【作者】 秦波；

【导师】 朱筱娟；

【作者基本信息】 东北师范大学 ， 遗传学， 2006， 硕士

【摘要】 马铃薯X病毒(potato virus X, PVX)的P25蛋白是参与病毒在细胞和细胞之间运动的一个重要蛋白,它在转录后水平的基因沉默中起到抑制作用,能够提高PVX病毒侵染植物的成功率。本研究对植物病毒沉默抑制子P25进行原核表达、纯化并制备该抑制子的多克隆抗体,为下一步研究抑制子在基因沉默中的分子机理奠定基础。通过体外DNA重组技术,将来源于pBIN61-p25质粒上的目的基因p25片段在T4 DNA连接酶的作用下,插入到原核表达载体pET-28a(+)的BamHⅠ和SacⅠ酶切位点之间,构建重组质粒pET-28a(+)-p25。经PCR扩增、酶切鉴定以及DNA序列分析,插入的目的基因p25的序列正确,N端与6×His标签形成融合蛋白,无移码现象。将重组质粒pET-28a(+)-p25转化到大肠杆菌BL21(DE3),加入终浓度为1.0 mM的异丙基-β-D-硫代半乳糖苷(isopropyl-β-D -thiogalactoside, IPTG)进行诱导,诱导后表达出分子量大约为28.6 kDa的P25融合蛋白。该P25融合蛋白以可溶形式存在,扩大培养按1:40的比例浓缩后在变性条件下经Ni离子亲和层析得到纯品P25融合蛋白。用Bradford法测定给该融合蛋白含量达180μg/ml。用纯化的P25融合蛋白免疫三只6-8周龄的BALB/c小鼠(15μg/只,次)三次后,进行ELISA检测,获得的P25融合蛋白的多克隆抗体以1:12000的比例稀释后仍可检测到0.25μg左右的抗原(融合蛋白P25)。P25融合蛋白与其多克隆抗体的Western-blot经AEC化学显色后,在硝酸纤维素膜上显示出单一的特异性条带。以上结果表明通过原核表达系统在大肠杆菌中表达出了P25融合蛋白,并且该蛋白具有良好的抗原性和免疫原性。获得的这种植物病毒沉默抑制子P25的多克隆抗体,可以进一步用于基因沉默机理的研究。更多还原

【Abstract】 The 25 kDa movement protein (P25) of potato virus X (PVX),a suppressor ofposttranscriptional gene silencing, which can help PVX to succeed in infecting plant, wasessential for the cell-to-cell movement of PVX in its host. Protein P25 was expressed byprokaryotic expression system, purified in vitro, and be used as antigen for making itspolyclonal antibodies. The p25 gene from the plasmid p25, was inserted into the pET-28a (+)vector between the site of BamH Ⅰ and Sac Ⅰ , to create a recombinant plasmidpET-28a(+)-p25. Then the recombinant pET-28a (+)-p25 was confirmed by PCR, restrictionenzyme digestion and DNA sequencing,showing that the p25 gene was fused with 6×Histag and expressed as fusion protein. The recombinant plasmid was transferred into thecompetent BL21 (DE3) cells and approximately a 28.6 kDa fusion protein was induced by theaddition of isopropyl-β-D -thiogalactoside (IPTG) (1.0 mM) during cultivation. The solublefusion protein P25 was purified after expending cultivation by Ni-NTA affinitychromatography under the denaturing condition. The concentration of the fusion protein P25was about 180 μg /ml determined by Bradford assays. The purified fusion protein P25 wasinjected into three 6 to 8 weeks old BALB/c mice (15 μg/mouse, time) three times to makepolyclonal antibody. The antibody titration was up to 1:12000 determined by ELISA assays.Western hybridization of antibody and P25 showed a single band in the nitrocellulose (NC)membrane. These results showed that we have obtained the P25 protein by in vitro expressionsystem and the expressed P25 showed the expected antigenicity and immunogenicity. Thepolyclonal antibody against the RNA silencing suppressor (P25) has been obtained, and willbe used in the research of the mechanism of gene silencing.更多还原