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Immunological and molecular characterization of the STX-10 antigen

University of British Columbia

The STX-10 antigen, recognized by the monoclonal antibody HSA-10, was found to be present in human placenta and in human sperm. This antigen was isolated from placenta and sperm extracts by immunoaffinity chromatography, it was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by western blotting to be a 75±5 kD protein. By enzyme-linked immunosorbent assay, the isolated protein was found to react specifically with rabbit anti-STX- 10 serum and with HSA- 10 antibody. The immunoreactivity of the STX- 10 protein was quantitatively determined by sandwich enzyme immunosorbent assay, with HSA-10 as the capturing and detecting antibody. By indirect immunofluorescent assay, the STX-10 antigen in sperm was found to be localized to the inner acrosome of human sperm. Rabbit anti-STX-10 sera were used to screen a Xgtl 1 human placenta eDNA library. Following isolation and purification of positive cDNA clones, the cDNA inserts were analyzed by restriction enzyme digestions, and PCR amplifications. One clone, 1 kb in size, was obtained. The cDNA insert of the 1 kb clone was sequenced and found to be mainly composed of the 3’untranslated region, including an mR.NA degradation signal. In the 3’-untranslated region, an identical sequence was found to 411 bp of a 449 bp clone isolated from the male liver HepG2 carcinoma cell line. The 1 kb cDNA clone contained a possible open reading frame of 168 amino acids in size. The full length of the open reading frame for the STX-10 gene still remains to be elucidated. In Northern blots, the 1 kb cDNA fragment was used as the probe. The mRNA derived from human carcinoma BeWo cell line, known to express the STX-10 antigen, was found to be 5.5 kb in length. In conclusion, the STX- 10 antigen which is recognized by monoclonal antibody HSA- 10, is an unique antigen that should be strongly considered for the development of an immunocontraceptive vaccine.

Thesis/Dissertation

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2009-03-02

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http://hdl.handle.net/2429/5349

Reproductive and Developmental Sciences

University of British Columbia

UBCV

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