雄兔垂体GnRHR的序列测定与生物信息学分析

目的探讨GnRH-A激动剂(阿拉瑞林)主动免疫对垂体GnRHR基因表达的影响和生物信息学特性。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(100μg/mL)阿拉瑞林抗原,EG-Ⅱ于20 d以原剂量加强免疫1次;从兔垂体中提取总RNA,PCR扩增GnRHR基因,进行反转录、克隆和测序;实时荧光定量PCR分析垂体中GnRHR、FSH-β和LH-βmRNA的表达,用DNAman、Tm-pred、Signal P 3.0、Target P 1.1、Expasy、PSORT II prediction等生物信息学分析软件和在线工具,对GnRHR序列及其蛋白的理化特性、跨膜结构、信号肽和二级结构等进行分析与预测。结果①雄兔GnRHR的核苷酸为1179 bp,同源性达96%。ssDNA分子量362.66×103,dsDNA 726.78×103。②GnRHR二级结构中151(40.27%)个氨基酸组成α-螺旋,15(4.00%)个氨基酸组成β-折叠。③GnRHR由389个氨基酸组成,蛋白质的序列长度为375;理论等电点(pI)5.02,不稳定指数58.08,脂肪指数28.67,疏水性平均值(GRAVY)0.869,表明该蛋白为不稳定的疏水性蛋白。④GnRHR蛋白TM螺旋长度为17～23,有34个强跨膜螺旋区,从内到外有35个螺旋。⑤GnRHR信号肽的概率0.999,最可能的酶切部位在17和18位点。结论阿拉瑞林免疫可以明显降低垂体GnRHR、FSH-β和LH-β基因表达,加强免疫效果更佳。GnRHR是一种含有信号肽序列的不稳定的疏水性跨膜蛋白,具有明显的生物信息学特征。

Expression, sequencing and bioinformatics analysis of GnRHR in the male rabbit pituitary

Objective To explore the expression of gonadotropin releasing hormone receptor（GnRHR） in pituitary and the bioinformatics properties of GnRHR following active immunization with GnRH agonist alarelin in male rabbits.Methods Thirty rabbits（Oryctolagus cuniculus）were divided into three groups.EG-Ⅰ and EG-Ⅱ were injected with 1.0 mL（100 μg /mL）alarelin antigen emulsion,EG-Ⅱ were re-injected at the same dosage on 20th d.Nothing was injected in the control group（CG）.All rabbits were killed at the experimental end（70 d）,and the pituitaries were collected aseptically.Total RNA was extracted,and GnRHR were amplified by RT-PCR,cloned and sequenced.Gene expressions of Gn-RHR,FSH-β and LH-β mRNA in pituitaries were also assayed by real time PCR.GnRHR sequences,protein physicochem-ical properties,membrane structure,signal peptide and secondary structural characteristics were analyzed and predicted with bioinformatics software and online tools of DNAman,Tmpred,SignalP 3.0,TargetP 1.1,Expasy,PSORT Ⅱ prediction.Results（1） GnRHR nucleotide was 1179 bp with 96% homology,the nucleotide contained A 338（28.7%）,C 293（24.9%）,G 224（19.0%） and T 324（27.5%）.Molecular weight of ssDNA and dsDNA were 362.66 kDa and 726.78kDa,respectively.（2） The patterns and secondary structure diagram of GnRHR sequence were obtained.The secondary structure consisted of 151（40.27%） amino acid α-helixes and 15（4.00%） amino acids β-sheets.（3） GnRHR consis-ted of 389 amino acids,and the protein sequence length was 375.The isoelectric point（pI）,instability index,aliphatic in-dex and average hydrophobicity（GRAVY） were 5.02,58.08,28.67 and 0.869,respectively,indicating that GnRHR pro-tein is an unstable hydrophobic protein.（4） The transmembrane（TM） helix length of GnRHR protein was 17-23,and comprised of 34 TM helix areas.（5） Signal peptide probability of GnRHR protein was 0.999,max cleavage site probability was in 17 and 18 positions.Conclusion Active immunization using alarelin can significantly decrease the gene expressions of GnRHR,FSH-β and LH-β in rabbit pituitary.GnRHR is an unstable hydrophobic transmembrane protein contai-ning signal-peptide sequence,and has distinct bioinformatics characteristics.