Journal of Okayama Medical Association
Published by Okayama Medical Association

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ヒト培養肝癌細胞の産生するHBs抗原の精製方法とその物理化学的特性について

Oda, Munehiro
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Abstract
HBs antigen was purified from the culture fluid of hepatoma huGK-14 cell line and its physico-chemical properties were studied. The purification consists of following steps: concentration of culture fluid by membrane filtration, affinity column chromatography (anti-HBs monoclonal antibody column and anti-human serum albumin antibody column), and ultracentrifugation (isopycnic centrifugation in CsCl density gradient and rate zonal centrifugation on sucrose gradient). Highly purified (purity>99%) HBs antigen was isolated with an overall yield of about 40%. The HBs antigen showed uniform spherical particles (diameter: 23.2±2.9nm) and had a specific gravity of 1.20g/cm3. The purified HBs antigen yielded, in SDS-PAGE (under reducing conditions), four protein bands with apparent molecular weights of 22,000 and 26,000 (the two major bands), and 44,000 and 47,000. The two proteins of molecular weights of 26,000 and 47,000 are likely to be glycosylated, as these were several fold reduced when the cells were cultured in the presence of Tunicamycin. Amino acid analysis, Edman degradation, carboxypeptidase digestion, and ultraviolet absorption spectrum indicated that the HBs antigen from hepatoma cells is very similar to that derived from human plasma.
Keywords
Hepatoma
Cell culture
HBs antigen
Purification
Characterization
ISSN
0030-1558
NCID
AN00032489