【作者】 李中奎；

【导师】 李云龙；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2003， 硕士

【摘要】 鸡贫血病毒（chicken anemia virus,CAV）是鸡传染性贫血病（chickeninfectious anemia,VIA）的病原，属于圆环病毒科（Circoviridae）。CAV基因组共编码三种病毒蛋白质：VP₁（51.6KD）、VP₂（24.OKD）和VP₃（13.6KD）。其中，VP₃蛋白质是鸡贫血病毒的致病因子，能选择性的诱导肿瘤细胞和转化细胞发生凋亡，但对人和鼠的正常二倍体细胞无影响，称为凋亡素（apoptin）。凋亡素诱导的肿瘤细胞凋亡不依赖于肿瘤抑制因子P53，并且不受原癌基因bcl-2表达产物的抑制，相反，过表达的Bcl-2蛋白质对其还有促进作用。凋亡素已成为肿瘤治疗的新希望，有望发展成为新的抗肿瘤制剂并在肿瘤预测与诊断中具有重要价值。 本研究根据已报道鸡贫血病毒凋亡素基因两端的特异性序列设计合成一对引物，通过PCR方法对分离到的鸡贫血病毒哈尔滨病毒株基因组进行扩增，将扩增得到的PCR反应产物进行琼脂糖凝胶电泳并回收凋亡素基因相应区带。回收的扩增产物经T₄DNA聚合酶末端补平后与用限制性内切酶Sma Ⅰ消化的pUC18质粒连接，构建重组质粒pUC-vp₃。重组质粒pUC-vp₃转化感受态大肠杆菌DH5 α，经蓝白斑筛选并提取质粒进行酶切鉴定，证明成功进行了基因的克隆和转化。通过测序，证明本实验克隆的基因由366个核苷酸组成，编码121个氨基酸。将克隆的基因与Genbank中收录的凋亡素基因的核苷酸和蛋白质的氨基酸序列进行比较，结果表明，其基因的核苷酸和蛋白质的氨基酸序列组成都具有已报道凋亡素序列组成的典型特点，证明本实验克隆的基因的确为凋亡素基因。序列分析结果表明，本实验克隆的凋亡素基因的编码产物可能具有较强的诱导肿瘤细胞发生凋亡的活性。 用限制性内切酶Nde Ⅰ、EcoR Ⅰ从重组质粒pUC-vp₃上切下vp₃基因，琼脂糖凝胶电泳并回收凋亡素基因相应区带。回收的凋亡素基因与用Nde Ⅰ、EcoR Ⅰ消化的pET30（a）质粒连接，构建原核表达载体pET-vp₃。回收的凋亡素基因连接到用限制性内切酶BamH Ⅰ、EcoR Ⅰ消化的pcDNA（3.1）质粒上，构建真核表达载体pcDNA-vp₃。原核表达载体pET-vp₃转化感受态大肠杆菌DE3并用IPTG化学诱导表达，表达产物经SDS-PAGE鉴定，证明凋亡素基因在DE3中获得了大量表达。表达的蛋白质产物经葡聚糖凝胶过滤和亲和层析两级纯化，纯化产物经SDS-PAGE鉴 凋亡素基因克隆、原核表达及蛋白质预测定，证明纯化得到了凋亡素蛋白质的纯品，从而进一步证实了凋亡素基因在DE3中的表达。这些结果为凋亡素功能的深化研究和医药开发创造了条件。 目前，对凋亡素的研究主要集中在其促进肿瘤和转化细胞选择性凋亡的功能方面，而对其作用机理和空间结构方面的报道较少。本实验利用An theprot和DNAstar软件对凋亡素的理化性质和高级结构进行了预测，并在Genbank中进行了 BLAST分析。从调亡素与蛋白质结构数据库（Pdb）的 BthST分析结果来看，与凋亡素存在形成相似结构序列片段的蛋白质主要是蛋白质酶类。这预示着凋亡素可能是通过发挥某种酶的作用来参与到细胞凋亡信号通路中，最终诱发肿瘤细胞凋亡的。尤其值得注意的是BLAST结果中具有磷脂酶活性、核糖核苦酸还原酶活性和尿音酸激酶活性的几种蛋白质多肽。凋亡素与它们的长序列相似性表明，它可能具有磷脂酶活性，在细胞信号通路中起作用；或者具有核糖核苦酸还原酶和尿苦酸激酶活性而对hnRNA发挥作用；或者是与其中具有某种活性的蛋白质存在竞争性抑制作用。对凋亡素蛋白质的预测分析将对凋亡素的结构与功能的进一步研究具有一定的指向意义。更多还原

【Abstract】 Chicken anemia virus (CAV) is the pathogen of chicken infectious anemia (CIA). CAV belongs to circoviridae. CAV genome encodes three kinds of protein: VP1 (51.6KD), VP2 (24. OKD) and VP3 (13.6KD). Apoptin (VP,) can selectively induce apoptosis in human transformed and malignant cells but not in normal diploid cells. Apoptin induces a p53-independent, Bcl-2-insensitive type of apoptosis in various human tumor cells. Apoptin is becoming a potential candidate as a therapeutic and diagnostic tool in cancer treatment.A pair of primers were designed and synthesized according to the conservative sequence of vp3 gene. The fragment amplified by PCR technique was enriched by electrophoresis and jointed into plasmid pUC18. The recombined plasmid pUC18-vp3 was got. The sequence of the fragment amplified by PCR was analyzed with F primer. It was found that it had well homology to the reported apoptin genes by comparing the gene with all genes in Genbank and Shandong strain SJ1 of China.The vp3 gene was cloned into pET30(a) and pcDNA(3. 1) vectors separately. The recombined plasmids pET30-vp3 and pcDNA-vp3 were got. The pET30-vp3 was induced to express in E. coli DE3 by IPTG, then the expression was purified by G-75 filtration and affinity chromatography. It was proved that the protein was got by SDS-PAGE. The successful cloning and expressing of vp3 gene become the basis for the study and exploiture of apoptin.The physical and chemical characters and secondary structure of apoptin were predicted with softwares Antheprot and DNAstar. According to the result from BLAST, apoptin maybe take part in the signal pathway of programmed cell death with activity of enzyme. Especially, apoptin is partially similar with ribonucleotide reductase, uridylate kinase and phosphotriesterase in the amino acid sequence. The similarity between them indicates that apoptin maybe takes part in the signal pathway of apoptosis with phosphotriesterase activity or operates on hnRNA with ribonucleotide reductase or uridylate kinase activity.更多还原