Cloning Of DDX3Y Gene And Preparation Of DDX3Y Polyclonal Antibody Of Dairy Goat

DEAD-box helicase 3 Y-linked(DDX3Y)belongs to ATP-dependent RNA helicase.DDX3Y is involved in RNA metabolism,ribosome synthesis,transcriptional regulation and cell cycle regulation.DDX3Y is located on Y chromosome and it is a specific gene of male,which play an important regulatory role in spermatogenesis.However,the studies of DDX3Y mainly reported in human and mouse,there is few literatures reported in goat.Antibodies are important tool to study gene function.Therefore,obtaining specific DDX3Y antibody of dairy goat is essential to investigate its functional mechanism in dairy goat spermatogenesis.The whole sequence of DDX3Y CDS region of dairy goat was obtained by cloning in this study,then the protein fragments which are suitable for antigens were selected by bioinformatics analysis.The expression of DDX3Y truncated antigen protein of dairy goat was induced by prokaryotic system.The 3-month-old female New Zealand White rabbits were immunized with the antigen purified by Ni-NTA Resin,the highly titer dairy goat specific DDX3Y protein polyclonal antibody was prepared,which provide a basis material for studying the function of DDX3Y in spermatogenesis of dairy goat.The main results of this study were showed as following:1. The whole CDS sequence of DDX3Y was obtained by PCR and sequencing.The full length of DDX3Y CDS region is 1 983 bp,which codes for 660 amino acids.Bioinformatics analysis found that the 1-120 amino acids in N-terminal and the 570-660 amino acids in C-terminal of DDX3Y protein are suitable for using as antigens for prokaryotic expression.The gene sequences of these two antigen fragments were obtained by PCR,which were 360bp(1-360 bp)at the 5’end of DDX3Y CDS region and 273 bp(1 710-1 983 bp)at the 3’end of DDX3Y CDS region.The gene sequence of DDX3Y 633 bp which codes for truncated antigen protein was obtained by overlap PCR.2. The prokaryotic expression vector of p ET32a(+)-DDX3Y was constructed,the DDX3Y truncated antigen protein with molecular weight of 23 ku was expressed.DDX3Y truncated antigen protein was affinity purified with Ni-NTA Resin,and the purification result was well by SDS-PAGE detection.The mass concentration of protein was≥2.5 mg/m L.3. Dairy goat specificity DDX3Y protein polyclonal antibody was prepared by immunizing New Zealand White rabbits with the DDX3Y truncated antigen protein.The titer of antibody was as high as 1:10~5showed by I-ELISA assay.The result from western blot showed that DDX3Y polyclonal antibody is high specificity,which can be used as a molecular tool to study the function of goat DDX3Y gene.Western blot analysis the proteins of mature sperm and spermatogonial stem cell of dairy goat revealed that DDX3Y protein was not expressed in mature sperm of dairy goat,but was obviously expressed in spermatogonial stem cell.In summary,the DDX3Y truncated antigen protein of dairy goat was produced and purified successfully in this study.Moreover,the highly titer dairy goat specific polyclonal antibody against DDX3Y protein was prepared,to provide important antibody material to further study the regulatory mechanism of DDX3Y in dairy goat spermatogenesis.