【作者】 刘庆友；

【导师】 石德顺；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 本研究主要探讨人TPO基因牛体细胞定点整合载体构建及其进行细胞转染的可行性。参照奶牛、绵羊、山羊等的β-酪蛋白序列设计了一对引物P1、P2，以本地水牛和内蒙古绵羊基因组为模板，采用高保真Ex Taq酶，扩增得到水牛、绵羊β-酪蛋白基因的5′调控区序列(分别长4.5Kb、4.1Kb)。将扩增产物克隆到pMD-18T上，经酶切分析、部分序列测定和同源比较确认得到了所需的基因片段。测序结果与水牛和绵羊的相应序列同源性分别达到98％(833bp)、98％(735bp)。利用已克隆的TPO基因(5.5Kb)、奶牛as1-酪蛋白基因同源臂序列(6.0Kb和1.78Kb)、水牛和绵羊β-酪蛋白5′调控区序列和ploxp质粒骨架，经多步酶切-连接-转化-筛选-鉴定基因重组过程(连接片段不经过脱磷酸化处理)，最终建成两个人TPO基因牛体细胞打靶载体：ploxp-6.0-BβCN-TPO-1.78和ploxp-6.0-SβCN-TPO-1.78，长达25Kb。采用PCR和酶切分析对构建载体进行全面验证，确认得到了设计的打靶载体。采用脂质体法，将线性化的打靶载体ploxp-6.0-SβCN-TPO-1.78导入体外培养到第3代的奶牛成纤维细胞，采用G418+GANC进行筛选，筛选到第24天时，得到抗性细胞。 结果表明：(1)应用LA-PCR技术，采用高保真Ex Taq酶能扩增得到绵羊和水牛beta-casein基因的5′调控区序列。(2)绵羊、水牛β-酪蛋白基因的5′调控区(启动子)、人血小板生成素(TPO)基因(目的基因)，靶向奶牛as1-酪蛋白基因的长、短同源臂(靶向基因)等基因元件不经脱磷酸化处理也可成功连接到ploxp质粒载体骨架上，构建成两个长25Kb的人TPO基因牛体细胞定点打靶载体。(3)构建得到的人TPO基因牛体细胞定点打靶载体线性化后用脂质体法进行细胞转染，长期筛选后能得到抗性细胞。更多还原

【Abstract】 This study was undertaken to explore the feasibility of constructing a vector to integrate human TPO gene to as 1-casein locus of bovine genome and transfecting this vector to bovine somatic cells through lipofectamine. Consulting beta-casein sequences of bovine, ovine and goat, a pair of primers was selected to amplify the 5 ’regulation region of beta-casein gene of buffalo and ovine through LA-PCR, which are about 4.5Kb, 4.1Kb separately. These two fragments were cloned into the pMD-18T vector for the partial sequencing and restriction enzyme analysis. Homologous sequence alignment indicated 98% homology with that of sequences in GeneBank and the restriction enzyme digestion results were consistent with the design. Two TPO gene bovine somatic cells targeting vectors, ploxp-6.0-B|3CN-TPO-1.78 and ploxp-6.0-SpCN-TPO-1.78, had been constructed, which are about 25Kb and are consisted of five parts: ploxp skeleton, 6.0Kb homologous left arm, 1.78Kb homologous right arm, 5.5Kb tpo gene and 4.1Kb, 4.5Kb 5’ regulation region. Analysis by LA-PCR and restriction enzymes digestion proved that the genetargeting vectors were identical to the design. The lined ploxp-6.0-SpCN-TPO-1.78 wastransfected into the fetal bovine fibroblast cells by lipofectamine and the transfected cellswere selected by G418 and GANC, a few of Aanti-neomycin cells were gotten after 24 daysof selection culture.In conclusions, 1) LA-PCR and Ex Taq can be used to amplify the 5’regulation region of buffalo and ovine. 2) Two 25Kb human TPO gene bovine somatic cells targeting vectors can be constructed by linking buffalo and ovine 5’ regulation region (promotor), human TPO gene (purpose gene) and bovine as 1-casein gene homologous elements (targeted gene) into the ploxp plasmid vector without de-phosphatization. 3) Aanti-neomycin cell clone can be obtained by targeting these vectors (ploxp-6.0-SpCN-TPO-1.78) into bovine fetal fibroblasts through lipofectamine and long time culture selection.更多还原