Artificially generated male-specific rabbit antibody against DBY-multi-epitope fusion protein and its immunoreactive examination

DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DBY or Ddx3y) is a candidate gene for male-specifi c antigen. The DBY gene detected in capacitated mouse sperm codes putative ATP-dependent RNA helicase. The objective was to produce anti- predicted DBY multi-epitope fusion protein antibody, which could be used to determine male specifi city of DBY. Epitope prediction is to aid the design of molecules that can mimic structure and function of a genuine epitope, is a useful tool in protein molecule design. This study predicted the DBY epitopes, prepared rabbit poloclonal antibody against DBY multi-epitope fusion protein, then investigated its immunoreactivity. The fusion protein used as the antigen consisted of three regions of DBY with greatest divergence from other family members, cloned together in-frame (with a His tag to facilitate purifi cation). The resulting antibody recognized both the DBY1-2-3 fusion protein and an endogenous DBY protein of the same size. Furthermore, DBY protein was present (Western blot) in testis, male mouse splenocytes and brain, whereas a weaker band was present in the female brain and splenocytes, and fi nally, ovary produced only a barely visible protein band. Optical density of DBY protein was higher for males versus corresponding tissues from females. Finally, positive signals of DBY1-2-3 antibody were present on only ~60% of mature murine sperm (based on immunofl uorescent staining and fl ow cytometry), in accordance with the expected proportion of Y-bearing sperm. We hypothesized that our antibodies recognized a specifi c epitope present in subpopulations of mouse sperm. Therefore, we concluded that anti-DBY1-2-3 antibody could be an alternative way of producing antibodies to DBY protein. Furthermore, this novel DBY antibody against a multi-epitope artifi cial antigen has potential for both investigating male-specifi c binding of DBY and as a new method of sex selection.