大鼠Trail基因慢病毒载体的构建及鉴定

发布时间：2018-05-26 22:19

本文选题：慢病毒 + 载体　；参考：《泸州医学院》2012年硕士论文

【摘要】：目的:本实验用PCR法扩增出的Trail基因引入慢病毒载体系统，生产Trail慢病毒颗粒并进行病毒滴度检测。方法:1.Trail基因的获得：根据GenBank中大鼠Trail基因序列(NM145681.1)，设计出该基因的特异性引物Trail-Agel-F和Trail-Agel-R，并且使用Agel酶的切位点。用PCR法从大鼠cDNA文库中扩增出目的基因。2.重组质粒的构建：采用In-Fusion技术，将酶切回收后的PCR产物线性交换连接入Agel酶切的真核表达载（GV218），产生目的重组质粒。连接产物质粒转化大肠杆菌DH5a感受态细胞，扩增目的重组质粒。3.连接产物的酶切鉴定：收集扩增后的连接产物，酶切后经电泳发现得到片段长度均与预期相符，基因测序结果亦正确，从而证明克隆Trail基因已成功。4.重组质粒转染293T细胞：用脂质体Lipofeetamine2000包裹构建的重组质粒和辅助包装载体，，三质粒共同转染293T细胞，产生含有表达Trail蛋白的Lentivirus病毒颗粒。5.病毒检测：重组质粒转入293T细胞,荧光显微镜下观察GFP的表达，行Western blot鉴定及使用Real-time定量PCR法检测病毒滴度。结果:成功得到Trail目的基因，重组质粒测序结果与GenBank中Trail基因序列(NM145681.1)比较，证实Trail基因序列正确；三质粒转染293T细胞后荧光显微镜观察到绿色荧光；WesternBlot检测观察到58KD附近处有特征条带，其大小和目的基因融合蛋白相吻合；孔稀释法及实时荧光定量PCR病毒滴度测定法测定结果为2E十8TU/ml。结论:成功克隆大鼠Trail基因和成功构建慢病毒载体系统并检测其滴度。为后续把有杀灭肿瘤的基因（Trail）转导进神经干细胞奠定基础。
[Abstract]:Aim: the Trail gene amplified by PCR method was introduced into the lentivirus vector system to produce Trail lentivirus particles and to detect the virus titer. Methods: 1. Obtaining the gene of Trail: according to the sequence of rat Trail gene NM145681.1 in GenBank, we designed the specific primers Trail-Agel-F and Trail-Agel-Rand used the restriction site of Agel. The target gene. 2. 2 was amplified from rat cDNA library by PCR. Construction of recombinant plasmid: by using In-Fusion technique, the recovered PCR products were linearly exchanged into the eukaryotic expression of Agel digested with GV218G, and the target recombinant plasmid was produced. The recombinant plasmid was transformed into Escherichia coli DH5a competent cells and the target recombinant plasmid was amplified. Identification of ligation products by enzyme digestion: the ligation products were collected and amplified. The results of electrophoresis showed that the length of the ligand fragments were in accordance with the expected results, and the sequencing results were correct, which proved that the cloned Trail gene had been successfully cloned. 4. The recombinant plasmid was transfected into 293T cells: the recombinant plasmid and the auxiliary package vector were constructed by liposome Lipofeetamine2000. The three plasmids were co-transfected into 293T cells to produce Lentivirus virus particles. 5. Virus detection: the recombinant plasmid was transferred into 293T cells. The expression of GFP was observed under fluorescence microscope. Western blot identification and Real-time quantitative PCR assay were used to detect the titer of the virus. Results: the target gene of Trail was successfully obtained, and the sequence of the recombinant plasmid was compared with the sequence of Trail gene in GenBank (NM145681.1), which confirmed that the sequence of Trail gene was correct. After the transfection of the three plasmids into 293T cells, the Western blot analysis showed that there were characteristic bands near 58KD, the size of which was consistent with the target gene fusion protein. The results of pore dilution method and real-time quantitative PCR titer assay were 2e + 8TU / ml. Conclusion: the rat Trail gene was cloned and the lentivirus vector system was successfully constructed and its titer was detected. To lay the foundation for the subsequent transduction of tumor killing gene Trail into neural stem cells.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392-33

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