Kun Xu

Date of Award

12-2001

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

James Weber

Second Committee Member

Charles Moody

Third Committee Member

Michael Vayda

Abstract

Three series of experiments were conducted to: 1) optimize the conditions for the production of pUC19 plasmid and of biotinylated pUC19 fragments; 2) optimize the conditions for the production of protein A-streptavidin chimera (chimeric protein); and 3) detect soluble antigens [bovine serum albumin (BSA) and interleukin-6 (IL-6)] and membrane-bound antigens [insulin receptor and epidermal growth factor receptor (EGFr)] of mouse embryos by immuno-polymerase chain reaction (I-PCR). The first experimental series, which included bacterial culture, chimeric protein purification, and chimeric protein functional experiments, was performed to investigate the effects of IPTG (isopropyl-β-D-thiogalactopyranoside) induction time and temperature, bacterial culture medium, and protein purification procedure on chimeric protein yield. The yield of chimeric protein was affected by IPTG induction time, but not by induction temperature. The yield of chimeric protein from TB broth culture was five to ten times higher than from either M9 or LB medium culture. Eliminating a centrifugation step just before the biotin affinity chromatography increased the yield of chimeric protein three times more than when the centrifugation step was included. Data demonstrated that our chimeric protein was fully functional. The second series, including culturing and biotinylation experiments, was conducted to investigate the effects of culture conditions [Lennox L broth (LB broth) versus Terrific broth (TB broth) and 15 versus 28 hours] on pUC19 plasmid yield from pUC19 plasmid-transformed Escherichia coli (E. coli), and the effect of incubation conditions on the efficiency of pUC19 biotinylation. Culture medium but not culture time affected pUC19 plasmid yield. pUC19 biotinylation at 37°C for 30 min seemed to be superior compared to 15 min either on ice or at room temperature. Data demonstrated that biotinylated pUC19 fragments were hlly hnctional. The third series, including BSA, IL-6, insulin receptor, and EGFr assays, was performed using I-PCR and enzyme-linked immunosorbent assay (ELISA) assays to determine the absolute sensitivity of I-PCR technique and to compare the relative sensitivity between I-PCR and ELISA for detecting these antigens. The sensitivity limits of I-PCR were 5.8 BSA and 5.8 x l03 IL-6 molecules immobilized on the surface of a microtiter plate, which were six and seven orders of magnitude more sensitive than ELISA, respectively. By using I-PCR technique, we were able to detect insulin receptors on two mouse embryos and EGFr on only one mouse embryo at the morula stage. Data from these studies should be valuable for the future development of a non-invasive, I-PCR-based assay of individual embryo viability.

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