犬瘟热病毒H基因5’端的表达纯化及抗原性分析

摘要

为了获得高纯度的犬瘟热病毒5’端H蛋白,本研究通过参考GenBank中发表的CDV H蛋白基因序列,用Oligo6.0软件设计一对特异性引物,从犬瘟热病毒疫苗毒株CDV3和野毒株CDVSD株提取总RNA,经RT-PCR扩增得到388bp的基因片段,将目的基因与原核表达载体pET28a(+)连接构建了pET28a-CDV3-H5和pET28a-CDVSD-H5重组质粒,转化到大肠杆菌Bl21(DE3)中进行鉴定、测序、IPTG诱导表达.重组表达载体pET28a-CDVSD-H5在大肠杆菌中成功的表达,目的重组蛋白为22kD,而pET28a-CDV3-H5未能表达.经Ni-NTA洗脱纯化后,rCDVSD-H5蛋白被成功纯化.Western blot分析表明,表达产物能够被犬抗CDV阳性血清所识别,具有良好的抗原性.为血清学诊断方法的建立和基因工程疫苗的研究奠定基础.%The aim of this research is was to get high purified 5' terminal H protein of canine distemper virus. One pair of the special primers was designed by 01igo6. 0 software according to the hemagglutinin(H) protein gene sequences of canine distemper virus (CDV) published in GenBank. A 388 bp CDV H protein gene fragment was amplified by RT-PCR from the total RNA template CDVs and CDVSD strains. The 5' terminal H protein gene from CDV was cloned into expression vector pET28a(+) and the recombinant plasmid was transformed into E. Coli L21 (DE3) cells for sequencing, identifying and expressed by IPTG Induced. The constructed pET28a-CDVSD-H5 was successfully expressed at 22kD and purified by Ni-NTA; but the constructed pET28a-CDV3-H5 was not expressed. Western-blotting analysis was shown that could be recognized by CDV positive serum. This research laid the foundation of sero-diagnosis method and genetically engineering vaccine.