【作者】 钱莉；

【导师】 季明春； 李厚达；

【作者基本信息】 扬州大学 ， 动物学， 2004， 硕士

【摘要】 慢性髓性白血病（Chronic myeloid leukemia, CML）是一种起源于多能干细胞的恶性增生性疾病，其最重要的细胞遗传学特征是存在特异性的Ph染色体，第9号染色体上的原癌基因c-ab1易位到第22号染色体的断点集中区（bcr）形成bcr-ab1融合基因，编码bcr-ab1融合蛋白(p210^bcr-ab1)。p210^bcr-ab1具有高酪氨酸激酶活性，与细胞恶性转化密切相关，而p210^bcr-ab1的抗原性限定在其融合位点内，这段氨基酸序列在正常细胞中不存在，因此该序列可作为肿瘤特异性抗原。本研究从以下三个方面探索利用bcr-ab1融合基因片段制备CML基因疫苗的研究。 一、慢性髓性白血病bcr-ab1融合基因片段的克隆与原核表达的研究 以慢性髓性白血病细胞株K562细胞总RNA为模板，采用RT-PCR技术扩增包含bcr-ab1融合位点周围的基因片段，定向克隆到pGEX-6P-1载体谷光甘肽-S-转移酶（GST）的下游。将重组质粒转化大肠杆菌BL₂₁菌株，以1.0mmol／l IPTG诱导bcr-ab1融合基因片段的表达，其产物经聚丙烯酰胺凝胶电泳鉴定，结果证实该融合蛋白分子量约42KD。用该表达产物免疫ICR小鼠，所制备的抗血清可与K562细胞中特异性抗原结合，表明该bcr-ab1／GST融合蛋白具有bcr-ab1基因产物的特异抗原性。 二、慢性髓性白血病bcr-ab1融合基因片段真核细胞表达的研究 根据ber一abl（b3aZ）融合基因序列设计一对引物，以CML K562细胞总RNA为模板，通过Rl’- pCR扩增包含忱r.abl融合位点周围的450bP的基因片段，并将其克隆进pGEM一T easy载体。经序列测定证实其阅读框架正确后，将b叮‘abl融合基因片段正向插入真核表达载体peDNA3 .1。以脂质体介导重组质粒PcDNAbe卜abl转染CHO细胞，间接免疫荧光检测结果显示，peDNAbcr-abl转染CHO细胞的免疫荧光强度高于K562阳性对照细胞。用该重组质粒DNA免疫BALB/c小鼠可产生特异性抗体。 三、稳定表达忱r‘abl融合基因片段的人一鼠嵌合肿瘤细胞系的建立 从重组克隆载体pGEMber.abl中酶切出bcr.abl融合基因片段，并将其亚克隆进反转录病毒载体pLxsN中。脂质体介导重组反转录病毒载体pLXsNber.abl转染包装细胞PT67，G418筛选后获得稳定产病毒的包装细胞。收集病毒上清感染sPZ/o细胞（H一Zd），经G418筛选获得稳定表达忱卜abl融合基因片段的Sp刀0细胞株。PCR证实bcr-abl融合基因片段已稳定整合于SP2/0细胞基因组中，RT-PCR证实感染后的SPZ/0细胞能稳定表达b二.abl融合基因片段，从基因组整合和基因表达水平证实我们获得了能稳定表达加卜abl融合基因片段的人一鼠嵌合肿瘤细胞系，作为研究be卜abl融合基因疫苗的实验工具。更多还原

【Abstract】 The crucial genetic event in Chronic myeloid leukemia is the generation of a t (9;22)(q34;qll) reciprocal chromosomal translocation in a hematopoietic stem cell. This translocation creates a new gene, bcr-abl on the 22q" or Ph chromosome, and the reciprocal abl-bcr on the derivative 9q+. The final product of this genetic rearrangement is a 210 kDa cytoplasmic fusion protein, or p210 bcr"abl. The bcr-abl protein is leukemogenic because its Abl-derived tyrosine kinase is constitutively activated. This leads to malignant transformation by interference with basic cellular processes. The chimeric p210 fusion protein is a potential antigen as the junction regions contain a sequence of amino acids that is not expressed on any normal cell. Therefore, the unique sequence can be considered truly as a tumor specific antigen of CML. The study focused on the bcr-abl fusion gene vaccine, and consisted of three sections as the following:1. Cloning and expression of CML bcr-abl fusion gene fragment in proeukaryotesThe 450 bp of bcr-abl fusion gene was amplified from total RNA of K562 cells by RT-PCR and was cloned into the dowmstream of GST gene in pGEX-6P-l vector. The recombinant vector was transformed into E.coli BL2i cells for expression. Fusion protein was 42 KD identified by SDS-PAGE. The ICR mice were immunized with the fusion protein. The polyclone antibody was combined to K562 cells which express p210 bcr-abl through indirect immune fluorescence immunoassay. This result suggested that the fusion protein had specific immunogenicity of p210 bcr-abl.2. Molecular cloning and expression of CML bcr-abl fusion gene fragment in eukaryotesBased on the gene sequence encoding bcr-abl (b3a2) fusion gene from chronic myelgenous leukemia K562 cell line, we designed a pair of primers, obtained the 450bp gene fragment of bcr-abl by using RT-PCR, and inserted the gene into plasmid pDNA3.1 via pGEM-T easy vector to construct recombinant plasmid. The recombinant plasmid pcDNAbcr-abl was transfected into CHO cells with liposome. The recombinant plasmid was transiently expressed the fragment of bcr-abl fusion protein hi cytoplasm of CHO cells confirmed by indirect immune fluorescence immunoassay. The BALB/c mice inoculated with pcDNAbcr-abl by intramusular injection could elicit to produce corresponding antibodies to K562 cells expressing p210 bcr-abl.3. Establishment chimeric human-murine tumor cell lines which satably expressed bcr-abl fusion fragmentThe bcr-abl fusion gene fragment was subcloned into retroviral vector pLXSNfrom pGEMbcr-abl. The recombinant retroviral vector pLXSNbcr-abl was transfected into PT67 packaging cells with the help of lipofectamine. The positive clones were selected out and cultured after G418 selection. Then viral supernatant was collected. The SP2/0 cells were infected with the collected viral supernatant. After G418 selection, we observed that the bcr-abl fusion gene fragment was integrated into the chromosome of SP2/0 cells infected with recombinant retrovirus stably confirmed by PCR and expressed in SP2/0 cells confirmed by RT-PCR respectively. Chimeric human-murine tumor cell lines expressing the fragment of bcr-abl fusion protein were established and would be used as experimental cell model for anti-CML immunotherapy.更多还原