Construction And Immunization For Gonadotropin Inhibitory Hormone-C3d DNA Vaccine

To solve the non-breeding season problem of Sheep, we have tried to apply Inhibin gene vaccine to immune Sheep that be built in the early stage, though in the breeding season which can promote Follicle stimulating hormone、Estradiol hormone secretion and Follicle development, but it fails to induce estrus ovulation, Speculated that which is related to gonadotropin-releasing hormone and luteotropic hormone pulse frequency and low concentration.Gonadotropin inhibiting hormone is the only elucidated neuropeptide that plays a negative regulatory role on gonadotropin-releasing hormone and luteotropic hormone. As a result, it can inhibit animal reproduction function during non-breeding period. This provide an opportunity for inducing the Sheep estrus at anestrous season by reproductive immune principle to neutralization gonadotropin inhibiting hormone,to adjust the gonadotropin-releasing hormone and luteotropic hormone. Therefore the research has established the indirect detection method through gonadotropin inhibiting hormone antibody, built the INH-GnIH fusion gene vaccine and has studied the post-immunization sheep’s reproductive hormone expression patterns at the non-breeding season. 1.Establishment and optimization of GnIH antibody indirect ELISA methodWith purified GnIH synthetic peptide(1~28a) for the envelope antigen, successfully established detection method of indirect ELISA to detect antibody of GnIH: the linear detection range was 0.4 ug/ml, and 5% skimmed milk powder as the optimal sealing fluid, and 1:20 000 HRP-conjugated raabit anti sheep serum albumin antibody.The sample and HRP-conjugated antibody reaction time were both 60 min,and the chromogenic time wa 15 min, this method has good repeatability, which could be used detection the antibody of GnIH.With synthetic GnIH polypeptide epitope vaccine immune(immune dose:300 ug/ml, immune number:2) Sheep at different stages, found that the antibodies are rising to the peak in five weeks, suggests GnIH(1~28) has good immunogenicity, and can be used as GnIN-INH gene vaccine candidate B cell antigen epitope 2. Cloning and expression of GnIN-INH-Follistatin fusion antigen epitopeWith the artificial synthetic INH alpha(1~ 32), bovine GnIH(1 ~ 28) and Follistatin(305 ~ 314) and sheep C3 d gene fragments insert into the cloning of pMD18, then load to the prokaryotic expression vector of pET28 a. and using endonuclease BamHI and XhoI cloning technology to constructe pET28aGnIN-INH-Follistatin-3(C3d) and pET28 a INH- 3(C3d) prokaryotic expression vector. INH-3(C3d) fusion can success expression protein after IPTG induction, but GnIN-INH-Follistatin-3(C3d) failed,the induction program still need to further optimize. 3.The construction of GnIH-INH-Follistatin fusion gene vaccineWith the GnIH-INH-Follistatin fusion of pET28a-GnIH-INH-Follistatin-3(C3d) prokaryotic expression vector as the foundation, then GnIH-INH-Follistatin-3(Cd3) was transferred into eukaryotic expression vector vaccine pSEC-tag2 a to build pSEC-tag2a-GnIH-INH-Follistatin-3(Cd3) gene vaccine.With the plasmid to transfection HEK293 cells, after 24 hours, to collecting cells, then do the takara fluorescence quantitative PCR in 2x premix for gene RT-PCR test to verify, the result show that pSEC-tag2a-GnIH-INH-Follistatin-C3d3 plasmid can be stability expressed in HEK293 cells after transfection HEK293 cells. 4. The influence of GnIH-INH-Follistatin fusion gene vaccine active immune Sheep to the reproductive hormones in non-breeding seasonThis gene immune text was carried out in the the Sheep(2-3 years) at the non-breeding season(February), using enzyme-linked immunoassay to detect GnIH antibodies and hormone level in different periods. The antibody levels rised after active immune with GnIH-INH fusion gene vaccine, the peaked at 28 days(OD: 1.2073±0.64)(P<0.05).After immunization, follicle stimulating hormone and luteinizing hormone average content were higher than the control group(P<0.05). GnIH-INH fusion gene vaccine can not only promote the secretion of FSH, but can significantly improve the LH level in the non-breeding season of Sheep.In conclusion, this study established indirect ELISA detection method and successfully constructed the GnIH-INH fusion prokaryotic expression vector and gene vaccine. Follicle stimulating hormone and luteinizing hormone content were higher than the control group after active immune Sheep with GnIH-INH fusion gene vaccine in non-breeding season.GnIH-INH fusion gene vaccine can not only promote the secretion of FSH, but can significantly improve the LH level in the non-breeding season of Sheep, it lay a foundation for the multiplets immunization work in the non-breeding season.