山羊IGFBP-2基因的克隆、序列分析及其组织表达

【目的】克隆获得南江黄羊IGFBP-2（Insulin-like growth factor binding protein -2）基因的CDS区全序列，对其进行生物信息学分析，并分析IGFBP-2的mRNA和蛋白组织表达特征，为深入研究该基因在出生后山羊生长和发育过程中的作用以及表达调控提供基础资料。【方法】下载NCBI的GenBank数据库中公布的绵羊（Ovis aries，NM_001009436）和牛（Bos taurus，NM_174555）的IGFBP-2基因的mRNA序列，经序列比对后采用保守区域序列并利用Primer Premier 6.0软件设计克隆引物，经PCR扩增后采用TA克隆技术获取含有阳性克隆子的菌液，送公司测序得到山羊IGFBP-2基因的完整编码区序列，并利用EditSeq、DNAMAN 6.0、MEGA 6.0和ExPASy在线平台等软件对CDS区序列和其编码的氨基酸序列进行生物信息学分析；获得山羊IGFBP-2基因CDS区序列后，利用该序列设计实时荧光定量PCR（Q-PCR）引物，采用Q-PCR和蛋白质免疫印迹杂交（Western blotting）方法检测了IGFBP-2的mRNA和蛋白在出生后南江黄羊不同组织（心脏、肝脏、肺、背最长肌、半膜肌和臂三头肌）和不同发育阶段（3、30、60、90和120 d）的表达情况。【结果】①克隆得到954bp的南江黄羊IGFBP-2基因的CDS区全序列，碱基组成为GC含量69.39%，AT含量30.61%，共编码317个氨基酸残基，预测的山羊IGFBP-2蛋白分子量大小为33.8808 kD，等电点为7.82，二级结构由无规卷曲（68.14%）、α-螺旋（18.30%）和延伸链（13.56%）三种形式组成；蛋白结构域分析发现，IGFBP-2蛋白序列包含保守的IB（IGFBP homologues）和TY（Thyroglobulin type Ι repeats）结构域，IB结构域位于第37-125位氨基酸处，TY结构域位于第250-302位氨基酸处；②磷酸化位点预测发现，IGFBP-2蛋白中存在Ser（5）和Thr（7）共12个磷酸化位点；糖基化位点预测发现，IGFBP-2蛋白序列中存在10个N-糖基化位点和2个O-糖基化位点；③CDS区序列相似性比较发现，山羊IGFBP-2与绵羊、牛、猪、人和小鼠的相似性分别达到98.99%、97.73%、87.12%、78.33%和76.26%；氨基酸序列相似性比较发现，山羊IGFBP-2与绵羊、牛、猪、人和小鼠的相似性分别达到99.24%、98.10%、87.07%、70.27%和73.00%；④系统进化树分析发现，山羊IGFBP-2与绵羊和牛的亲缘关系最近；⑤组织表达分析表明，IGFBP-2在肝脏中的mRNA和蛋白的相对表达量均最高(P<0.01)，背最长肌次之；在不同发育阶段的肝脏组织中，IGFBP-2 mRNA和蛋白相对表达量均呈现一直上升的趋势；在不同发育阶段的背最长肌组织中，IGFBP-2的mRNA表达水平呈现一种“上升-下降-上升”的波动平衡。【结论】获得了山羊IGFBP-2基因完整的编码区序列和组织表达特征，生物信息学分析发现IGFBP-2基因的编码区序列具有物种间的保守性，肝脏是山羊IGFBP-2 的mRNA和蛋白表达的主要组织，同时在山羊不同发育阶段的肝脏和背最长肌组织中，IGFBP-2基因的mRNA和蛋白表达丰度呈现一定的规律性，表明IGFBP-2基因可能在出生后山羊的早期生长发育过程中发挥着重要的作用。

Cloning,Sequence Analysis and Tissue Expression of IGFBP-2 Gene in Goat (Capra hirus)

【Objective】The objectives of this study were to clone goat IGFBP-2 (Insulin-like growth factor binding protein -2) coding sequence (CDS), to analyze its sequence characteristics, mRNA and protein expression profiles. It could provide data for further investigation on the function and expression regulation of IGFBP-2 gene in the growth and development of postnatal goat. 【Method】The IGFBP-2 mRNA sequences of Ovis aries (NM_001009436) and Bos taurus (NM_174555) were downloaded from GenBank database, and the primers were designed according to the conserved regions by using Primer Premier 6.0 software after sequences alignment, then PCR amplification and the positive clones were obtained using TA clone technology. A complete coding sequence of IGFBP-2 gene was obtained from sequencing. The CDS and coding amino acid sequences were analyzed by using EditSeq, DNAMAN 6.0, MEGA 6.0 and ExPASy online software. The real-time quantitative PCR (Q-PCR) primers were designed using the acquired CDS sequence of the IGFBP-2 gene. The expression level of the IGFBP-2 gene and protein in postnatal Nanjiang brown goat’s different tissues (heart, liver, lung, longissimus dorsi muscle, semimembranosus muscle and triceps brachii muscle) and development stages (3 d, 30 d, 60 d, 90 d and 120 d) were detected using Q-PCR and western blotting. 【Result】A full-length CDS of 954bp was cloned for goat IGFBP-2, and encodes a protein of 217 amino acid. The GC and AT content is 69.39% and 30.61%. The molecular weight of goat IGFBP-2 protein is 33.8808 kD, and its theoretical isoelectric point is 7.82. The secondary structure of the protein contains random coil (68.14%), α-helix (18.30%) and extended strand (13.56%). Protein structure domain analysis showed that its amino acid sequence contains conserved IB (IGFBP homologues) and TY (Thyroglobulin type Ι repeats) domains, IB domain in the 37-125 amino acids and TY domain in the 250-302 amino acids. Phosphorylation sites prediction indicated that there are five Ser phosphorylation sites and seven Thr phosphorylation sites existsin IGFBP-2 protein sequence. Glycosylation sites prediction found that there are ten N-glycosylation sites and two O-glycosylation sitesexistsin IGFBP-2 protein sequence. Sequence analysis indicated that the IGFBP-2 CDS sequence of the Nanjiang brown goat is similar with those of the sheep (98.99%), cattle (97.73%), pig (87.12%), human (78.33%) and mouse (76.26%). Sequence analysis revealed that the IGFBP-2 amino acid sequence of the Nanjiang brown goat is similar with those of the sheep (99.24%), cattle (98.10%), pig (87.07%), human (70.27%) and mouse (73.00%). Furthermore, goat IGFBP-2has the closest phylogenetic relationship to the sheep and cattle IGFBP-2 in amino acids sequences. The mRNA and protein levels of IGFBP-2 in the liver were significantly higher than that in other tissues (P＜0.01), and LD were the second (P＜0.01); in the liver, the mRNA and protein levels of IGFBP-2 were upregulated from 3 day to 120 day. In addition, the mRNA level of IGFBP-2 showed an “up-down-up” expression pattern during postnatal longissimus dorsi muscle development. 【Conclusion】IGFBP-2 was cloned and its tissue expression patterns were investigated.The sequence characteristics of IGFBP-2 is conserved in species, and liver is the main expression tissue of goat IGFBP-2 mRNA and protein. The expression of IGFBP-2 mRNA and protein occurred with some regularity and indicated that IGFBP-2 may play an important role in early growth and development of postnatal goat.