<abstract xmlns="http://www.w3.org/1999/xhtml"><p>DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (<italic>DBY</italic> or <italic>Ddx3y</italic>) is a candidate gene for male-specific antigen. The <italic>DBY</italic> 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 specificity 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 purification). 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 finally, 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 immunofluorescent staining and flow cytometry), in accordance with the expected proportion of Y-bearing sperm. We hypothesized that our antibodies recognized a specific 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 artificial antigen has potential for both investigating male-specific binding of DBY and as a new method of sex selection.</p></abstract>

DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DBY or Ddx3y) is a candidate gene for male-specific 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 specificity 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 purification). 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 finally, 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 immunofluorescent staining and flow cytometry), in accordance with the expected proportion of Y-bearing sperm. We hypothesized that our antibodies recognized a specific 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 artificial antigen has potential for both investigating male-specific binding of DBY and as a new method of sex selection.

Artificially Generated Male-Specific Rabbit Antibody Against DBY-Multi-Epitope Fusion Protein And Its Immunoreactive Examination

College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan Province 410128, PR China

Acta Veterinaria

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

ISPITIVANJE IMUNOREAKTIVNOSTI VEŠTAČKI DOBIJENOG ANTITELA SPECIFIČNOG ZA MUŽJAKE KUNIĆA NA DBY MULTIEPITOPNI FUZIONI PROTEIN

DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DBY or Ddx3y) is a candidate gene for male-specific antigen. The DBY gene detected in capacitated mouse...

Artificially Generated Male-Specific Rabbit Antibody Against DBY-Multi-Epitope Fusion Protein And Its Immunoreactive Examination

Published Online: Sep 30, 2015

Page range: 328 - 338

Received: Dec 31, 2014

Accepted: May 04, 2015

DOI: https://doi.org/10.1515/acve-2015-0027

Keywords
DBY, antibody, spermatozoa, gender selection

© Naidong Wang et al.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Artificially Generated Male-Specific Rabbit Antibody Against DBY-Multi-Epitope Fusion Protein And Its Immunoreactive Examination

Published Online: Sep 30, 2015

Page range: 328 - 338

Received: Dec 31, 2014

Accepted: May 04, 2015

DOI: https://doi.org/10.1515/acve-2015-0027

KeywordsDBY, antibody, spermatozoa, gender selection

© Naidong Wang et al.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Keywords
DBY, antibody, spermatozoa, gender selection