CAJ | 학술논문

目的：探讨阿拉瑞林主动免疫绵羊对垂体和卵巢FSHR表达的作用，分析绵羊FSHR的生物信息学特点。方法：42只5～6月龄母绵羊随机分为6组（n＝7），EG-Ⅰ、EG-Ⅱ和EG-Ⅲ分别于0 d和14 d皮下注射阿拉瑞林抗原200μg、300μg和400μg；EG-Ⅳ和EG-Ⅴ分别皮下注射阿拉瑞林抗原200μg、300μg，0、7、14、21 d各一次，共4次；对照组在0 d和14 d皮下注射抗原溶媒2.0ml。于70 d（实验结束时）屠宰动物，无菌采集垂体和卵巢。从绵羊垂体中提取FSH，进行PCR扩增、克隆和测序，荧光定量PCR分析，将得到的序列运用生物信息学软件行分析，用Western blot检测卵巢FSHR蛋白表达。结果：实验组垂体FSHR mRNA的2-ΔΔCt均低于对照组， EG-Ⅰ、EG-Ⅱ和EG-Ⅲ的2-ΔΔCt值逐渐下降；而且EG-Ⅳ低于EG-Ⅰ，EG-Ⅴ低于EG-Ⅱ，即注射阿拉瑞林4次后FSHR mRNA的2-ΔΔCt值分别低于注射2次后的值。各实验组卵巢FSHR蛋白表达量随阿拉瑞林注射剂量的增加而逐渐增加，EG-IV和EG-V卵巢FSHR蛋白显著高于对照组相比（P＜0.01）。 FSHR序列为1091 bp，开放阅读框（0RF）780 bp，位于41～820位点，其半衰期、理论等电点、不稳定指数、疏水性平均值和脂肪指数分别为1.2 h、5.05、47.75、0.836和30.61。结论：阿拉瑞林免疫可抑制垂体FSHR mRNA的表达，但是增加卵巢FSHR蛋白表达；FSHR蛋白为不稳定的疏水性蛋白。
목적：탐토아랍서림주동면역면양대수체화란소FSHR표체적작용，분석면양FSHR적생물신식학특점。방법：42지5～6월령모면양수궤분위6조（n＝7），EG-Ⅰ、EG-Ⅱ화EG-Ⅲ분별우0 d화14 d피하주사아랍서림항원200μg、300μg화400μg；EG-Ⅳ화EG-Ⅴ분별피하주사아랍서림항원200μg、300μg，0、7、14、21 d각일차，공4차；대조조재0 d화14 d피하주사항원용매2.0ml。우70 d（실험결속시）도재동물，무균채집수체화란소。종면양수체중제취FSH，진행PCR확증、극륭화측서，형광정량PCR분석，장득도적서렬운용생물신식학연건행분석，용Western blot검측란소FSHR단백표체。결과：실험조수체FSHR mRNA적2-ΔΔCt균저우대조조， EG-Ⅰ、EG-Ⅱ화EG-Ⅲ적2-ΔΔCt치축점하강；이차EG-Ⅳ저우EG-Ⅰ，EG-Ⅴ저우EG-Ⅱ，즉주사아랍서림4차후FSHR mRNA적2-ΔΔCt치분별저우주사2차후적치。각실험조란소FSHR단백표체량수아랍서림주사제량적증가이축점증가，EG-IV화EG-V란소FSHR단백현저고우대조조상비（P＜0.01）。 FSHR서렬위1091 bp，개방열독광（0RF）780 bp，위우41～820위점，기반쇠기、이론등전점、불은정지수、소수성평균치화지방지수분별위1.2 h、5.05、47.75、0.836화30.61。결론：아랍서림면역가억제수체FSHR mRNA적표체，단시증가란소FSHR단백표체；FSHR단백위불은정적소수성단백。
Objective:To investigate the effects of GnRH agonist on the expressions of FSHR proteins in pituitary and ovaries in ewes,also to analyse the bioinformatics characteristics of ovine pituitary FSHR.Methods: Forty-two prepubertal ewes (Ovis aries) were randomly assigned to six groups (n=7).The ewes in experimental group EG-Ⅰ,EG-Ⅱand EG-Ⅲwere subcutaneously injected with 200μg,300μg and 400 μg alarelin antigens twice (at day 0 and 14),respectively.Ewes in EG-Ⅳand EG-Ⅴwere injected sub-cutaneously with 200μg and 300μg alarelin antigen four times (at day 0,7,14 and 21),respectively.Ewes in the control group (CG) were subcutaneously injected with 2.0 ml solvent twice (at day 0 and 14).Fluorescence quantitative RT-PCR was used to detect gene expression of FSHR in pituitary glands.The expression of FSHR protein in the ovaries was detected using Western blot.Results:The 2-ΔΔCt values of GnRHR,FSHR and LHR mRNAs in the pituitary gland of EG were lower than that in control group (CG).The 2-ΔΔCt values in EG-Ⅳ( P<0.05 ) and EG-Ⅴ( P<0.05 ) were lower than those in EG-Ⅰand EG-Ⅱ.Expressions of FSHR proteins in EG ovaries increased.Levels of FSHR proteins in EG-Ⅳand EG-Ⅴ( P<0.05 ) were higher than CG.The length of sheep FSHR sequence of nucleotides were 1 091 bp,the homology of FSHR sequences was 100% with that reported in NCBI.The theory isoelectric point , half-life,unstable index,fat index,hydrophobic average were respectively 1.2 h,5.05,47.75,0.836 and 30.61.Conclusion:Alarelin active immunization can inhibit the expression of FSHR mRNA in the pituitary gland ,but increase FSHR expression in ovarian of ewes.FSHR is an unstable hydrophobic protein.