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      • 합성펩타이드를 이용한 영양배엽세포-특이 가토 다클론 항혈청의 제작

        이희섭,오재민,김정중,문형배,김원신,이황희 원광대학교 생명공학연구소 1995 생명공학연구소보 Vol.3 No.1

        Within the last few years, a different approach to generating protein-reactive antibodies has been developed that has several advantages over conventional immunization. This involves synthesizing short peptide sequences, coupling them to immunogenic carrier molecules, and immunizing animals with the conjugates. 3βHSD(3β-hydroxy-5-ene steroid dehydrogenase; EC 1.1.1.145) is the enzyme of the plasma membrane of human trophoblast and it's cDNA sequence was identified by Nickon et al(Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3β-hydroxy-5-ene steroid dehydrogenase. J Reprod Fert 1991;149;156). For the production of trophoblast-specific antibody, we synthesized three oligopeptides that are epitope sites chosen from cDNA sequence of 3βHSD. Oligopetides were coupled with KLH(keyhole limpet hemocyanin) under 25% glutaraldehyde. The trophoblast-specific rabbit polyclonal antisera was produced by conventional methods. This antisera reacts with a 43kDa protein in human placental lysate by Western blotting analysis and The syncytiotrophoblasts and cytotrophoblasts from villi are stained positively with this antisera by immunohistochemistry. Villous trophoblasts were cultured in methionine-free media for 1 hour and [^(35)S]-Methionine for 24 hours. Media and cell lysate were immunoprecipitated with this antisera and 12% SDS-PAGE was performed. In fluorography, bend was not noted in media and 43kDa band was noted in medis and 43kDa band was noted in lysate. It was concluded that anti-3βHSD antibody produced by synthetic peptide was specific to trophoblasts and 3βHSD was membrane-bound protein of trophoblasts.

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