小鼠α1,3-半乳糖基转移酶RNA干扰重组慢病毒的构建与鉴定  

Constructing and identifying a recombinant lentiviral vector of RNA interference carrying murine alpha-1, 3-galactosyltransferase gene

作　　者：吕承育[1] 谷雅川[2] 戚峰[1] 朱理玮[1] 

机构地区：[1]天津医科大学总医院,天津市300052 [2]郑州大学第三附属医院,河南省郑州市450052

出　　处：《中国组织工程研究与临床康复》2009年第31期6089-6092,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30500489)~~

摘　　要：背景:研究表明,通过抑制α1,3-半乳糖基转移酶的生成而减少甚至避免供体Galα(1,3)Gal的生成,是渡过器官移植后超急性排斥反应的一条切实可行的方法。目的:构建小鼠α1,3-半乳糖基转移酶基因的RNA干扰慢病毒载体,有效沉默小鼠血管内皮细胞的α1,3-半乳糖基转移酶基因表达,以期为有效控制异种移植超急性排斥反应提供稳定的转染细胞载体。设计、时间及地点:单一样本观察,于2006-07/2007-05在天津医科大学总医院普外研究所完成。材料:慢病毒载体系统(四质粒)购自Tronolab公司,包装细胞293T细胞株购自中科院上海细胞所。方法:在线软件设计小鼠α1,3-半乳糖基转移酶基因发卡小分子干扰RNA片段。合成的双链DNA片段并将其连接到Tele-sh003/U6.2载体的黏性末端,产生重组质粒Tele-sh003/U6.2-shRNA/α1,3-半乳糖基转移酶。对干扰质粒做测序分析。用磷酸钙法将得到的阳性重组子与慢病毒包装质粒共转化293T细胞,72h后收集含病毒上清液,超速离心后得到重组慢病毒。用实时定量聚合酶链反应测定病毒滴度。主要观察指标:干扰质粒的测序分析,慢病毒颗粒的包装和滴度测定。结果:小鼠α1,3-半乳糖基转移酶基因小发卡RNA片段被成功克隆进Tele-sh003质粒中,测序结果显示干扰质粒小干扰RNA编码序列与设计片段的序列完全一致。所得慢病毒亦为阳性克隆,经定量聚合酶链反应测定病毒滴度为2×108Tu/mL。结论:成功构建出小鼠α1,3-半乳糖基转移酶基因的小发卡RNA慢病毒RNA干扰表达载体,为进一步控制异种移植超急性排斥反应提供了稳定的转染细胞的载体。BACKGROUND： To inhibit the generation of α-1, 3-galactosyltransferase （α-1, 3-GT） in order to reduce or even inhibit the production of Gala（1,3）Gal is a feasible method to prevent hyperacute rejection following organ transplantation. OBJECTIVE： To construct a recombinant lentiviral vector of RNA interference （RNAi） on murine α-1, 3-GT and to effectively silence the gene expression in vascular endothelial cells in order to well control hyperacute rejection following xenotransplantation and provide stable transfected cell vector. DESIGN, TIME AND SETTING： A single sample study was performed at General Surgery Institute, General Hospital of Tianjin Medical University from July 2006 to May 2007. MATERIALS： Lentiviral vector system was provided by Tronolab Company, and 293T cell strain was provided by Shanghai Cytology Institute of Chinese Academy of Sciences. METHODS： α-1, 3-GT shRNA sequence of mouse was designed by on-line designer software. Double strand oligonucleotides were synthesized and cloned onto the Tele-sh003/U6.2 plasmid to obtain Tele-sh003/U6.2-shRNA/α-1,3-GT. DNA sequencing was used to examine the recombinant plasrnid. 239T cell line was transfected by the positive clone and the packing plasmids of lentivral vector. The supernatant with lentMrus was collected and centrifuged for 72 hours to get positive lentivirus. The virus titer was determined by real time PCR. MAIN OUTCOME MEASURES： Sequence analysis of interference plasrnid, packing of lentivral vector, and virus titer. RESULTS： α-1, 3-GT shRNA expression fragment was successfully inserted into the Tele-sh003 plasmid. The result of DNA sequencing analysis showed that the coding sequence of the shRNA in interfering plasmid was identical with the designed one. The lentiviral vectors were all positive clones, and the virus titer was 2×10^8 Tu/mL. CONCLUSION： Lentiviral shRNA expression vector of murine α-1, 3-GT gene for RNAi was constructed successfully. It provided an effective vector to control the hyperacute re

关 键 词：Α1 3-半乳糖基转移酶 RNA干扰 质粒 慢病毒 小鼠 

分 类 号：R394.2[医药卫生—医学遗传学]