Background:
The human hepatitis B virus(HBV) is an enveloped DNA virus which causes acute and chronic infection of the liver in humans. The HBV associated subpopulations are consisted of virions, spherical and filamentous subviral lipoprotein particles
referied
to as hepatitis B surface antigen(HBsAg). The purpose of this study is to clarify the HBV particle by ultracentrifugation and to investigate the changes of SDSPAGE patterns according to the density of each HBV-fractions.
Methods:
For HBV sources, 500mL plasma, which was positive for HBsAg by radioimmunoassay(RIA), was separated by plasma exchange from patient diagnosed axs systemic lupus erythematosus(SLE). Virus particles were primarily pelleted by polyethylene
glycol(PEG)
precipitation method. The precifpitates were then ultracentrifuged by cesium chloride(CsCl) and sucrose density gradient method. Each fractions layered by centrifugation was measured for density and analyzed by sodium dodecyl
sulfate-polyacrylamide
gel
electrophoresis(SDS-PAGE). SDS-PAGE added 10% urea(urea-SDS-PAGE) was also performed to investigate low molecular weight viral protein. For the evaluation of HBV envelope protein components, glutamic acid specific protease(Sigma, miles, USA) was
added
to ultracentrifuged fractions.
Results:
Two polypeptide bands were separated by SDS-PAGE from the fraction with CsCl density 1.011 g/mL and their molecular weights were estimated as 24kd and 27kd. Whereas the fraction with CsCl density 1.108 g/mL revealed 4 polypeptides bands
containing
two additional 42kd and 55kd bands. Urea-DSD-PAGE showed more distinct separating bands in polypeptides with low molecular weight. After digestion with glutamic acid specific protease, the original 55kd and 66kd d bands were not observed, while
new
bands, which were 11kd, 24kd, 27kd, 33kd, and 36kd, were additionally detected.
Conclusions:
Different SDS-PAGE patterns according to fractionated density suggest that various subpopulations of HBV particles can be clarified by ultracentrifugation. Disappeared 55kd and 66kd polypeptide bands after reaction with glutamic acid specific
protease are considered to be oligomers of HBV polypeptides.
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