Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The first aim was to study the expression of individual IFNW, IFNB, and IFNX genes. In order to accomplish this goal, punch biopsies from the nose and blood samples were collected from nineteen calves immediately prior to vaccination and approximately 26 hours post-vaccination. The buffy coat was isolated from the blood samples after centrifugation. The RNA was isolated from the nose biopsies and buffy coats by following the protocol for the TriReagent kit (Sigma, St. Louis, MO, USA) and genomic DNA was removed through the Turbo DNase-free kit (Ambion, Austin, TX, USA). RNA was converted into cDNA through incubation at 50 C for 60 minutes in a mixture of 5 mM MgCl2, RT Buffer (1X), 0.5 mM DNTP mixture, 10 pM oligo dT, 10 mM DTT, Rnase Inhibitor, DEPC treated water, and Superscript III (Invitrogen, Carlsbad, CA, USA), the reverse transcriptase. Semi-quantitative PCR on paired pre-vaccination and post-vaccination samples from three calves verified dimorphic expression patterns and may help focus later expression analysis on specific IFN genes during quantitative RT-PCR. The DNase-free RNA samples were submitted to the University of Missouri DNA Core for high-throughput sequencing. At the DNA core's request, additional sample preparation to improve the high-throughput sequencing analysis is currently underway. The second aim of the study was to study differences in induced gene expression after treatment of cells lines with purified interferon-chi (IFNX). The coding regions of the two bovine IFNX genes were first subcloned into a GST-fusion bacterial expression vector (pGEX-4T-1). The fusion proteins were expressed in E. coli BL21 (DES) cells (Novagen) after induction through IPTG. After the production protocol was optimized, the bacteria were broken by means of a French press, and the soluble protein run through a glutathione-Sepharose column (Pierce) to bind GST-IFN fusion proteins. After elution, the fusion protein was be cleaved by thrombin and the mixture. Several methods to remove the GST were attempted including passing the cleaved protein over a glutiothione-sepharose column. At this time, an appropriate means to separate the GST fusion tag from the IFNX is still being determined. The final aim of the study was to study protein expression of IFNX through immunohistochemistry The IFNX-GST fusion protein from the longer IFNX variant was produced and purified as described in aim 2. A stable emulsion of fusion protein and Freud's adjuvant was injected subcutaneously on a rabbit. The rabbit received a second and third boost of antigen approximately every two weeks. Pre-immunization and post-immunization blood was collected with a 19 gauge needle through the central ear artery. The antiserum was removed from after allowing the blood to clot and clarify through centrifugation. The antiserum had high affinity for IFNX on Western blots, but also cross-reacted with other E.coli proteins from the same source that initially produced the recombinant protein. Therefore, a sepharose matrix was biotin-avidin crosslinked to E. coli cell lysate. The antiserum was passed over the crosslinked matrix twice to remove non-specific antibodies. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This work will improve animal health and, thereby, production efficiency in cattle and related ruminant species by utilizing data acquired from our annotation of the Type I IFN (interferon) locus as part of the International Bovine Genome Project. The preliminary results from semi-quantitative RT-PCR in the gene expression study from pre and post-vaccinated calves have already indentified some Type I IFN that contribute to the innate immune system as a first response to viral infection and have been used to prevent and treat viral diseases in human and veterinary medicine. The use of high-throughput sequencing of these samples will greatly enhance our understanding in this area. Since this was only the first year of a two year project, the results from this work, particularly in regard to the second and third aim, have not all been realized.
Publications
- Evolutionary Pressure On Multigene Type I Interferon Subfamilies Angela M Walker and R. Michael Roberts. 2010. Plant and Animal Genome Meeting January 9-13, 2010, San Diego CA; Abstract P230.
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