甲型肝炎病毒衣壳蛋白融合基因植物表达载体的构建 与遗传转化柑桔的研究

基因工程领域的研究进展使得植物体成为具有重要经济价值的药用蛋白的生产体系。以含甲型肝炎病毒结构基因cDNA的克隆载体pCDNAⅡA16为模板,用甲型肝炎病毒衣壳蛋白融合基因特异引物进行PCR扩增,得到全长2.2kb衣壳蛋白融合基因序列。经测序鉴定后正向克隆于植物表达载体pBI121中,衣壳蛋白融合基因位于pBI121质粒T-DNA左右边界区间内,处于CaMV35S启动子控制之下。经限制性内切酶分析和PCR鉴定后利用冻融法将重组质粒pBI121-A导入根癌农杆菌LBA4404。以锦橙 (Citrus. Sinensis Osbeck) 上胚轴为转化材料,通过根癌农杆菌介导法将衣壳蛋白融合基因转 化到植物基因组中。120株转化外植体经卡那霉素50 mg/L筛选,其中13株生长状况良好未出现白化现象的拟转化芽微嫁接到实生砧木继续培养。PCR分析证明,13株拟转化植株中有5株植物基因组中已导入甲型肝炎病毒衣壳蛋白融合基因,转化率为4.1%。此研究是对遗传转化柑桔表达外源蛋白的初步探讨,为进一步研究食用疫苗开辟了新途径。Abstract: The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter. The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404. The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments. 13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings. The presence and integration of the transgene had been verified by PCR analysis. The result showed that five transformants were integrated and the transformation efficiency was 4.1%.     

人核受体Nurr1基因的克隆和表达及产物纯化(英文)

核受体相关因子 1(nuclearreceptor relatedfactor 1,Nurr1)是主要表达于中脑黑质及腹侧被盖区多巴胺能神经元的一种转录因子 ,属于核受体超家族成员 ,其功能性配体尚未被确认 .研究表明 ,Nurr1对中脑多巴胺神经元的发育、存活以及成熟后功能的维持具有特殊重要意义 .如能找到它的特异性配体 ,将为最终筛选出治疗帕金森病等中枢多巴胺失调性疾病的药物或化学合成先导物打下基础 .为了获取Nurr1蛋白以标定其配体以及研究蛋白质间的相互作用 ,采用RT PCR技术 ,从人胚中脑组织特异性扩增及克隆了人Nurr1cDNA ,并获得一个在氨基端缺失 35 0bp碱基的Nurr1突变体 .将正常的Nurr1基因片段亚克隆至表达载体pET2 8a ,分别在TNTRT7偶联网织红细胞溶胞系统和大肠杆菌BL2 1(DE3)中获得表达 ,均以可溶性形式存在 ,且产自于体外转录 翻译系统的真核表达Nurr1蛋白已标记上同位素3 5S .Western印迹分析表明 ,所表达的重组目的蛋白具有特异的免疫反应性 .经Ni NTA亲和层析 ,得到了初步纯化的rhNurr1蛋白 .     

体细胞克隆牛和转基因体细胞克隆牛的遗传学分析(英)

分析了来自同一细胞系的体细胞克隆牛甜甜、庆庆、浒娃及来源同一培养转基因体细胞系转基因体细胞克隆牛九妹、乐娃和1个妊娠8个月转基因流产胎牛8C2以及随机抽取的1头鲁西黄牛(LX)、1头褐斯坦牛(HS)在24个微卫星位点标记牛的基因型.结果表明24个多态位点均表现出多态,等位基因数为1～5个,平均为3.17个.根据网上公布的数据,按其最高频率计算,甜甜、庆庆、浒娃、九妹、乐娃、8C2与培养细胞系、转基因细胞系间匹配概率为1.17×10^－36,根据本研究观察到的数据计算,匹配概率为1.90×10^－23;而与随机抽取的1头鲁西黄牛及褐斯坦牛的基因型分别在23和20个位点上完全不同.     

乳腺癌中雌激素受体α表达水平调节的分子机制

雌激素受体α(ERα)在乳腺癌的发生发展中扮演重要角色,因而ERα成为乳腺癌治疗的分子靶标。ERα的表达水平在乳腺癌患者中差异较大,即使同一患者,在乳腺癌的不同阶段也可能有很大的差别。乳腺癌内分泌治疗的疗效以及预后都与ERα表达水平密切相关。影响ERα表达水平的分子机制复杂,众多调节分子在染色质、转录、转录后、翻译和翻译后等水平参与ERα表达水平的调节。在染色质和转录水平,许多分子通过直接或间接地与ERα启动子的相互作用改变ERα的转录;在转录后/翻译水平,一些microRNA通过诱导ERαmRNA的降解和/或抑制其翻译降低ERα的水平;在翻译后水平,许多分子通过泛素-蛋白酶体途径调节ERα蛋白水平。文章从不同水平,对这些调节分子的调节机制进行简要综述。     

Hypoxia-induced erythropoietin expression in human neuroblastoma requires a methylation free HIF-1 binding site

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH-SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth-associated protein-43 (GAP-43), and neuron-specific enolase (ENO). NB cells with a more epithelial phenotype like SH-SHEP and LAN-5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up-regulation of hypoxia-inducible factor-1 (HIF-1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3' enhancer was only present in the SH-SHEP and LAN-5 cells but not in SH-SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.     

Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species

Summary Virus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.     

Genetically variable nucleopolyhedroviruses isolated from spatially separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) in Orkney

Here we report a lepidopteran system in which a pathogen is both abundant and genotypically variable. Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles to investigate the prevalence of a pathogen, O. brumata Nucleopolyhedrovirus (OpbuNPV), within the natural system. Virus was recorded in 11 of the 13 winter moth populations sampled, with two populations suffering mortality due to virus at levels of 50%. The virus genome from 200 single insect isolations was investigated for variation using restriction endonuclease digests. Twenty-six variants of OpbuNPV were detected using SalI. The polyhedrin gene of the virus was partially sequenced, allowing the relationship between the 26 variants to be portrayed as a cladogram. The phylogenetic relationship between OpbuNPV and other known baculovirus polyhedrin gene sequences was also established. The discovery of virus at such high prevalence is discussed with reference to occurrence and genetic variation of pathogens in other lepidopteran host populations. This study shows encouraging results for further studies into the role of pathogens in the regulation of host insect populations.