[Expression, purification of the recombinant protein fragment of the monkey epididymis expressed gene ESc-615 and preparation of polyclonal antisera to it].
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 ac id 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. Immunostaining results showed that Esc-615 was a secreted protein and could bind sperm s, which was similar with other carboxylesterases in male reproductive system; t he 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.
Hu
Zhi-Hong
ZH
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, Chin a. yonglz@sunm.shcnc.ac.cn
Li
Bin
B
Liu
Qiang
Q
Zhang
Yong-Lian
YL
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004822:Epididymis / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D000882:Haplorhini; D007106:Immune Sera / Q000276:immunology*; D008297:Male; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013094:Spermatozoa / Q000378:metabolism
D007106:Immune Sera; D011994:Recombinant Proteins