| In late 1960s,Hormone immuno-nutralization techniques emerged on the foundation of immunology,biochemistry and endocrinology.Hormone immuno-nutralization techniques offered a new regulation method in animal reproduction.Recently,the studies show that the inhibin play an important role in the animal reproduction,and have a large potentiality to promote the animal fertility.However,recently,the inhibin vaccine prepared in previous studies appear to have a number of practical drawbacks,including incomplete efficacy.In order to find immunogen that has good immune effect,INHαgene of Xinjiang fine-wool sheep was cloned and expressed in E.coli BL21(DE3) strain.The INHαsubunit recombination fusion protein contained two epitope.Then,we undertaken to determine the effects of active immunization of SD rats with the INHαsubunit recombination fusion protein on the ovarian function and endocrinology of the estrous cycle,with the aim of the present study is to provide data for future inhibin immunization studies.Firstly,we have investigated the effects of inhibinαsubunit on antibody titers and hormone profile.Ten adult female rats were randomly assigned to two groups of five animals each.The first group was immunized with 1.0mg inhibinαsubunit recombinant expression protein using CFA as the adjuvant and received booster immunization on the days 14d and 28d later,using FA as the adjuvant.The other group was injected with PBS and served as untreated control.Blood samples were collected on day of the first inoculation(taken as day 0) and on day 14,28,and 38,respectively,thereafter,blood samples were collected every 6h for 4 days(from day 34 to 37) for determination of FSH, LH,and progesterone level in blood.Blood antibody titer against inhibin on day 0,14,28, 38 was determined by an enzyme linked immunosorbent assay(ELISA).Plasma concentrations of FSH,LH,progesterone were measured by radio immunoassay(RIA). The results indicated that:After the immunization,the antibody titers(P/N) increased steadily in inhibin-immunized rats,the antibody titers in immunized animals reached highest level(P/N=6.62±0.66) on day 28 after immunization,and were significantly higher than that on day 14(P/N=2.27±0.29)(P＜0.05).Circulating FSH levels showed an impulse pattern throughout the estrous cycle.A peak of serum FSH was observed at 24.00 h on the day of pro-oestrus.The peak of serum FSH became significant higer(28.25±0.83 mIU/mL) than that in the control(12.47±2.71 mIU/mL)(P＜0.05).The average level of FSH during the estrous cycle was 13.83±0.79 mIU/mL,and was significant higer than that in the control(5.36±0.57 mIU/mL).On the other hand,plasma LH at the oestrus in the immunized group was similar to that in the control.The peak of P concentrations in serum during oestrus cycle(51.34±9.30 ng/mL) was significantly higer than that in the control (30.86±0.92 ng/mL)(P＜0.05).Secondly,with the aim of studing the dose effect of INHαsubunit recombination fusion protein immunization,twenty five adult female rats were randomly assigned to one of five treatments(1-5)as follows:0.1mg(group1,n=5);0.5mg(group2,n=5);1.0mg (group3,n=5);2.0mg(group4,n=5);0mg(control,n=5) inhibinoαsubunit recombinant expression protein using CFA as the adjuvant,followed by two booster immunization with FA on days 14d,28d of the experiment.Blood samples were collected on day of the first inoculation(taken as day 0) and on day 14,28,and 38,thereafter,blood samples were collected every 6h for 4 days(from day 34 to 37) for determination of FSH,LH,and progesterone level in blood.Blood antibody titer against inhibin on day 0,14,28,38 was determined by an enzyme linked immunosorbent assay(ELISA).Plasma concentrations of FSH,LH,progesterone were measured by radio immunoassay(RIA).The results indicated that there was significant effect of dose of INHαsubunit recombination fusion protein on antibody titers.Antibody titers in all immunized animals reached their highest level on day 28 after immunization.And the antibody titres on day 28 after immunization in the 1.0mg dose group(P/N=6.62±0.66) were significantly higher than that in the 0.1mg,0.5mg or 2.0mg group(P/N=2.49±0.70,2.55±0.34,3.72±0.85,respectively)(P＜0.05).There was a significant dose effect on serum FSH and Plevels,but there was no dose effect on serum LH levels.The peak of serum FSH levels during oestrus cycle after immunization in the 1.0mg or 2.0mg dose group(28.25±0.83 mIU/mL,31.68±6.85 mIU/mL) were significantly higher than that in the 0.1 mg,0.5mg group or control(19.70±4.55mIU/mL, 23.21±7.50 mIU/mL,12.47±2.71 mIU/mL,respectively)(P＜0.05).The peak of serum LH levels during the oestrus cycle after immunization in the four dose groups were 11.90±0.92mIU/mL,12.67±0.39 mIU/mL,14.91±1.45 mIU/mL and 14.79±1.36 mIU/mL,respectively,which were similar to that in the control(14.20±0.76 mIU/mL). During the oestrus cycle,the peak of serum P levels after immunization in the 1.0mg or 2.0mg dose group(51.34±9.30 ng/mL,52.13±3.77 ng/mL) were significantly higher than in the 0.1ing,0.5mg group or control(40.53±8.66ng/mL,38.10±6.73 ng/mL,30.86±0.92 ng/mL,respectively)(P＜0.05).And the number of follicles≥0.8mm in 1.0mg dose group(31.0±2.65) were significantly higher than that in 0.5mg,2.0mg or control(20.4±2.31,22.25±3.42 and 15.33±0.67)(P＜0.05).Analyzing the correlation between ab titer and follicular mumbers,a remarkable positive correlation was observed between ab titer and follicular mumbers(r=0.73,P＜0.01).After all,the above results identified that the recombinant expression protein inhibin could induced rats to produce antibody against inhibin and within the dose-range studied, there was a significant of dose effects of INHαon the antibody titer,hormone levels and ovarian follicullar development,and the 1.0mg dose is optimal,which was useful for future inhibinimmunization studies or future practical application.
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