A Polyclonal Antibody Against the C Subunit of Porcine Aminopeptidase N Expressed in Escherichia coli
A Polyclonal Antibody Against the C Subunit of Porcine Aminopeptidase N Expressed in Escherichia coli
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大肠杆菌表达的猪氨肽酶N C亚基多克隆抗体
DOI：
10.1089/hyb.2011.0042
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发表时间：
2011-10-01
期刊：
影响因子：
--
通讯作者：
Wu, Rui
中科院分区：
文献类型：
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作者：
Sun, Dongbo;Wang, Yueqiang;Wu, Rui
从新生仔猪肠道刷状缘膜提取总RNA，用RT-PCR方法扩增出猪氨基肽酶N(PAPN)基因的全长。将pAPN基因扩增产物克隆到载体pMD18-T中，构建成重组表达载体pMD18-T-pAPN。以pMD18-T-pAPN为模板，用带有His标签的表达载体pET-32a表达pAPN的C亚基(pAPN-C)。在确认重组蛋白pAPN-C与天然pAPN抗体的反应性后，以纯化蛋白为免疫原，在兔体内制备了抗重组蛋白pAPN-C的多克隆抗体。Western印迹分析表明，重组蛋白pAPN-C所产生的抗体能识别天然的pAPN。这些数据表明，pAPN-C重组蛋白及其多克隆抗体可以为进一步的受体拮抗剂提供一定的依据。
The entire pig aminopeptidase N (pAPN) gene was amplified by RT-PCR using total RNA extracted from intestinal brush border membrane of a newborn piglet. The amplified products of the pAPN gene were cloned into the vector pMD18-T, generating a recombinant plasmid pMD18-T-pAPN. The C subunit of pAPN (pAPN-C) produced by PCR from the plasmid pMD18-T-pAPN was expressed in Escherichia coli using vector pET-32a with His tag. After confirming reactivity of the recombinant protein pAPN-C to antibody against native pAPN, polyclonal antibody against the recombinant protein pAPN-C was prepared in rabbit using purified protein as immunogen. In Western blot analysis, the antibody elicited by the recombinant protein pAPN-C could recognize the native pAPN. These data demonstrate that the pAPN-C recombinant protein and its polyclonal antibody can provide some basis for further receptor antagonist.