Sun Dongbo, Wang Yueqiang, Wu Guojun, Zhang Hong, Zhu Qinghe, He Xianjing, Guo Donghua, Wu Rui
Department of Veterinary Clinical Medicine, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, P.R. China.
Hybridoma (Larchmt). 2011 Oct;30(5):457-62. doi: 10.1089/hyb.2011.0042.
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.
使用从新生仔猪肠刷状缘膜提取的总RNA，通过逆转录聚合酶链反应（RT-PCR）扩增整个猪氨肽酶N（pAPN）基因。将pAPN基因的扩增产物克隆到载体pMD18-T中，产生重组质粒pMD18-T-pAPN。使用带有His标签的载体pET-32a，从质粒pMD18-T-pAPN通过PCR产生的pAPN的C亚基（pAPN-C）在大肠杆菌中表达。在确认重组蛋白pAPN-C与抗天然pAPN抗体的反应性后，以纯化蛋白作为免疫原在兔中制备针对重组蛋白pAPN-C的多克隆抗体。在蛋白质印迹分析中，重组蛋白pAPN-C引发的抗体可以识别天然pAPN。这些数据表明，pAPN-C重组蛋白及其多克隆抗体可为进一步研究受体拮抗剂提供一些依据。
