Date of Award

Spring 1993

Document Type

Thesis

Department

Life & Environmental Sciences

First Advisor

John Addis

Second Advisor

James Murphy

Third Advisor

Marilyn Schendel

Abstract

Acquired pure red cell aplasia (PRCA) is an autoimmune disease characterized by poor red blood cell formation. In no instance has the molecular target of the autoimmunity been identified. In one case, a unique antibody, binding to a membrane protein of 130 kDa (P130) has been shown on immunoblots of mouse erythroleukemia (MEL) cells and other erythroid progenitor cells. Because P130 may be important in red cell development, the cloning and sequencing of its cDNA is of interest.

Initially, I reproduced the detection of this protein with patient antisera using an indirect immunoblotting technique and enhanced chemiluminescence (ECL) detection. Experimenting with a variety of conditions, I found that pl30 could best be visualized on autoradiograms using 1:50 dilution of the patient’s plasma to blocking buffer and by performing three ten-minute washings at 30°C between the additions of each subsequent antibody. I began the molecular cloning of the pl30 cDNA by plating on a Clontech MEL cDNA expression library (using the phage lambda gtll expression vector) on E. coli strain Y1090. Nitrocellulose lifts of the plates were made, and these were screened with the patient’s antibody using the same conditions described above. Several positive plaques were identified, potentially indicating the desired fusion protein. These plaques remain to be screened repeatedly to obtain the purified protein.

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