Cloning, Prokaryotic Expression And Purification, Preliminary Function Study Of Mouse Epididymial Specific Defb48、fam12b

This study focuses on the defb48 and fam12b that belong to unigene database in the NCBI, and fam12b has homologues in human. Protein analysis software predict that Defb48 domain withβ-defensin, and Fam12b belongs to RNase A superfamily. Previous studies show that these two genes transcripts could be detected in the mouse epididymis, and that eukaryotic expression vector consisted with the two genes respectively were transfected into COS-7 cells confirmed that the proteins encoded by these two genes can be secreted to the extracellular. So we speculate Defb48 and Fam12b may play a role in sperm maturation, but whether the two proteins work in sperm maturation and the role of the mechanism is still unknown.In order to clarify the function of epididymis secretory protein Defb48 and Fam12b in sperm maturation, we carry out the following research:1. The gene defb48 and fam12b were clone from mouse epididymis, we constructed prokaryotic expression vector pET28 (a)-defb48, pET28 (a)-fam12b and the aimed protein were expressed in E. coli Rosetta (DE3) .At last we gain purified fusion protein in high purity.2. We immune ten weeks male mousse with the two purified fusion protein respectively, After this process, the males were allowed to mate with normal females. The number of offspring were counted. The results showed the experimental group with Defb48 were significant in the number of offspring compared with the control group and the sperm motility also declined(data unshown); Another experimental group with Fam12b fusion protein made no significant difference with the control group.3. Western-blot and Immunofluorescence were applied to observe whether the normal sperm bind with Defb48. As the mouse anti-serum is a complex, we didn’t obtain expected result. But western-blot detection with normal male epididymal fluid showed a single band which was around 10kD much than the predicted molecular weight, and Protein analysis software predicts that Defb48 protein may have a glycosylation.4. To test whether the antiserum was available with the endogenous protein, we construct eukaryotic expression vector pcDNA3.1-defb48 and transfected into 293T cells .This try failed since 293T cell was in high mortality rate after transfection.This study explored the functions of mouse epididymis-specific Defb48 and Fam12b in sperm maturation, The results show Defb48 may influence sperm maturation. Our work paves way to vaccine preparation in mouse.