods include ELISA and FAT using kidney tissue • Unable to detect nonlethally at subclinical levels Can we develop a nonlethal and noninvasive method that detects the bacteria? How could infections be detected? SELEX is a method that selects for an RNA that binds tightly with the protein. Optical properties of nanoparticles may make it possible to detect bacteria in water samples. MSA • Renibacterium salmoninarum produces large quantities of an extracellular protein, p57, also known as Major Soluble Antigen or MSA • Protein is acidic and large in size (57 kDa) • Native p57 exists as a monomer • Does not have a transmembrane domain, but is secreted and then bound to the exterior of the cell • In vitro, p57 reassembles onto strains of R. salmoninarum lacking the protein Wiens and Kaattari, 1990 MSA and disease • MSA is a virulence factor of Rs; it is secreted into the extracellular space and causes agglutination of host leucocytes • Causes long term immunosuppression if present in fish eggs • Causes agglutination of salmonid spermatozoa • Restores cell surface hydrophobicity • Forms fimbrial structures and functions as an adhesin for bacterial attachment to cellular receptors – may allow intracellular invasion by the bacteria • Also acts to suppress antibody production by the host Wiens and Kaattari, 1990 MSA proteins Full Length (27-558) R1a (27-155) R2a (172-356) R2b (172-333) R2c (228-331) R3a (357-558) IPT Domain • Protein was sectioned into three regions and constructs formed of varying lengths • Regions 1 and 2 have been shown to be exposed on the cell surface when bound to bacteria (Wien and Kaattari, 1990) Cloning MSA Protein purification • Two methods for protein purification • GST bead purification: – protein bound to beads and eluted with glutathione • Anionic exchange Q column: – negatively charged protein binds the positively charged column and is eluted off at high NaCl concentrations Fraction 8 Fraction 7 Fraction 6 Fraction 5 Fraction 4 Flow through Eluted supernatant Eluted beads Bound beads Wash #1 Cell lysate Protein ladder R2c protein gel 70 kDa 27 kDa 37 kDa R2c protein SELEX • Method that isolates RNA aptamers that bind with high affinity to a protein (MSA) • A 90 bp RNA with a 40 bp randomized sequence is created using PCR and transcription 90 bp RNA aptamer N = random nucleotide constant N40 constant • RNA is run through several SELEX rounds to isolate the most effective binding species constant 1014-1015 Random Sequence RNA’s N40 constant GST-MSA Bind RNA pool to protein Bind RNA to immobilized protein RT-PCR and transcribe to amplify RNA Remove unbound RNA Elute bound RNA A potential detection scheme • Use colorimetric approach based on gold nanoparticles • Gold nanoparticles change color based on their aggregation state – red - dispersed – blue/purple – aggregated • RNA/nano complexes bound to MSA will turn blue in color Liu, J., Lu, Y. 2006. Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes. Nat. Protoc.1, 246-252. To be continued… • Finish purifying R3a and R2b protein fragments • Begin SELEX on the proteins – begin with proteins spanning region 2 since the region appears to be exposed on the cell surface – locate the best RNA aptamer for binding • Conjugate RNA aptamer with gold nanoparticles • Develop a method to detect color changes in nanoparticles Potential problems • Radioactivity: no facilities at EOU – working to find other staining methods • Full length construct doesn’t seem to be soluble; how can it be purified? • No sensor for detecting color changes in gold nanoparticles Acknowledgements Berglund Lab: – – – – Andy Berglund Julien Diegel Amy Mahady Bryan Warf, Jamie Purcell, Leslie VanOs, Rodger Voelker, Devika Gates, Paul Barber Sarah Servid Anna Cavinato, Eastern Oregon University SPUR Peter O’Day Chelsie Fish