| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 85 === In this study, a 1228bp cDNA previously isolate from a
carp ovariancDNA library and encoding a 331 residue
polypeptide homologous toCysteine protease was used as a
probe to study its gene expression inovary. In situ
hybridization showed that Cysteine protease mRNA arepresent
in oocytes and follicle cells. Transcription of Cysteine
proteasegene starts very early during oogensis. Antibody
against the recombinantCysteine protease was used to
investigate the tissue distribution andbiochemical nature
of c arp ovarian Cysteine protease.
Byimmunohistological study, the Cysteine protease was found
majorly incortical alveoli and minorly in zona pellucida of
oocytes, and also thefollicle cells surrounding developing
oocytes. During the corticalreaction of egg, the Cysteine
protease in the cortical alveoli wasreleased to the
perivitelline space. From western blot analysis, theCysteine
protease in the PBS-soluble fraction of carp ovary and two
form,26/8kDa and 30.2kDa,while those in the PBS-insoluble
fractio n are morethan two form,26.8kDa and 30.2kDa and those of
larger molecular weights. To study the gene expression
during oocyte maturation and ovulation,ovapim was injected to
mature female carp. Northern blot analysis showedthat ovarian
Cysteine protease mRNA(0.98KB) was significantly increased4hr
after ovaprim injection and decreased thereafter. In
addition, the26.8kDa Cysteine protease in the PBS-soluble
fraction was increasedgradually during maturation and
ovulation while the content of 30.2kDaform was not changed.
However, both the 26.8kDa and 30.2kDa Cysteineprotease in the
PBS -insoluble fraction were gradually increased afterovaprim
injection. They were decreased after 7hr. postinjection.
In order to purify carp ovarian Cysteine protease, TSK-DEAE 650
anionexchanged chromatography and Cystatin-affinity
Agarose columnchromatography were employed. A partially
purified Cysteine protease of30.2kDa without protease activity
were obtained. Further experiment needto be done for the
purification of the biologically active Cysteineprotease.
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