Production of polyclonal antibodies to late erythroblast-3 (LEB-3), a protein expressed during erythropoiesis and spermatogenesis
Kamath, Rashmi M.
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Previous studies in the lab identified a number of partial messenger RNAs (mRNAs) that are differentially expressed at the later as opposed to early stages of the production of red blood cells (terminal erythroid differentiation). Partial nucleic acid sequences obtained for one of these clones, called Late Erythroblast 3 (LEB-3) were compared to the mouse genome sequence database and were found to match a novel gene with no known function. LEB-3 mRNA was also found to be strongly expressed in the testis and was localized to a region of the seminiferous tubule where secondary spermatocytes and early spermatids are found. This suggests a role for LEB-3 in spermatogenesis as well as terminal erythroid differentiation. A first step in understanding the function of LEB-3 would be to localize it within erythroblasts using immunofluorescence or western blot. The purpose of this study was to make recombinant LEB-3 protein for use in the production of antibodies to LEB-3. A previously prepared LEB-3 cDNA was ligated into the expression vector pPROEX-HT and used to transform E. coli. Recombinants resulting from the transformation reaction were screened by plasmid miniprep isolation and restriction digestion with EcoRI and BamHI. A plasmid isolated from one of the recombinants that gave the correct fragment sizes after restriction digestion was sequenced and found to contain the LEB-3 cDNA in the correct orientation for expressing a recombinant fusion peptide. No protein of the correct size or in sufficient quantity for isolation was expressed in E. coli after induction of the recombinant peptide, though bands of molecular weights different than expected were expressed. A crude lysate obtained from E. coli that were induced to express the recombinant LEB-3 peptide was used to immunize two New Zealand rabbits. Preimmune sera collected from the rabbits and absorbed with an acetone powder prepared from E. coli not expressing recombinant LEB-3 peptide showed no reaction with the crude lysate used for immunization. Sera collected after the first boost of immunogen and after the final bleed of the rabbits gave varying results on western blots and are still being evaluated to determine if any specific antibody was made. It was hoped that the determination of the cellular localization of LEB-3 would aid in understanding the role played by this protein in normal erythroid terminal differentiation and spermiogenesis, but further studies will be required to answer these questions.