| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
We have isolated cDNA clones which are specifically expressed during androgen-dependent regrowth of rat ventral prostate by using PCR-based subtractive hybridization technique. One of the clones, AIEG322 (tentatively designated as prostaspanin), showed significant sequence homology with those of the members of tetraspanin superfamily. Among rat tissues examined, highlevels of prostaspanin mRNA expression was detected in the prostate and seminal vesicle. To investigate physiological function of prostaspanin in the prostate and to evaluate the possibility of utilization of the serum prostaspanin concentration as a marker of prostatic cancer, we set the highest priority for preparing anti-prostaspanin antibodies. Prostaspanin cDNAs were amplified from cDNAs derived from rat and human prostates, and the expression vectors for rat and human prostaspanin with a GST- and a hexahistidine-tag were prepared. The recombinant proteins were expressed in E. coli. However, the expressed proteins could not be solubilized, because of the formation of inclusion bodies. Due to the difficulties in using the recombinant proteins as antigens, we started to produce anti-oligopeptide antibodies. The use of an synthetic oligopeptide, FTQVWNTTM, corresponding to one of the extracellular domains of prostapanin (EC2), resulted in the production of high titer antiserum in rabbits. The antiserum recognized recombinant rat and human prostaspanin in Western blot analysis. Western blot analysis using rat tissue extracts and membrane fractions, however, showed that the antiserum could not react with native prostaspnin. Glycosylation at the aspargine residue in the nativ prostaspanin may explain the observation. Currently, we are trying to prepare anti-prostaspanin antibodies using a synthetic oligopeptide corresponding to different regions of the protein.
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