猪β_2肾上腺素能受体基因克隆及穿梭表达质粒的构建  

Gene Cloning and Shuttle Expression Plasmid Construction of Pig β_2 Adrenergic Receptors

作　　者：王健[1,2] 王静[1] 王淼[1] 孔祥雅 祝凤彬 邵小雪 

机构地区：[1]中国农业科学院农业质量标准与检测技术研究所/农业部农产品质量安全重点实验室,北京100081 [2]河北北方学院食品科学系,河北张家口075000 [3]北京勤邦生物技术有限公司,北京102206

出　　处：《中国农业科学》2014年第18期3691-3699,共9页Scientia Agricultura Sinica

基　　金：国家公益性行业(农业)科研专项(201203094)

摘　　要：【目的】利用重组β2肾上腺素能受体(β2AR)检测β2激动剂类药物,是弥补传统免疫学检测方法不足、实现该类违禁物多残留的快速检测手段。获得高纯度、高亲和力的重组受体是该技术的核心和难点。本文克隆猪β2 AR并构建其重组穿梭表达质粒,为筛选最适宜的受体表达系统和表达条件提供基础材料。【方法】克隆猪β2AR并进行序列分析,完成重组穿梭表达质粒构建。首先采集新鲜猪肝组织,提取总RNA,根据GenBank已登录的猪β2AR核苷酸序列(AF000134),设计1对引物并进行RT-PCR扩增。扩增产物切胶回收后与pMD-18T载体于4℃、T4连接酶作用下连接过夜。连接产物转化DH5α感受态细胞,经蓝白斑筛选后提取克隆质粒并对其作PCR鉴定、双酶切鉴定及测序分析。然后对所获得的基因及编码的氨基酸序列进行在线BLAST分析、系统进化树构建和疏水性分析。为了提高受体蛋白的表达量及受体配体间的亲和力,对该克隆基因作N端截短186个核苷酸改造;为使表达蛋白C端携带6×His标签,对克隆基因C端进行去除终止子改造。将改造后的克隆基因分别插入pTriEx-1.1 Hygro穿梭表达载体,连接产物转入NovaBlue感受态细胞,经Amp抗性筛选,挑取单菌落提取质粒并进行鉴定。【结果】经分光光度计检测及琼脂糖凝胶电泳分析,所提猪肝细胞总RNA的纯度、浓度及完整性可满足后续试验要求。RT-PCR产物电泳结果显示,在1 200 bp左右出现1条特异性条带,与预期结果一致。克隆质粒pMD-18T-β2AR经鉴定,证实为阳性重组质粒。测序结果显示,所得猪β2AR基因cDNA序列全长1 257 bp,编码418个氨基酸。该序列已提交至GenBank,登录号为KF023571.1。Compute pI/Mw在线软件预测该蛋白分子质量为46.73 kD。与已发表的猪β2AR(AF000134)相比,基因序列一致性为99.68%,4处发生碱基突变,编码的氨基酸序列一致性为99.28%,3处发生突变,且配体与�[ Objective ] Receptor assay is a new method suitble for rapid multianalysis for detection of β-agonists compared to traditional immunoassay. The key problem of this method is acquiring pure recombinant receptor with high affinity and selectivity. To provide the basic material for screening of optimal expression system and expression condition, the specific recombinant shuttle expression plasmid was constructed with pig fleAR gene. [Method] Total RNA was extracted from fresh pig liver and a pair of primers was designed and synthesized according to the published pig β2AR gene sequence in Genbank（AF000134）. The recovery of RT-PCR product from agarose gel was connected with cloning vector pMD-18T by T4 DNA ligase at 4℃ overnight. The ligation product was then transformed into competent cell DH5t and the plasmid was extracted after blue and white spot selection. And that the plasmid was confirmed by PCR, double enzyme digestion and sequencing analysis. The DNA sequence and deduced amino acid sequence were firstly analyzed by BLAST, and then phylogenetic tree construction and hydrophobicity were performed, respectively. In order to enhance the expression and binding affinity of receptor protein, N-terminal 186 bp of the cloned gene were tnmcated, and 6 x His-tag was added at C-terminal. Finally, the genetically modified genes were respectively cloned to pTriEx-1.1 Hygro vector, and the ligation products were transformed into competent cell NovaBlue. The recombinant plasmids were extracted from single colonies and identified after Amp resistance screening. [ Result ] The purity, concentration and integrity of the extracted total RNA could meet the requirements of successive test by UV spectrophotometer testing and agarose gel electrophoresis. The RT-PCR product was 1 257 bp by sequencing, which encods 418 amino acids. The sequence has been submitted in Genbank as accession number KF023571.1. The deduced protein was predicted to have a computed molecular mass of 46.73 kD by Compute pI/Mw. Compared with the publi

关 键 词：猪 Β2肾上腺素能受体 基因克隆 序列分析 穿梭表达质粒 

分 类 号：Q78[生物学—分子生物学]