GnRH-A免疫对GnRHR、FSH-β和LH-β表达及生物信息学特性的影响

摘要

Objective:To explore the effects of GnRH-A on gene expression of gonadotropin releasing-hormone receptor (GnRHR),FSH-β and LH-β in pituitary and GnRHR bioinformatics properties after active immunization with alarelin in rabbits ,further to study the manchanism of GnRH-A.Methods :30 male rabbit( Oryctolagus cuniculus )were divided randomly into three groups ,each of 10 rabbits.EG-Ⅰ and EG-Ⅱwere injected with 1.0 ml(100 μg/ml)of alarelin antigen ,and EG-Ⅱ was re-injected at the same dosage on 20 d.Nothing was injected in control group.The pituitary was collected on 70 d.Total RNA was extracted ,and the expression of GnRHR ,FSH-β and LH-β mRNA was analyzed by real-time quantitative-PCR.They were reveasely transcribed and sequenced.The gene expression ,physicochemical properties ,signal peptide and secondary structure of GnRHR protein were assayed and predicted using bioinformatics softwares and online tools of Tmpred ,SignalP ,TargetP and Expasy.Results :GnRHR mRNA in EG-Ⅰ and EG-Ⅱ were less than in control group ( P＜0.01 ).Meanwhile GnRHR mRNA in EG-Ⅱwas lower than in EG -Ⅰ ( P＜0.05 ).GnRHR nucleotide was 1 179 bp and homology 96％ with NM-001082738.The nucleotide consisted of 28.7％ A ,24.9％ C ,19.0％ G and 27.5％ T.Mitochondrion transit peptide ,signal peptide and subcellular location changed too.Compared with NM-001082738 ,the nucleotide numbers ,amino acids ,molecular weight ,aliphatic index and GRAVY of GnRHR mRNA were less than that of NM-001082738 ,but instability index ,α-helixes and β-turn increased.Conclusion :Active immunization of Alarelin could significanly decrease gene expressions of GnRHR ,FSH-β and LH-β mRNA in rabbit pituitary ,and also influence the bioinformatics properities of GnRHR.GnRHR is an unstable hydrophobic transmembrane protein.%目的:探讨GnRH-A主动免疫对垂体GnRHR、FSH-β和LH-β基因表达和生物信息学特性的影响,进而研究GnRH-A的作用机理.方法:30只日本大耳白兔(Oryctolagus cuniculus)随机均分为三组,在EG-Ⅰ和EG-Ⅱ皮下注射1.0 ml(100 μg/ml)GnRH-A抗原,EG-Ⅱ于20天以相同剂量加强免疫一次,对照组不做任何处理.70天颈动脉放血致死实验兔,无菌采集垂体.从兔垂体中提取总RNA,GnRHR,FSH-β和LH-β mRNA基因扩增、克隆和测序,实时荧光定量PCR分析mRNA基因的表达.用DNAMAN、Tmpred、SignalP、TargetP、Expasy等生物信息学分析软件和在线工具,对GnRHR序列和蛋白的理化特性、跨膜结构、信号肽及二级结构等进行分析和预测.结果:EG-Ⅰ和EG-Ⅱ的GnRHR mRNA低于对照组(P<0.01),EG-Ⅱ又低于EG-Ⅰ(P<0.05);EG-Ⅰ和EG-ⅡFSH-β mRNA和LH-β mRNA极显著低于对照组(P<0.01),EG-Ⅰ与EG-Ⅱ间也具有显著差异(P<0.05).GnRHR核苷酸为1 179 bp,与NM-001082738的同源性达96%,核苷酸中含A 28.7%,C 24.9%,G 19.0%,T 27.5%.GnRHR的线粒体转运肽、信号肽及转运肽长度发生了明显改变.与NM-001082738的比较显示,GnRHR mRNA的核苷酸、氨基酸、分子量、脂肪指数和平均疏水性均变小,而不稳定指数、α螺旋和β折叠数均增加.结论:GnRH-A免疫可明显降低雄兔垂体GnRHR、FSH-β和LH-β mRNA基因的表达,而且对GnRHR的生物信息学特性具有一定的影响,加强免疫作用更明显;GnRHR是一种含有信号肽的不稳定的疏水性跨膜蛋白.