Construction Of Sp17-Eppin Fusion Protein And Study The Effects On Immunocontraception

Background:The world's population is growing at a tremendous rate and there is an urgent requirement for a better contraception method, which is reversible, safe and highly effective, easily accessible, inexpensive and suitable for all stages of reproductive life. Contraceptive vaccines may be the best choice, which can fulfill most of the properties of an ideal contraceptive. The sperm membrane antigen(SMAg) is the key marker in the spermatogenesis and mature, has closely relation with fertilization and early development of embryo, involves sperm capacitation, sperm–egg recognition, the acrosome reaction, sperm penetration of the zona pellucida, sperm-oocyte fusion, and oosperm fission. Then the occurrence of ASA(antisperm antibody) gives rise to the disease of"immunological infertility". So sperm antigens are particularly attractive candidates for the development of a highly effective, reversible, and safe contraceptive vaccines.Sperm protein 17 (Sp17) is a testes-specific protein, present on the equatorial surface of live and acrosome-reacted spermatozoa. Sp17 is involved in the interaction of spermatozoa with oocytes after acrosome reaction, and was shown to bind the sulphated carbohydrate components of the zona pellucida to initiate fertilization. Eppin(epididymis protease inhibitor) is specifically expressed in epididymis and testis. Human ejaculated spermatozoa are coated with Eppin over both head and tail regions before and after capacitation, which is involved in coagulum formation in the ejaculation. As the spermatozoa pass through the efferent ducts of epididymis, Eppin, secreted by caput and caudal, binds to the sperm surface at some specific site. Eppin has important function for liquefaction of ejaculated semen, sperm capacitation and motility.As demonstrated in many studies, Eppin vaccine has good effect on immunocontraception in the non-human primates. Many experiments also showed that some sperm membrane proteins have the reversible contraceptive effect, but all of them can't achieve 100% solely. To construct compound contraception vaccine , based on the different mechanism of some target antigens, may solve the problem. Because of the same combine site on sperm surface, the different block section and contraceptive mechanism, Sp17-Eppin is predicted to be a better contraceptive compound vaccine with lower toxicity and higher effect.Objective:In our study, the recombinant plasmid would be constructed and purificated, which could express effectively in pichia pastoris. when hSP17 and Eppin as the target antigens. Then, the mechanism and effect of the contraceptive vaccine would be discussed , the safety and the stability would be assessed.Methods :There are three parts in our study :①The construction of pPIC9K-Sp17-Eppin yeast expression vector.Firstly, the full 1ength cDNA encoding Sp17 and Eppin was amplified respectively from a man's testicle by RT-PCR method. Then mutated the aim amino acids by PCR get recombination gene.Finally,the recombination gene was subcloned into SnaB I and Not I sites of pPIC9K vector.The recombinant expression vectors were verified by DNA sequence test.②The Pichia yeast expression,purification of Sp17-Eppin protein and the primary test of its biological activity. The recombinant vector was transformed into Pichia strain GS115 by electroporation.The transformants with multiple inserts were screened in vivo by their resistance to G418, PCR analysis, and Mut screening.A time course study of expression was performed to optimize the protein yield.Large baffled flasks were used to scale up the expression,and the culture supernatant were harvested on the time point obtained from previous study.The culture supernatant containing the recombinant protein were demineralized and dialyzed,purified by Sephadex G-50 column,and frozen in -70℃.The purified protein was confirmed by SDS-PAGE and Western blot analysis . Its biological activity was tested by immunohistochemical assay.③Study immune contraception effects of the Sp17-Eppin fusion protein. The adult male Balb/c mice were immunized with the fusion protein,to detect the specific antibody by ELISA and testosterone(T) concentrations in mice serum,to observe histopathologic damage,pregnant rates and number of offSprings, and the inhibition test of that serum to spermatozoa was performed.Results:1. The primers were designed and synthesized, based on the biological information. The amplified full length cDNA encoding Sp17 and Eppin were exactly the same sequence as Genbank data.pPIC9K-Sp17-eppin were obtained and confirmed by sequence analysis.2. The verified recombinant vector was transformed into Pichia strain GS115 by electroporation. The purified fusion protein was confimed by SDS-PAGE and Western blot analysis.Its biological activity was tested by immunohistochemical assay.3. The adult male Balb/c mice were immunized with the fusion protein,control with PBS. The specific antibody IgG was detected in experiment group, and there is no significant difference on testosterone(T) between the groups. The fusion protein can effectively inducing specific humoral antibody response and effected on mice fertilization in experiment group.The sperm amount in convoluted tubule of testis and caudal of epididymis of experiment group was obviously reduced than control grouop. The fusion proteins can interfere with spermatogenesis and sperm motility. The pregnant rates and number of offsprings in experimental group are lower than that of control.Conclusion:Sp17-Eppin fusion protein can interfere with spermatogenesis and sperm motility, whose contraceptive effect is reversible and higher than Sp17. The successful preparation of Sp17-Eppin fusion protein will be useful for male immunocontraception.