PCBP1 RNAi重组慢病毒包装及筛选其稳定感染的神经细胞系

目的 PCBP1是真核细胞内的一种桥梁蛋白,具有多种生物学功能。本研究以慢病毒质粒构建PCBP1基因小干扰RNA(small interfering RNA,siRNA)表达载体,并筛选其稳定感染的神经细胞系,为进一步通过该载体系统探讨PCBP1的具体功能提供有力手段。方法设计合成针对人及小鼠的PCBP1基因的siRNA茎环结构的正反义序列,退火形成双链,与pLenti-Lox3.7载体的双酶切产物连接,以质粒PCR、酶切和DNA测序等方法,对重组克隆进行鉴定以确认目的片段的插入。之后,采用脂质体转染,将慢病毒载体系统的核心载体pLentiLox3.7和包装质粒共转染293T细胞,收获病毒颗粒,分别感染SH-SY5Y细胞,再通过流式分选获得感染不同病毒颗粒的细胞,提取RNA检测PCBP1 mRNA水平。结果检测结果显示PCBP1 siRNA茎环结构序列正确插入pLentiLox3.7载体,并且成功构建PCBP1基因敲低的神经细胞系。结论 PCBP1基因siRNA表达载体及其基因敲低稳定细胞系的成功构建,为进一步研究该基因在相应细胞中的功能奠定了基础。

Packaging PCBP1 RNAi-lentivirus and Screening the Infected SH-SY5Y Cells

Objective PCBP1 poses multiple functions and it is an important member in eukaryocytes.To further determine the important function of PCBP1 in neurocytes,we constructed the recombinated RNAi-lentivirus and infected SH-SY5Y cells with the corresponding virus supernatant.Methods Four pairs siRNAs of PCBP1 were devised,synthesized and annealed to generate double strands.And then the four kinds of double strands were connected with pLentiLox3.7 to form recombinated plasmids.After identified with PCR,enzymic method and sequencing,transfacting 4 kinds of packaging plasmids into 293T cells was used to producing virus supernatant.Then SH-SY5Y cells were infected with different virus supernatant and the infected cells were sorting with aseptic flow cytometry.At last,total RNAs were extracted to detected PCBP1 mRNA levels with real-time PCR.Results PCBP1 shRNAs were correctly inserted pLL3.7 palsmid,and PCBP1 knocked-down neurocytes were successfully constructed.Conclusion PCBP1 siRNA recombinated plasmid and SH-SY5Y cells with PCBP1 knocked-down provided a platform to study this gene function in neurocytes.