Date of Graduation

12-2011

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor/Mentor

Yanbin Li

Committee Member

Gisela F. Erf

Second Committee Member

Young Min Kwon

Keywords

Biological sciences, Aptamer, Avian influenza virus, Selex

Abstract

Over $10 billion losses in the poultry industry were caused by avian influenza (AI) so far. Rapid and specific detection of avian influenza virus is urgently needed with the concerns over the outbreaks of highly pathogenic H5N1 influenza virus and cases of animal and human infection. Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule with good affinity. They show better thermal stability than antibodies. The goal of this research was to select DNA-aptamers as the specific recognition element of AI H5N1virus to be used in detection assays specific for field application. In this study, Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to select DNA aptamers targeted to hemagglutinin (HA) and neuraminidase (NA) proteins of AI H5N1 virus. In the first four cycles of selection, aptamers were selected by incubating HA proteins with a DNA library starting from 1014 molecules randomized at central 74 nt and subsequent nitrocellulose filtration. Then aptamers were eluted from filters and amplified by PCR. Single stranded DNA aptamers were derived from these double stranded DNAs by &lambda digest and were used as input for the next selecting cycle. In the following 9 cycles of selection, H5N1 virus was incubated as a substitute of NA proteins with aptamers pool in SELEX process. After 13 cycles of isolation, 115 bp DNA-aptamers were screened out and three apatmer sequences were obtained after cloning. Results of Dot ELISA and Dot Blot showed that these DNA-aptamers have stronger binding specificity and affinity to AI H5N1 subtype compared with their binding to H5N2, H5N3, H5N9, H2N2, H7N2 and H9N2. SPR test detected binding affinity of aptamer O16 to HA protein KD is 4.65×10-9 M and a linear equation between y (SPR signal) in RU and x (virus titer) in HAU was described as: y=208.39x +2.2347 (R2=0.99). But SPR results showed aptamers had weak cross-reaction with H5N2. Theses selected aptamers could be applied to detection of AI H5N1 virus in the future.

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