猪β2肾上腺素受体基因克隆及重组酵母表达质粒的构建
Cloning of β2 adrenergic receptors c DNA and construction of yeast expression plasmid
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摘要: 克隆猪β2AR基因并进行序列分析,构建其重组酵母表达质粒。采集新鲜猪肝组织,提取总RNA,经RT-PCR扩增、克隆及鉴定,获得猪β2AR的c DNA序列,并对该序列及编码的氨基酸序列进行分析。同时将目的基因插入p PICZαA酵母表达载体,构建重组表达质粒。克隆获得猪β2AR基因c DNA序列1257 bp,Genbank登录号为KF023571.1,编码418个氨基酸,蛋白分子质量46.73 ku。生物信息学分析表明,该基因与其他物种的β2AR基因相似性较高,均在83%以上,具有较高的同源性。经PCR和双酶切鉴定以及测序分析,证明成功构建了重组酵母表达质粒p PICZαA-β2AR。本研究为猪β2AR基因在毕赤酵母系统的表达及建立基于受体的β激动剂多残留检测方法奠定了基础。Abstract: This study aimed to clone pig β2AR gene,analysize its sequence and construct its recombinant yeast expression plasmid. Total RNA was extracted from pig liver, and then RT- PCR was conducted based on published pig β2AR gene sequence(AF000134). By cloning of the PCR product and identification,the c DNA fragment of pig β2AR was obtained. Bioinformatical tools were adopted to analyze the gene sequence and amino acid. The target gene was cloned to p PICZα A vector,constructing recombinant expression plasmid.The c DNA fragment of pig β2AR(KF023571.1) was 1257 bp,and encodeed 418 amino acids. The deduced protein was predicted to have a computed molecular mass of 46.73 ku. There were high similarity(more than83%) and homology in β2AR gene between pig and other species. Finally,recombinant plasmid named p PICZαA- β2AR was constructed successfully after verification by PCR, restriction enzyme analysis and sequencing. This research will serve as a base for expression of pig β2AR gene in yeast expression system and its application in multiresidue determination of β2-adrenoceptor agonists.
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