本生烟病程相关蛋白PR-10的原核表达及抗血清制备

病程相关蛋白(Pathogenesis-related proteins,PRs)是植物受生物或非生物胁迫后诱导并积累的一类蛋白质,具有抗病抗逆的多种功能。为检测本生烟病程相关蛋白10(Nicotiana benthamiana Pathogenesis-related protein 10,NbPR10)的表达,制备其抗血清,并用以探究其可能的特性和功能。将已有的NbPR10基因构建到原核表达载体p GEX-KG中获得重组质粒p GEXKG-NbPR10,转化大肠杆菌BL21(DE3),经0.2 m M IPTG诱导表达分子量约44 k Da的融合蛋白(含GST标签),用GST亲和柱纯化获得纯化的融合蛋白。以纯化的融合蛋白为抗原免疫家兔制备NbPR10蛋白的抗血清,经Western blot测定抗血清效价达1∶10 000,能至多检测到稀释比例为1∶320的转基因本生烟叶片中的PR10蛋白或1 ng原核表达的蛋白,且能检测到本生烟根和茎中的PR10蛋白,证明PR10在本生烟根和茎中表达量较在叶部高,为进一步研究NbPR10蛋白的功能提供参考。

Prokaryotic expression of pathogenesis-related protein 10 from Nicotiana bentha-miana and the preparation of antiserum against the protein

Pathogenesis-related proteins (PRs) are a class of proteins that was induced and accumulated in plants during pathogens infection or under abiotic stress, and show multifunction related to disease and stress resistance. In order to test expression of PR10 in Nicotiana benthamiana (NbPR10) and explore the possible cha-racter and function, specific antiserum against the protein was prepared. NbPR10 gene was inserted into the prokaryotic expression vector pGEX-KG. Recombinant plasmid pGEXKG-NbPR10 was transformed into the Escherichia coli strain BL21 (DE3), 44 kDa GST-fused NbPR10 protein was induced by using 0.2 mM IPTG followed by purification with GST affinity column and immunization of the rabbits. The titer of the antiserum was about 1∶10 000 as evidenced by Western blot analysis. Meanwhile, we can even detect the PR10 in 1∶320 diluted leave samples of PR10-transgenic N. benthamiana or 1 ng prokaryotic expressed protein by Western blot. In addition, NbPR10 in roots and stems of N. benthamiana can be detected by the antiserum, indicating that NbPR10 accumulated more in these two tissues compared with the leaves. The NbPR10-specific antibody prepared in our work should provide the basis for further investigating the function of NbPR10.