Construction Of A New Vaccine Anti-GnRH And Its Immune Efficacy Evaluation

Gonadotrophin releasing hormone (GnRH), which is a hypothalamic decapeptide hormone,could stimulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary, which in turn regulates gonadal steroidogenesis and gametogenesis.The immunogen of GnRH conjugated to carrier proteins or T-helper epitopes,coule induce animal to generate antibody against GnRH,which neutralize the endogenous GnRH making the level of bioactive GnRH to decrease,and the reproduction is inhibited.An important direction of immuncastration is the designing of new vaccines against GnRH and the screening of effective adjuvants. The study were divided into four parts as follows:1. Selection of adjuvants for vaccine against GnRH3-4weeks male rats were vaccinated with a GnRH chemical synthetic peptide(Th-4GnRH) using different adjuvants such as white oil, aluminum hydroxide Freund’s adjuvant or the adjuvant combination of CpG、sodium fluoride/CaCl2、Imiquimod and white oil or aluminum hydroxide. Then the immune efficacy was evaluated by detecting the antibody response, testosterone concentration and the testis index. The results showed that white oil is the best adjuvant with the most fast and highest antibody response,the lowest testosterone level and testis weight compared with other groups(P<0.05); the adjuvant combination of Imiquimod and aluminum hydroxide enhancmented the immune response better with higher antibody level,lower testosterone concentration and testis index than singal aluminum hydroxide, while adjuvant combination of CpQsodium fluoride/CaCl2,Imiquimod and white oil did not display the cooperativity;The testis development and spermatogenesis were markedly inhibited in tested mouse with testis seminiferous tubules epithelium atropic and layer of different spermatogenic cells is not clear,no or little sperm in the seminiferous tubules.2. Prokaryotic expression, purification and identification of GnRH vaccine molecule.A synthetical gene fragment encoding Th epitope and four repeated GnRH was inserted into pET32a(+).The positive recombinat plasmid pET-Th-4GnRH was transformed into the competent cells of E.coli BL21, and the recombinant bacterium were induced by IPTG, Then the expression of target protein Th-4GnRH was analyzed by SDS-PAGE and Western-blot. Resluts show that the target protein was obtained which was measured to be27kDa by SDS-PAGE; The fusion protein was purified by Ni-sepharose affinity chromatography,and displayed a brown strap by Western-blot.It demonstrated that Th-4GnRH protein was expressed correctly in E.coli.3.Construction and identification in vitro of GnRH eukaryotic expression plasmidThe gene fragments of Th-4GnRH were amplified and inserted into the pVAX1vector.The recombinant plasmid pVAX-Th-4GnRH was transfected into Vero cells, and the expression in vitro was tested by indirect immunofluorescence.The results showed that the Vero cells transfected by pVAXl-Th-4GnRH recombinant plasmid showed specific green fluorescence,while no specific green fluorescence in pVAXl plasmid transfected cells.It indicated that the purposes protein Th-4GnRH was expressed correctly in Vero cells,which lay the foundation for further study twords GnRH DNA vaccine.4.Research on immunogenicity of GnRH recombinant protein and its DNA vaccineThe purified recombinant protein Th-4GnRH and its inclusion body form expressed in E.coli, as well as pVAX-Th-4GnRH eukaryotic expression plasmid, immunized3-4weeks male mouse. And DNA prime-protein boost strategy was used to enhance the immune effect of DNA vaccine.The immune efficacy was evaluated by detecting the antibody response, testosterone concentration, the testis index and histological change of testis. The results show that, both the purified protein and inclusion bodies of Th-4GnRH protein induced antibody response; there is a rapid upgrade of the antibody induced by mouse immunized with DNA primer and protein boost after boosting immunization,which reach the same level with both purified proteins and inclusion bodies rapidly.The testosterone concentration and testis weight of mouse vaccinated both with inclusion bodies, purified proteins or DNA+proteins were lower than the mouse in control group; Testicular histological examination revealed that testis development and spermatogenesis were markedly inhibited in tested mouse above-mentioned three groups with testis seminiferous tubules epithelium atropic and layer of different spermatogenic cells is not clear,no or little sperm in the seminiferous tubules.