Molecular Cloning, Prokaryotic Expression And Polyclonal Antibody Preparationof The Sheep Equatorin As Well As Its Localization In The Sperms Of Pre-And Post-Acrosome Reaction

The success of the mammalian fertilization is the premise of an animal race continues and genetic evolution.The plasma membrane adhesion and fusion of sperm and egg is the most important step to complete the fertilization. In recent years, researches on sperm-egg plasma membrane adhesion and fusion have been performed continously by many scholars, using modern molecular biology methods, and theresults demonstrated that sperm-egg fusion is a process depending on the participation of multiple molecules and multiple receptors. It is generally accepted that the initiation site of sperm-egg fusion is located at the equatorial zone of sperm membrane. An protein on the sperm equatorial zonenamed equatorin was identified by the application of monoclonal antibody MN9on the protein identification of sperm. Injection of MN9antibody in vitro can significantly inhibit sperm egg fusion, thus implies the equatorin protein Eqtn is involved in the process of sperm-egg fusion. But how Eqtn exactly participates in the acrosome reactionand eventually mediated sperm egg fusion is not yet known.This experiment uses Inner Mongolia sheep as experimental animals to clone sheep Equatorin cDNA. First we compare the published Eqtn cDNA sequence of related species in GenBank to find out the conservative region for designing the specific primers of Eqtn cDNA. The total RNA of sheep testicular tissue was extracted and it’s the cDNA was obtained by reverse transcription (RT), and then the Eqtn cDNA was cloned with the total cDNA as template. After sequencing, new primers were designed based on the reference sequence to subclone the mature peptide fragment (19aa-336aa) into the prokaryotic expression vector pGEX-4T-1to construct prokaryotic expression plasmid pGEX-Eqtn by removing the region of signal peptide. The recombinant plasmid pGEX-Eqtn was then transformed into competent E. coli BL21(DE3) cells. A large amount of GST-Eqtn fusion protein was expressed under the optimal conditions,37℃,4h, and was purified by the reductive GSH peptide affinity column. The purified GST-Eqtn fusion protein was concentrated and mixed with freund’s adjuvant to make antigen emulsion and was injected into Kunming male mice by the way of subcutaneous multi-point, and immune strengthening was performed once every10days. After immunization of35days, S180ascite tumor cells were injected. When the abdomens of immunized mice obviously summon up, polyclonal antibody in ascites and serum were collected respectively. The specificity and titer of the polyclonal-antibodies were separately determined by the Western blotting and indirect ELASA. Finally serial sections of sheep testicular tissues were prepared and intact sperm cells and acrosome-reacted sperm cells were treated with the polyclonal antibody to observe the location of Equatorin protein by immunohistochemical staining and immunocytochemical staining, respectively. Our observation showed that Eqtn proteins are mainly distributed aroud every kind of cells in the testicular. In the sperm before acrosome reaction, Eqtn mainly distributes in the acrosome, but after the acrosome reaction, the remaining Eqtn reposition to the equatorial region of the sheep sperm.