Expression, Purification of the Recombinant Protein Fragment of the Monkey Epididymis Expressed Gene ESc-615 and Preparation of Polyclonal Antisera to It

( State Key Laboratory
of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai
200031, China )

Abstract

ESc-615 is a gene which was specifically expressed in
epididymis, newly discovered in our laboratory. To elucidate the function of ESc-6
15 in sperm maturation and fertilization at the protein level, its C-terminal 310 amino acid residues fragment was expressed in E.coli and used to immunize rabbits to obtain polyclonal antiserum with titer of 100 000. The antisera could detect th
e antigen as low as 3 ng. A 63 kD protein in rat epididymis protein extracts
could be detected in Western blot by using this particular antiserum, in accordance
with the size of Esc-615 homologue polypeptide deduced from its cDNA. Immunost
aining results showed that Esc-615 was a secreted protein and could bind sperms,
which was similar with other carboxylesterases in male reproductive system; the
positive staining was eliminated after antigen absorption. All these results showed that this antiserum was not only effective but also highly specific,
providing a useful tool in further functional studies of Esc-615.