Studies On Extraction And SDS-PAGE Of Wapiti(Cervus Elaphus)Sperm Membrane Proteins

Mature sperms had many kinds of Sperm membrane proteins (SMPs) in its membrane. Some of them were sperm-specific, others binded to the sperm surface during the period of sperm maturation. SMPs were important symbols of the mature progress of sperm. By the development of life science, people recognized that these SMPs play an important role in the maintenance of sperm's shape and structure, the metabolism of sperms and the reproduction, especially in the fertilization. Some of the SMPs as antigen could induce the engine body generate relevant antibody, and caused fertilization failure or immunosterility or to both.With the sperm of wapiti (Cervus elaphus) as the experiment material, we screened out the optimum method of extracting the SMPs of wapiti; established the most ideal system of the extraction solution of the SMPs; determined the best condition of SDS-PAGE of the SMPs, and. analyzed some physicochemical properties of them. The main experimental results of the present work were shown as follows:1. Screened out the optimum method to extract the wapiti SMPsThe SMPs were extracted from the surface of wapiti sperms by three different methods. Base on the result of SDS-PAGE and BCA, the extraction solution with NP-40 was the optimum method of the wapiti SMPs2. Established the ideal system of the extraction solution of the wapiti SMPsThe SMPs were extracted from the surface of wapiti sperms by NP-40 extraction solution under different systems. Base on the result of SDS-PAGE and BCA, the extraction solution with 0.5%NP-40, 0.5%PMSF and 5 times volume was the most ideal system of the extraction solution of the wapiti SMPs.3. Determined the best condition of SDS-PAGE of the wapiti SMPsThe SMPs were separated by SDS-PAGE under 4 different kinds of concentration. Base on the result of SDS-PAGE, the best concentration of the SDS-PAGE for the wapiti SMPs was 12%.4. The analyze of the physicochemical property of the wapiti SMPs(1)8 kinds of SMPs in different Molecule Weight (MW) were extracted from the sperm of wapiti;(2) The concentration of the wapiti SMPs was 468.12μg/ml;(3) There were three kinds of glycoprotein, the MWs of them were 98.4 kDa, 67.6 kDa and 47.3 kDa.5. 2-DE gel electrophoresis of the SMPs of wapitiThe wapiti SMPs was separated by 2-DE technique, the effects of various sample preparation methods were studied .The satisfied result was obtained when two step method of urea-hydrochloride lysed sperm. About 823 protein spots were detected by the imaging analysis system. The molecular weight and isoelectric point (PI) of most protein spots were 10 to 100 kDa and 4.0 to 9.0 respectively.