中国农业科学 ›› 2014, Vol. 47 ›› Issue (18): 3691-3699.doi: 10.3864/j.issn.0578-1752.2014.18.017

• 专题:农产品安全 • 上一篇    下一篇

猪β2肾上腺素能受体基因克隆及穿梭表达质粒的构建

王健1,2,王静1,王淼1,孔祥雅3,祝凤彬3,邵小雪3   

  1. 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
  • 收稿日期:2014-03-31 修回日期:2014-05-27 出版日期:2014-09-16 发布日期:2014-09-16
  • 通讯作者: 王静,w_jing2001@126.com
  • 作者简介:王健,E-mail:xuanyuanjian0228@126.com
  • 基金资助:
    国家公益性行业(农业)科研专项(201203094)

Gene Cloning and Shuttle Expression Plasmid Construction of Pig β2 Adrenergic Receptors

WANG Jian1,2, WANG Jing1, WANG Miao1, KONG Xiang-ya3, ZHU Feng-bin3, SHAO Xiao-xue3   

  1. 1Institute of Quality Standard and Testing Technology for Agri-products, Chinese Academy of Agricultural Sciences/Key Laboratory of Agrifood Safety and Quality, Ministry of Agriculture, Beijing 100081
    2Department of Food Science, Hebei North University, Zhangjiakou 075000, Hebei
    3Beijing Kwinbon Biotechnology Co., Ltd, Beijing 102206
  • Received:2014-03-31 Revised:2014-05-27 Online:2014-09-16 Published:2014-09-16

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,且配体与受体结合位点处的氨基酸均得到正确克隆。在线BLAST分析表明,猪与其它物种的β2AR及氨基酸序列一致性均在80%以上,具有较高的同源性。由系统进化树分析可知,所克隆的猪β2AR与领西猯Pecan tajacu(JN 633666.1)的亲缘性较近,与大仓鼠Tscherskia triton(AY683091.1)和草原田鼠Microtus ochrogaster(XM 005356183.1)的亲缘性较远。氨基酸疏水性分析表明,该受体蛋白N端以疏水性氨基酸为主,C端以亲水性氨基酸为主。经PCR和双酶切鉴定以及测序分析证明,成功构建重组穿梭表达质粒pTriEx-1.1Hygro-β2AR1-418和pTriEx-1.1Hygro-β2AR63-418。【结论】所获克隆基因与已发表猪β2AR高度一致,且活性位点处的氨基酸均得到正确克隆。此外,pTriEx-1.1 Hygro穿梭表达载体适宜用作大肠杆菌、昆虫细胞和哺乳动物细胞表达系统的启动子,是研究目标基因在不同表达系统表达效果的良好材料。本文所构建的穿梭表达质粒pTriEx-1.1Hygro-β2AR1-418和pTriEx-1.1Hygro-β2AR63-418均可用于以上3种表达系统中β2AR蛋白的表达纯化研究。

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

Abstract: 【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 β2AR 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 DH5α 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 truncated, and 6×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 published pig β2AR gene sequence (AF000134), the identity of nucleotide between them was 99.68%, and the rate of deduced amino acid was 99.28%. Furthermore, all of the amino acids at the ligand binding sites were cloned correctly. BLAST analysis indicated that there was a high homology of β2AR between pig and some species. From phylogenetic tree analysis based on β2AR gene, Sus scrofa is more close to Pecan tajacu and not closely related to Tscherskia triton and Microtus ochrogaster. Hydrophobicity analysis illustrated that the N-terminal and C-terminal of the receptor were dominated by hydrophobic amino acids and hydrophilic amino acids, respectively. Recombinant plasmid named pTriEx-1.1Hygro-β2AR1-418 and pTriEx-1.1Hygro- β2AR63-418 were constructed successfully after verification of PCR, double enzyme digestion and sequencing. 【Conclusion】 The β2AR gene and the amino acids at active sites were highly consistent with the published records. Besides, pTriEx-1.1 Hygro vector contains promoters suitble for Escherichia coli, insect cells and mammalian cells, respectively. It is a good material to explore the expression effect of target gene in different expression systems. Consequently, both of the recombinant shuttle expression plasmid pTriEx-1.1Hygro-β2AR1-418 and pTriEx-1.1Hygro-β2AR63-418 can be applied to the further studies on β2AR expression and purification in all three of expression systems.

Key words: pig, β2 adrenergic receptor, gene cloning, sequence analysis, shuttle expression plasmid