CX3CL1 RNA干扰慢病毒载体的构建包装与鉴定

摘要 目的：构建包装趋化因子CX3C的配体1（CX3CL1）RNA干扰慢病毒载体。方法：参考目的基因CX3CL1序列，设计PCR引物扩增相应的干扰序列，然后将干扰序列连接至pLVX-shRNA2酶切后的线性化载体上，通过酶切及测序鉴定获得阳性克隆，即为构建成功的pLVX-shRNA2-CX3CL1慢病毒干扰载体。将构建好的慢病毒干扰载体同慢病毒包装载体共同转染293T细胞，收集上清，纯化浓缩后即为pLVX-shRNA2-CX3CL1慢病毒。最后将慢病毒感染BMSCs细胞，QPCR检测慢病毒的干扰效率。结果：经过酶切能切出大小约为6500bp和1350bp的两条带，获得与预期结果相一致的DNA片段，并通过测序验证了序列的准确性，成功构建CX3CL1 RNA慢病毒干扰载体。然后经过包装、纯化浓缩后得到pLVX-shRNA2-CX3CL1慢病毒。QPCR结果表明干扰组明显抑制了CX3CL1mRNA的表达，干扰效率在70%以上。结论：成功构建包装CX3CL1干扰慢病毒载体，并证实其显著沉默了BMSCs细胞CX3CL1的表达，为CX3CL1在BMSCs移植的大鼠缺血性脑卒中炎症反应的机制研究奠定基础。

Construction, Packaging and Identification of CX3CL1 RNAi Lentiviral Vector

ABSTRACT Objective: To construct and pack CX3CL1 RNAi lentiviral vector. Methods: With reference to the target gene CX3CL1 sequence, PCR primers were designed to amplify the corresponding interference sequence. Then the interference sequence was connected to pLVX-shRNA2 linearized vector.Positive clones were obtained by identification of PCR and digestion, which was a successful construction of pLVX-shRNA2-CX3CL1 Lentiviral interference vectors. The constructed RNAi lentiviral vector was transfected into 293T cells together with the packaging vectors. The supernatant was collected, purified and concentrated to pLVX-shRNA2-CX3CL1 lentivirus. Finally, BMSCs were infected with lentivirus, and the interference efficiency was detected by QPCR. Results: After digestion, two bands with 6500bp and 1350bp were obtained consistent with the expected results, and the sequence accuracy was verified by sequencing. The CX3CL1 RNAi lentiviral vector was successfully constructed.After packaging, purification and concentration, the pLVX-shRNA2-CX3CL1 lentivirus was obtained. QPCR results showed that the interference group significantly inhibited the expression of CX3CL1mRNA, and the interference efficiency was above 70%. Conclusion: The CX3CL1 RNAi lentiviral vector was successfully constructed and packed and significantly silenced the expression of CX3CL1 of BMSCs, which laid the foundation for the study of the mechanism of CX3CL1 on inflammatory response of ischemic stroke in BMSCs transplanted rats.